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Substrate and inhibitor interactions of glyceraldehyde 3-phosphate dehydrogenase and intramolecular general base-catalyzed alcoholysis of amides and esters

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Substrate and inhibitor interactions of glyceraldehyde 3-phosphate dehydrogenase and intramolecular general base-catalyzed alcoholysis of amides and esters

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Content SUBSTRATE AND INHIBITOR INTERACTIONS OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE AND INTRAMOLECULAR GENERAL BASE CATALYZED ALCOHOLYSIS OF AMIDES AND ESTERS by Bruce Monte Benjamin A D isse rtatio n Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In P a rtia l F u lfillm e n t of the Requirements fo r the Degree DOCTOR OF PHILOSOPHY (Biochemistry) June 197A UNIVERSITY OF SOUTHERN CALIFORNIA T H E GRADUATE S C HO O L U N IV E R S IT Y PARK LOS A N G E LE S , C A L I F O R N IA 9 0 0 0 7 This dissertation, written by B ruce .Monte Benj\ami n................................. under the direction of k.l?.... Dissertation Com mittee, and approved by a ll its members, has been presented to and accepted by The Graduate School, in partial fulfillm ent of requirements of the degree of D O C T O R O F P H IL O S O P H Y f ' T n Dean DISSERTATION C O M M ITTEE j J & g A j Chairman ACKNOWLEDGEMENTS I wish to thank Professor Thomas H. F ife , whose ideas and guidance have been invaluable as an example in my graduate education. Also, the laboratory post-doctorates— Tadaaki R ikih isa, Edwin Anderson, and Jon Hutchins—were a ll generous with th e ir time and knowledge with problems o f laboratory technique and procedure. However, my greatest debt is to my w ife , Susan, whose confidence inspired my continued determination to reach this goal. TABLE OF CONTENTS Chapter Page Number ACKNOWLEDGEMENTS.................................................................................................. ii LIST OF TABLES....................................................................................................... v LIST OF ILLUSTRATIONS v ii i INTRODUCTION........................................................................................................... 1 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE.................................. 2 The Reactions of G PD Subunit Interactions Objectives of Study oC-CHYMOTRYPSIN ESTER AND AMIDE HYDROLYSIS............................... 7 The Chymotrypsin Reaction Model Studies Objectives of Study PART I . SUBSTRATE AND INHIBITOR INTERACTIONS OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE............................ II EXPERIMENTAL........................................................................................... 12 Enzyme Preparations M aterials and Syntheses Rate Measurements and Spectra Calculations RESULTS........................................................................................................... 19 DISCUSSION.................................................................................................... 55 Summation PART I I . INTRAMOLECULAR GENERAL BASE CATALYZED ALCOHOLYSIS OF AMIDES AND ESTERS........................................................................ 70 EXPERIMENTAL................................................................................................ 71 M aterials and Syntheses Rate Measurements and Spectra Calculations TABLE OF CONTENTS (CONTINUED) Chapter Page Number RESULTS................................................................................................................ 83 DISCUSSION........................................................................................................... 118 IMPLICATIONS FOR ot-CHYMOTRYPSIN CATALYZED REACTIONS.......................................................................................................... 140 REFERENCES CITED......................................................................................................... 1^6 LIST OF TABLES T ab le - Page No. 1. Reaction of Glyceraldehyde 3-Phosphate Dehydrogenase with Cinnamaldehyde in the Absence and Presence of Arsenate at 2 5 °........................... 23 2. In hib itio n of the Dehydrogenase A c tiv ity of Glyceraldehyde 3-Phosphate Dehydrogenase with Cinnamaldehyde by Trimethylacetyl Phosphate at 25 °.........................................................................• 24 3. Trimethylacetyl Phosphate In h ib itio n of the Cinnamaldehyde Reaction with Glyceraldehyde 3-Phosphate Dehydrogenase and the Effect of Arsenate......................................................................................... 32 4. Acyl Phosphatase A c tiv ity of Glyceraldehyde 3-Phosphate Dehydrogenase with 0 - (2-F u ryl) - acryloyl Phosphate and the Effect of Trimethylacetyl Phosphate In h ib itio n .................................. 38 5. Trimethylacetyl Phosphate In h ib itio n of the Acyl Phosphatase A c tiv ity of Glyceraldehyde 3-Phosphate Dehydrogenase at 25° with /3“ (2-Fury 1)acryloyl Phosphate............................................... 42 6. Reaction of /S— (2-Fury1)acrylaldehyde with Glyceraldehyde 3-Phosphate Dehydrogenase at 25° and the Effects of Trimethylacetyl Phosphate and Cyclic AMP............................................................. 45 7. Trimethylacetyl Phosphate In h ib itio n of the Reaction of /S-(2-Fury1)aerylaldehyde with Glyceraldehyde 3"Phosphate Dehydrogenase at 2 5 °......... 8. Cyclic AMP In h ib itio n of the Reaction of /s-(2-Furyl)acrylaldehyde with Glyceraldehyde 3-Phosphate Dehydrogenase at 2 5 °........................................... 52 9. K j j , Values for Various Substrates of Glyceraldehyde 3-Phosphate Dehydrogenase........................... 57 10. Analytical Analysis Results fo r the Ethyl Esters and Benzamides................................................................... 75 v LIS T OF TABLES (CONTINUED) T a b le Page No. 11. Experimental Wavelengths Used to Observe the C yclizatio n Reactions...................................... 80 12. Observed Pseudo-First-Order Rate Constants fo r the Imidazole-Catalyzed C yclizatio n o f Ethyl 2-Hydroxymethylbenzoate to P hthalide.............. 84 13. Observed Pseudo-First-Order Rate Constants fo r Buffer-Catalyzed C yclizatio n o f Ethyl 2-Hydroxymethyl benzoate................................................................. 88 14. Rate Constants fo r Spontaneous and General Base Catalysis of, the C y clizatio n o f Ethyl 2-Hydroxymethyl benzoate................................................................. 89 15. Rate Constants fo r Spontaneous and General Base Catalysis o f the C y clizatio n o f Ethyl 2-Hydroxymethyl-4-ni tro b en zo ate............................................. 96 16. Rate Constants fo r Spontaneous Catalysis o f the C y clizatio n o f 2-Hydroxymethyl-3-amino- benzamide to 4-Ami nophthal id e ................................................... 99 17. Rate Constants fo r Spontaneous and General Base C atalysis o f the C y clizatio n of 2-Hydroxymethyl-4-aminobenzamide to 5"Anino phthal id e ................................................................................................ 100 18. Rate Constants fo r Spontaneous and General Base Catalysis o f the C y clizatio n o f 2-Hydroxymethyl-6-aminobenzamide to 7”Anino phthal id e ......................... 101 19. Temperature Dependence and A ctivatio n Parameters fo r the pH-lndependent C yclizatio n of 2-Hydroxymethyl -3-ami nobenzami de............................................ 106 20. Observed Pseudo-First-Order Rate Constants fo r the Imidazole-Catalyzed C y clizatio n of 2-Hydroxymethyl-6-aminobenzamide to 7“Anino phthal id e 108 vi LIST OF TABLES (CONTINUED) T able Page No. 21. Rate Constants for Spontaneous and General Base Catalysis of the C yclization o f 2-Hydroxymethyl-5 -ami nobenzami de to 6-Aminophthalide.................................................................................. 113 22. Comparison of Rate Constants fo r Cyclization of Esters and Amides........................................... 117 23. K inetic Factors Responsible fo r the Difference Between the Hydroxide Ion and «c-Chymotrypsin Catalyzed Hydrolysis of N-Acetyl-L-tryptophanamide.................................................. \k$ v i i Fi gure 1. 2. 3. k. 5. 6. LIST OF ILLUSTRATIONS Page No. Comparison of u ltr a v io le t spectra fo r NADH (71), tra n s -cinnamovl-papai n (12), and /3- (2-fu ry 1)-GPD (5 7 )..................................................................... 17 Percentage o f retained a c tiv ity o f glyceraldehyde 3-phosphate dehydrogenase versus hours o f incubation o f the enzyme a t 25° before reaction with 5*67 x 10"3 M benzaldehyde in 0.3 M Tris b u ffe r, pH 7*85............................. 21 Plots o f 1/V versus 1 / (S) fo r reaction of cinnamaldehyde with glyceraldehyde 3-phosphate dehydrogenase in the absence and presence of 1.32 x <0“2 m arsenate........................................................... 27 Plots of 1/V versus 1 / (S) fo r reaction of cinnamaldehyde with glyceraldehyde 3- phosphate dehydrogenase at 25° In the absence and presence o f 2.73 m M and 5*65 ^ trim ethylacetyl phosphate................................................................................................ 29 Plot of 1/V versus trim ethylacetyl phosphate concentration fo r reaction o f 3*56 x 10"^ M cinnamaldehyde with glyceraldehyde 3"phosphate dehydrogenase a t 25° in the absence of arsenate................................................................................................... 33 Plot o f 1/V versus trim ethylacetyl phosphate con centration fo r reaction o f 3.56 x 10”^ M cinnamaldehyde with glyceraldehyde 3"Phosphate dehydrogenase at 25° In the presence of 0.0132 M arsenate.................. 35 Plot of 1/V versus 1 / (S) fo r the reaction o f ^ -(2 -fu r y l)a c r y lo y l phosphate w ith glyceralde hyde 3~phosphate dehydrogenase a t 25° i n the absence and presence of 2 .0 x 10"’ M trim ethylacetyl phosphate............................................................ 39 vi i i LIST OF ILLUSTRATIONS (CONTINUED) Figure Page No. 8. Plot o f 1/V versus trim ethylacetyl phosphate concentration for reaction o f 5 .0 x 10"4 M or 3.5 x 10"^ M Z 3 -(2 -fu r y l Jacryloyl phosphate w ith glyceraldehyde 3-phosphate dehydrogenase at 2 5 °...................................................................... 4-3 9. Plot of 1/V versus 1 / (S) fo r reaction of /3-(2-fu rylJacrylald eh yd e w ith glyceraldehyde 3-phosphate dehydrogenase at 25 in the absence of in h ib ito rs , with 5 .0 x 10“3 M trim ethylacetyl phosphate, or with 2 .0 x 10“2 M cyclic AMP................................................................................4-6 10. Plot of 1/V versus trim ethylacetyl phosphate concentration fo r reaction o f 1.0 x 10"4 M and 3*33 x 10"^ M /3 -(2 -fu ry l )acrylaldehyde with glyceraldehyde 3-phosphate dehydrogenase at 2 5 °........................................................................................................ 49 11. Plot o f 1/V versus c y c lic AMP concentration fo r reaction of 1.0 x 10"3 M 8 - (2 -fu ry l ) - acrylaldehyde with glyceraldehyde 3-phosphate dehydrogenase at 2 5 °...................................................................... 53 12. Scheme fo r the reactions and conformation changes in the reaction o f glyceraldehyde 3-phosphate dehydrogenase.......................................................... 63 13. Typical u ltr a v io le t spectra fo r the ester and amide substrates and th e ir products.............................. 78 14. Plot o f kobs versus the imidazole fre e base concentration fo r c y c liza tio n o f ethyl 2-hydroxymethylbenzoate to phthalide at 30° and at pH 7.13, 7.28, and pH 7 .6 1 ...................................85 15. Br^nsted plot o f log kB versus the pKa of the catalyzing base in the c y c liza tio n of ethyl 2-hydroxymethylbenzoate to phthalide at 30° in H 20 .........................................................................................90 ix LIST OF ILLUSTRATIONS (CONTINUED) Figure Page No. 16. Plot o f log k versus pH p ro file fo r c y c liz a tio n of ethyl 2 -fiydroxymethy1 benzoate and ethyl 2-hydroxymethyl-4-nitrobenzoate to phthalide and 5 _nitrophthal ide a t 30° in H 20 ....................................... 93 17. Brjrfnsted plo t of log kg versus the pKa o f the catalyzing base in the c y c liza tio n of ethyl 2-hydroxym ethyl-4-nitrobenzoate to 5 — n i trophthal i de at 30° in H2O................................................ 97 18. Plot o f log kQ versus pH fo r spontaneous c y c li zation of 2-hydroxymethyl-3-aminobenzamide, 2-hydroxymethyl-4-aminobenzamide, and 2-hydroxymethyl-6-aminobenzamide at 30° ............................ 102 19. Plot of k0tjS versus the imidazole fre e base concentration fo r c y c liza tio n of 2-hydroxy methyl -6-ami nobenzami de to 7-aminophthalide at pH 7*16 and 7-49 in H 2O at 30° and at pD 7.99 in D20 ..................................................................................... 109 20. Brj^nsted plot of log k(3 versus the pKa o f the catalyzing base in the c y c liza tio n o f 2 - hydroxymethy1-4-aminobenzamide to 5-amino phthal ide at 30° in H 20 ...................................................................I l l 21. Br0nsted p lo t o f log kg versus the pKa o f the catalyzing base in the c y c liza tio n o f 2 - hydroxymethyl-6-aminobenzamide to 7_amino phthal ide and 2-hydroxymethyl-5-aminobenzamide to 6-ami nophthal i de at 30° in H 20 ............................................114 x INTRODUCTION A large amount of biochemical research has had as a common goal the solution of certain basic questions of enzyme catalysis. What are the mechanisms of enzyme interaction? How do enzymes catalyze the commonly observed huge rate accelerations? Jencks (47) has pointed out that the problem of enzyme a c tiv ity can be studied through two d if f e r ent general approaches: (1) examination of the properties of the enzyme it s e lf (for example, enzyme-substrate complexes, the enzyme- catalyzed reaction, subunit in te ra ctio n s ); or (2) investigation of nonenzymatic reactions to e lu c ita te the basic chemistry of the enzyma tic reaction. This two-part d issertatio n presents an example of each approach. The enzyme-catalyzed reaction and subunit interactions resulting from the reaction of glyceraldehyde 3~phosphate dehydrogenase with various substrates and inhib itors to form an acyl-enzyme reveal that very complex regulatory mechanisms can occur in the a c tiv ity of this enzyme. On the other hand, considerable insight in the actual chemical reaction mechanism o f °c-chymotrypsin can be inferred from nonenzymatic study o f the cy c liza tio n of ester and amide derivatives of 2-hydroxymethylbenzoic acid. It is hoped this work w ill provide increased understanding of enzymatic and bio-organic problems. 1 2 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE The Reactions o f Glyceraldehyde 3 - Phosphate Dehydrogenase D-Glyceraldehyde 3-phosphate dehydrogenase (D-Glyceraldehyde 3“ phosphate: NAD oxidoreductase (phosphorylating), EC 1 .2 .1 .1 2 ) is a key enzyme o f carbohydrate metabolism, catalyzin g several d iffe r e n t reactions depending on reaction conditions (1 8 ). The physiological dehydrogenase reaction (21) involves the conversion o f D-glyceraldehyde 3-phosphate to 1,3-diphosphoglyceric acid in a reve rs ib le reac tio n , as + shown in Equation I . NAD is required as a cofactor and is converted to NADH in the reactio n . In the presence o f arsenate the product is 3 - phospho-D-glyceric acid . The reaction sequence involving oxidation O o 0 . ■ *. ii *r_u NflD N*DH c-o- p -o i V V 1 H -C -O W + HP0 ; a - • I r\~ ■ 1 1 ° C H ,~ 0 - P - C T C H . - 0 - P - 0 " H II O 0 and o x id a tiv e phosphorylation has been implicated as a major control point in glycolysis (32, 79)• In a d d itio n , an acyl phosphatase a c t iv it y has been noted in the presence of NAD+ (18, 40, 63, 6 6 ). The dehydrogenase and phosphatase a c tiv itie s appear to be independently in h ib ite d by ac tiv e s ite in h ib i to rs , as well as also being d iffe re n tia te d by pH optima (1 8 ). In acyl phosphatase a c t iv it y the C-0 bond is broken (63), and a cysteine residue is acylated at the active s ite of the enzyme (59)- Esterase a c tiv ity has been detected toward phenolic esters with an enzyme from which NAD+ has been removed (64). The same th io l-e s te r intermediate is apparently formed (37, 59) in reaction of the enzyme with acetyl phosphate and /°-nitrophenyl acetate. The evidence points strongly to involvement of a th io l group, and somewhat more ambiguous evidence implicates the imidazole ring of h is tid in e (31, 35)* A reaction scheme (Equation I I ) presented by Olson and Park (62) for the action of the enzyme is quite speculative but is in accord with the existing evidence. 11 . r-NAD* r-^N A D O ' — CH?S - C - R + U ' ' R ----------- 1 ^---------- H n^ nh L - W j O " — NADH " * » -CH^S-C-R _ \ = ~ / -NAD ■CH*S' N AH _x==y ‘ ® - R - C - O - P - O 0. — NAD o Hl V ^ N - C - R In the dehydrogenase acylation reaction of Equation I I , the aldehyde appears to react with a cysteine residue, forming a hemithio- acetal which is subsequently oxidized by NAD to y ie ld the th io l-e s te r. Reaction to form the th io l-e s te r has been shown to occur with acetaldehyde (39), proprionaldehyde, butyraldehyde (40) and glycer- 4 aldehyde (6 9 ). A mechanism involving general base cata ly s is by h i s t i dine has been suggested fo r deacylation o f the th io l-e s te r interm ediate in the esterase reaction on the basis o f the D 0 solvent isotope 2 e ffe c t (2 ), although th is is , o f course, highly ambiguous evidence (4 6 ). The r e v e r s ib ilit y o f the phosphorolytic cleavage o f the a c y l- enzyme (40) accounts fo r the acyl phosphatase a c tiv ity o f g lyceralde hyde 3- phosphate dehydrogenase. Subuni t Interactions Many enzymes in c r it ic a l positions o f metabolic a c t iv it y appear subject to regulation by in d ire c t interactions called a llo s te r ic effe c ts between s p e c ific binding s ite s . These interactions are pro duced by a conformation change induced or s ta b iliz e d in the protein when i t binds to an a llo s te r ic ligand. Thus, the binding can have a profound influence on the conformation of adjacent subunits or on neighboring parts of the same polypeptide, and may g re a tly a ffe c t binding of a second ligand and the a c tiv ity o f the enzyme. D iffe re n t models (52, 60) have been proposed to explain how cooperative changes in conformation take place. An understanding o f the s p e c ific e ffe cts o f cooperative conformation changes in modifying the binding o f m etabolites to the enzyme, and possibly the r e a c tiv ity of the enzyme, would be highly in s tru c tiv e in providing insight into the mechanism of th is enzyme. 5 Glyceraldehyde 3-phosphate dehydrogenase is composed of four identical subunits (37)* Cooperativity effects have been detected in the binding of NAD+ with both the yeast enzyme (49) and the muscle enzyme (19). At 40° plots of absorbance at 400 nm. versus NAD4* concentration are weakly sigmoidal with the yeast enzyme. Temperature- jump relaxation experiments on binding of NAD+ with the yeast enzyme could detect only two conformations. Thus, binding of NAD is in flu enced by a conformation change (49) in which a ll the subunits change a lik e . A d iffe re n t scheme involving many conformations (19) has been suggested for the muscle enzyme. Acyl phosphates with a high degree of alkyl group branching in the acyl group, such as trim ethylacetyl phosphate (Equation I I I ) , are not substrates fo r glyceraldehyde 3- phosphate dehydrogenase, but do bind strongly to the enzyme and are excellent inhibitors fo r both the ’ i I I I I . CS °- acyl phosphatase a c tiv ity and the normal dehydrogenase a c tiv ity . Plots of 1/V versus acyl phosphate in h ib ito r concentration are sigmoidal fo r the dehydrogenase reaction, and provide an excellent opportunity fo r elucidating the mechanism by which cooperative subunit interactions brought about by in h ib ito r binding can regulate enzyme a c tiv ity . 6 Objectives o f Study The glyceraldehyde 3~phosphate dehydrogenase mechanism is c le a rly a very complex set of reactions and subunit in te ra c tio n s . Determina tio n of the factors of importance in acyl phosphate in h ib itio n were studied through reaction w ith the aromatic chromophore cinnamaldehyde (Equation IV ). C H = C H — C IV. Study o f in h ib itio n by acyl phosphate and NAD+ analog in h ib ito rs on both the dehydrogenase and acyl phosphatase reactions is achieved through substrates forming a common acyl-enzyme interm ed iate. P a r t i c u la rly a v a ila b le fo r study are /^ -(2 -fu ry l)a c ry la ld e h y d e and @ - (2 -fu ry l)a c ry lo y l phosphate. Molecular structures are shown below in Equation V. V. P — OH C H = C.H — 0 It is hoped th at through such investigations an understanding can be gained of the in teractio n s among the binding s ite s fo r acyl + phosphates, aldehydes, and NAD which influence each other through th e ir binding e ffe c ts on protein conformation. oc-CHYMOTRYPSIN ESTER AND AMIDE HYDROLYSIS 7 The Chvmotrvpsi n Reaction The «<-chymotrypsin-catalyzed hydrolysis of ester and amide substrates involves formation of an acyl-enzyme interm ediate (14, 47) w ith release of the alcohol or amide portion of the substrate. Deacylation then follows to regenerate the ac tiv e enzyme (9 ). There is very strong evidence that the acylation and deacylation o f * - chymotrypsin involves the imidazole ring of h is tid in e -5 7 as a functional group important in the c a ta ly tic process. The acyl-enzyme intermediate is undoubtedly an ester of serine-195 at the active s ite . In the generally accepted mechanism shown in Equation VI h is tid in e -5 7 assists acylation of serine by classical general base c a ta ly s is , p a r t ia lly abstracting a proton from the serine hydroxyl as i t attacks the carbonyl during reaction. Imidazole probably also acts as a general base in deacylation, abstracting a proton from water which attacks the carbonyl of the acyl-enzyme to produce hydrolysis and free the carboxylic acid. VI + X ' 8 Model Studies Intramolecular chemical reactions bear a s trik in g resemblance to enzymatic reactions proceeding through an enzyme-substrate complex where the substrate is held in close proximity to the appropriate functional group(s) of the enzyme. In the decomposition of carbamate esters (42, 43), neighboring phenoxide and alkoxide ions have been studied as intramolecular nucleophiles. These reactions are very e ffic ie n t with e ffe c tiv e m olarities for the neighboring group of 10^ g to 10 M in comparison to the analogous bimolecular reactions. The e ffe c tiv e m olarity can be considered to represent the concentration of an alcohol ate ion in reaction with the unsubstituted compound necessary to give a value of k ^ of the magnitude observed in the intramolecular reaction. Thus, a neighboring oxygen anion is a powerful intramolecu la r nucleophile in reactions at the ester carbonyl. However, no buffer catalysis is observed in these studies. Despite the large volume of work done on the hydrolysis of esters and amides by chymotrypsin, there have been no systematic studies of the alcoholysis of esters or amides with which to provide a chemical model fo r the enzymatic reaction. Hydroxyamides are capable of under going acylation and deacylation reactions (14) in a manner analogous to those of chymotrypsin. A neighboring hydroxymethyl group has been studied as a p a rtic ip ie n t in the hydrolysis of amides (3, 13). In the hydrolysis of S-hydroxybutyramide, an accelerated rate in the alkaline and neutral pH regions in comparison to acetamide and butyramide is probably due to attack of the oxyanion on the neutral and protonated 9 amide. The only known example o f im idazole-catalyzed 1acto n izatio n of amides was demonstrated in the c y c liz a tio n o f 2-hydroxymethylbenzamide (3)* This reaction has been shown to involve a tetrah ed ral interm edi ate w ith a change in rate determining step at around pH 7 to 8 (61)- Both general acid and general base cata ly s is was reported. Ob iect i ves o f Study The goal of a ll organic model studies fo r enzymatic ca ta lysis is to separate and q u a n tita tiv e ly l i s t the factors producing the very e f f ic ie n t cata ly s is seen in enzyme reactions. Bender's analysis of the rate o f hydrolysis o f N-acety1-L-tryptophan amide (8) has been one of the best a v a ila b le to date, but s t i l l contains many broad approxi mations or o u trig h t guesses. The 2-hydroxymethylbenzamide model presented by Belke (3) provides an exc itin g system for*"Vurther study. There have not previously been any studies o f general base-catalyzed tra n s e s te rtfic a tio n reactions of esters analogous to the chymotrypsin acylatio n step. In th is work, the fin d in g o f such ca ta ly s is is reported fo r the c y c liz a tio n o f ethyl 2-hydroxymethylbenzoates to the corresponding phthalides (Equation V I I ) . VI I . 0 ? - ° " CHjOH 10 In addition, there have been no previous studies o f intramolecular alcoholysis reactions where catalysis by a general base is also in tr a molecular. Such an investigation would allow calculation of the e ffe c tiv e m olarity of a neighboring general base, an essential piece of information in any analysis o f °t-chymotrypsin rates in terms of individual mechanistic fa cto rs. Cyclization of 2-hydroxymethyl- 3- aminobenzamide (Equation V I I I) to 4-aminophthalide allows such an 0 n C - N H j analysis both in the amide system and in the corresponding ethyl ester system. PART I SUBSTRATE AND INHIBITOR INTERACTIONS OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE 11 12 EXPERIMENTAL Enzyme Preparations Glyceraldehyde 3-phosphate dehydrogenase, rabbit muscle enzyme, was obtained from Worthington Biochemical Corporation, Code 9FA. The enzyme stock solutions were prepared by the method of P h illip s and Fife (66), whereby the c ry s ta llin e suspension is spun fo r twenty minutes at 4° and 14,500 g. The supernatant is removed, and the enzyme crystals dissolved in the desired b u ffer. Any solid material is removed by respinning for ten minutes, and the preparation stored at 0°. The ratio of absorbance at 280 nm. to that at 260 nm. was.1.05 fo r a ll preparations. Enzyme concentrations were determined by measur- ment of the absorbance at 280 nm. o f a 1:50 d ilu tio n of the stock 7 -1 solution. An extinction co effic ien t of 1.002 cirrmg and the molecular weight of 140,000 employed by Fox and Dandliker (30) were used for a ll calcu latio n s. Materials and Syntheses A ll compounds and reagents were obtained commercially in reagent grade. Nicotinamide adenine dinucleotide (NAD, fre e acid, te tr a - hydrate) was purchased from Calbiochem. Adenosine 3 ' , 5 l_cyclic monophosphate (cyclic AMP) was purchased from Sigma Chemical Company. Buffers were prepared using analytical grade materials and deionized 13 w ater. Benzaldehyde, cinnamaldehyde, and 0 - (2- f u r y 1 )aery 1 aldehyde (also known as fu ran acro le in ) were p u rifie d by vacuum d i s t i l l a t i o n or r e c r y s ta lliz a tio n before use. Trim ethylacetyl phosphate was obtained as the sodium s a lt by the method of P h illip s and F ife (6 6 ). 0 - (2 -F u ry l)a c ry lo y l phosphate was synthesized as the cyclohexyl- ammonium s a lt as described by Malhotra and Bernhardt (57)- Treat 0 - (2-fu ry 1 )acryl i c acid w ith equivalents of isobutyl chloroformate and t r ie th y l amine in ice-cold tetrahydrofuran. A fte r twenty minutes, the mixed anhydride mixture is concentrated in a vacuum and added to an aqueous so lu tio n of phosphoric acid and p y rid in e . The reaction m ixture is adjusted to pH M a fte r another twenty minutes and the barium s a lt is p re c ip ita te d by add ition of barium c h lo rid e . This s o lid is thoroughly tr itu r a te d with an excess cyclohexylammoniurn s u lfa te so lu tio n , and then frozen and ly o p h iliz e d . 0 - ( 2 -Fury 1)acrylo yl phosphate is p u rifie d by e x tra c tio n with methanol and ether which p re c ip ita te s the inorganic s u lfa te and phosphate s a lts , and the f i l t r a t e is removed in a rotary evaporator. Analysis by the hydroxa- mic acid assay o f Lipmann and T u ttle (55) fo r acyl phosphates and comparison of spectral measurements with the known spectra (£ -max is 2.62 x 104 Acm \ A. is 307 nm.) (57) show the id e n tity and IllaX re la tiv e p u rity o f the product. The acyl phosphate is stored in a dessicator at 0° where I t is s ta b le fo r reasonable periods. Synthesis o f the s tr u c tu r a lly s im ila r compound, cinnamoyl phosphate, and its analysis was achieved by s im ila r procedures. 14 Rate Measurements and Spectra Reaction rates fo r a ll the enzymatic reactions were followed spectrophotometrically with a Beckman DU spectrophotometer equipped with a G ilford Model 2000 recorder. Temperature in a l 1 cases was controlled at 25 - 0.1° C. in the dehydrogenase reactions of aldehydes with glyceraldehyde 3-phosphate dehydrogenase, each reaction was in itia te d by adding 0.1 ml. of enzyme stock solution to 2 .9 ml. of solution in the reaction cuvette containing 0.1 ml. of NAD+ stock solution in w ater, 0.1 ml. of aldehyde in a c e to n itri1e, and 2 .7 ml. of buffer solution with 0.013 M mercaptoethanol and 0.005 M EDTA. Unless otherwise stated, the complete system fo r a ll enzymatic reactions contained 2.95 x 10_if M NAD+ and 3*33% ace to n itri1 e . Total enzyme concentrations were set at 0.9 mg./ml. (6.4 x 10“^ M). T ris , sodium b a rb ita l, or sodium pyro phosphate buffers were used for enzymatic rate measurements. The in it ia l velocity of benzaldehyde was determined by measuring the increase in absorbance at 340 nm. due to the NADH product. The extinction c o e ffic ie n t of NADH at 340 nm. was taken to be 6.22 x 10^ cm /mole (41). With cinnamaldehyde as the substrate, the reaction was followed at 340 nm. as with benzaldehyde and also at 310 nm. where the observed absorbance change was p a r tia lly due to formation of a th io l-e s te r interm ediate. Identical results were found at the two wavelengths. The high absorbance of cinnamaldehyde at 281 nm. (extinction c o e ffi cient of 22,600) precludes following oxidation of the aldehyde at that 15 wavelength. The wavelength of maximum absorbance fo r cinnamoyl- cysteine is 310 nm. and 326 nm. fo r cinnamoyl-papain which is also a th io l-e s te r (1 2 ). The spectra o f NADH and the cinnamoyl-enzyme therefore overlap, as seen in Figure 1. As a consequence, the i n i t i a l v e lo c itie s were calculated as the change in absorbance, which is proportional to product formed, per minute. The i n i t i a l v e lo c itie s o f the ^ - ( 2 - f u r y l Jacryloyl phosphate and ^ -(2 -fu ry l)a c ry la ld e h y d e reactions were determined by measuring the increase in absorbance at 360 nm. due to formation o f the stab le a c y l- enzyme. Spectral properties o f the acyl-enzyme have been well docu mented by Malhotra and Bernhard (57)* The absorbance maximum of the t h io l-e s te r interm ediate is “ ikk nm. The spectra of NADH and the 0- (2- fu r y 1 )aeryloyl-enzyme th erefo re overlap (Figure 1) as in the case of cinnamaldehyde. The i n i t i a l v e lo c itie s are expressed as the change in absorbance at 360 nm. per minute. ^ -( 2 - F u r y l )acryloyl phosphate was added in aqueous so lu tion d ir e c tly to the b u ffe r s o lu tio n , since the use o f a c e to n itr ile to increase s o lu b ility was unnecessary. Cyclic AMP and trim ethyl acetyl phosphate in h ib ito rs were dissolved in the b u ffe r systems at desired concentrations immediately before use. Calculations An 01ivetti-Underwood Programma 101 programmed w ith a lin e a r regression program, was employed to c a lc u la te the least squares slopes 16 and intercepts of lin ear data. In a ll illu s tra tio n s the lin e a r data are joined by a theoretical least squares lin e . 17 Figure 1. Comparison o f u ltr a v io le t spectra fo r NADH (71), tra n s -ci nnamov 1 -papai n (1 2 ), and /S -(2 -fu ry l )-GPD (57)* Wavelength, nm. Molar Absorptivity x 10”^, M~^cm * — ■ ro v *j o o o h o oo o o T J O T 1 81 19 RESULTS Cinnamaldehyde is a good substrate fo r glyceraldehyde 3 _phosphate dehydrogenase. This substrate w ill give ris e to a th io l-e s te r interm ediate which should absorb strongly a t 310 nm. The increase in absorbance was followed a t 310 nm., as well as at higher wavelengths, at substrate concentrations greater than the enzyme concentration. The ve lo c ity o f this rapid i n i t i a l increase in absorbance decreases w ith tim e. A fter th ir t y to fo r ty minutes, a slow lin e a r increase is obtained. Extrapolating th is lin e a r increase to zero tim e, and employing the extin c tio n c o e ffic ie n ts o f NADH (71) and cinnamoyl- cysteine (12), w ith the' assumption that the la t t e r is the same as the cinnamoyl-enzyme, i t can be calculated th a t at the highest substrate concentration employed approximately 61% o f an equivalent o f the enzyme is being acylated in the i n i t i a l re a c tio n . When equimolar q u a n tities of substrate and enzyme (1.5 x 10 ** M) were reacted, the slow lin e a r increase in absorbance a fte r the i n i t i a l reaction was not observed, the absorbance becoming constant. A c a lcu latio n then in d i cated that at completion o f the reaction the r a tio of acyl groups per tetram er was 1.4. Addition o f 0.013 M arsenate at th is point produced a slow decrease in absorbance at 310 nm. Presumably, arsenate acts to increase the steady-state deacylation by accelerating the rate of tu rn over of acyl-enzyme. 20 I n i t i a l l y i t was hoped to be able to determine pseudo-first-order rate constants fo r the formation o f the cinnamoyl-enzyme interm ediate. However, in activ atio n o f the enzyme at 25° occurs with an approximate h a l f - l i f e of nine hours as shown in Figure 2 . This in a c tiv a tio n is s u ffic ie n t to introduce s ig n ific a n t errors in the c a lc u la tio n o f rate constants. Employing only the in it ia l portion of these data, i n it ia l v e lo c itie s were calculated fo r reaction a t 310 nm. and at higher wave length where the observed reaction is predominantly the conversion of NAD+ to NADH. Tables 1 and 2 give the i n i t i a l v e lo c itie s fo r reaction of cinnamaldehyde with glyceraldehyde 3“Phosphate dehydrogenase at 310 nm. and 3^0 nm. In these and la te r ta b le s , the data are averages of several i n it ia l v elo city measurements. The Lineweaver-Burk double-reciprocal p lo t (5*0 makes use of a derived form of the Michael is-Menton equation (Equation IX ). _L_ _ J + JSd • (S) IX. V V V max max The Michael is-Menton equation is based on the assumption of reversible formation o f an enzyme-substrate complex followed by formation of the product, in this case, the acyl-enzyme (Equation X ). The Lineweaver- Burk d erivatio n allows a lin e a r plot of the reciprocals of in it ia l 21 Figure 2 . Percentage o f retained a c t iv it y o f glyceraldehyde 3-phosphate dehydrogenase versus hours o f incubation o f the enzyme at 25° before reaction w ith 5*67 x 10"^ M benzaldehyde in 0.3 M T ris b u ffe r, pH 7*85. Hours at 25 % Retained A ctivity to oo o vo to o o vn 0 0 to zz 23 Table 1. Reaction of Glyceraldehyde 3“Phosphate Dehydrogenase with O 3 Cinnamaldehyde at 25 in the Absence and Presence of Arsenate. Arsenate Substrate In it ia l Concentration Concentration Velocity x 10^ (Molar) (M illim o lar) (a A ^ g /m in .) 0.329 1.30 0.495 1.87 0.550 2.18 0.680 2.70 0.823 3.14 1.37 5.20 1.88 6.40 2.06 6.80 2.22 6.49 0,200 0.47 0.250 0.57 0.313 0.67 0.391 0.95 0.488 1.13 0.610 1.39 0.763 1.61 0.954 1.85 1.192 2.08 a Each reaction vessel contained 0.9 mg. protein per ml. and was 0.025 M in sodium barbital b u ffer, pH 7-85. 24 Table 2. In h ib itio n of the Dehydrogenase A c tiv ity of Glyceraldehyde 3-Phosphate Dehydrogenase w ith Cinnamaldehyde by Trimethyl acetyl o a Phosphate at 25 • ■ a Trim ethylacetyl Substrate Velocity x l ( r Phosphate Concentration Concentration (AA_.n/m in .) (M illim o la r) (M illim o la r) 0.385 2.88 0.440 3.41 0.495 3.28 0.550 5.12 0.823 5-74 1.10 7-41 1.37 8.76 1.61 9.68 0.290 0.59 0.395 0.88 0.580 1.10 0.815 1.32 1.135 2.23 1.685 3.23 1.975 3.66 Trimethyl acetyl Phosphate Concentration (Mi 11imolar) Substrate Concentration (Mi 11imolar) Veloci ty x 10^ (AAjio/rain.) 0.300 1.60 0.324 1.76 0.327 1.82 0.355 2.12 0.383 2.01 0.437 2.20 0.547 2.64 0.833 4.35 2.05 9.86 a Each reaction vessel contained 0.9 mg. protein per ml. and was 0.025 M in sodium barbital b uffer, pH 7*85- 26 v e lo c itie s and substrate concentrations ( l/V and 1 /S ), and accurate least squares analysis o f the data fo r c a ta ly tic constants. The abscissa in te rc ep t o f the double-reciprocal p lo t gives the reciprocal of V , the maximal v e lo c ity obtainable by elevating substrate concen- inci/s t r a tio n . (*Si/^max^ ' s derived ^rom the slope o f the p lo t, and 1^ can be o p e ra tio n a lly defined as the substrate concentration at which v e lo c ity is h a lf maximal. K is a complex o f several rate constants m and need not give any inform ation on binding. Figures 3 and 4 illu s t r a te double-reciprocal plots fo r the reaction of cinnamaldehyde with glyceraldehyde 3"phosphate dehydrogenase followed at 310 nm. and 340 nm, both plots y ie ld in g an id en tic al value of 5 .2 x 10“^ M in the absence of any in h ib ito rs . At low concentrations, arsenate exerts a small acceleratin g e ffe c t on the i n it ia l v e lo c ity o f the cinnamaldehyde reac tio n . This e ffe c t is maximal at an arsenate concentration o f approximately 0.002 M which gives a two-fold enhancement in the rate when cinnamaldehyde concen- tra tio n is 1.5 x 10 M. With arsenate concentrations g reater than 0.002 M an in h ib ito ry e ffe c t can be detected. In the presence of 0.013 M arsenate (Figure 3 ). the apparent K fo r cinnamaldehyde de n i -3 creases to 1.64 x 10 M. Trimethyl acetyl phosphate is a good in h ib ito r of the cinnamalde hyde reaction w ith glyceraldehyde 3“phosphate dehydrogenase. A p lo t of l/V versus 1 / (S) at in h ib ito r concentrations of 2.73 m M and 5*65 m M is given in Figure 4 . The plots are lin e a r w ith lines e x tra p o latin g to the same point on the abscissa axis as in the absence o f in h ib ito r , 27 Figure 3 . Plots of l/V versus 1 / (S) for reaction of cinnamaldehyde with glyceraldehyde 3- phosphate dehydrogenase in the absence ( o ) and presence ( © ) of 1.32 x 10“ M arsenate. Velocity is expressed as the change in absorbance at 3^0 nm. per minute at an enzyme concentration of 0.90 mg./ml. < 9 “ 0 1.0 2.0 3.0 (1/S) x 10^, Molar 29 Figure 4. Plots of l/V versus 1 / (S) fo r reaction of cinnamaldehyde with glyceraldehyde 3_phosphate dehydrogenase at 25° in the absence ( o ) and presence of 2.73 m M ( • ) and 5-65 m M ( © ) trim ethyl acetyl phosphate. Velocity is expressed as the change in absorbance at 310 nm. per minute at an enzyme concentration of 0.90 mg./ml. 30 OJ 0 2 3 (1/S) x 10"3 , Molar indicating noncompetitive in h ib itio n . Dissociation constants K. , which are true binding constants fo r trim ethyl acetyl phosphate binding, can be calculated using Equation XI which is v alid fo r noncompetitive in h ib itio n . Such calculations show K. to vary from 6.35 m M to 2 .0 m M max max fo r in h ib ito r concentrations of 2.73 and 5-65 mM, respectively, in d i cating that K. changes with in h ib ito r concentration. A p lot of l/V versus trim ethylacetyl phosphate concentration at a constant cinnamaldehyde concentration of 3*56 x 10“^ M in Figure 5 is sigmoidal with maximum in h ib itio n at an in h ib ito r concentration o f approximately 0.002 M. Sim ilar sigmoidal kinetics are seen at 3^0 nm. The kinetics exhibited in Figures b and 5 are obtained in the absence of arsenate. However, in the presence o f 0.013 M arsenate, trim ethylacetyl phosphate has no detectable in h ib ito ry e ffe c t at con centrations as low as 0.002 M. At higher concentrations in h ib itio n does occur in the presence o f 0.013 M arsenate, and as seen in Figure 6 a plot of l/V versus trim ethylacetyl phosphate concentration is lin e a r. I t w ill be noted in Figures 5 and 6 that the velo city at zero in h ib ito r concentration is less in the presence o f 0.013 M arsenate, illu s tr a tin g the inhib ito ry e ffe c t of arsenate at concentrations larger than 0.002 M. In addition , Figure 3 indicates that arsenate in h ib itio n is probably noncompetitive. 32 Table 3* Trim ethylacetyl Phosphate In h ib itio n o f the Cinnamaldehyde Reaction w ith Glyceraldehyde 3"Phosphate Dehydrogenase and the Effect o f Arsenate.3 In h ib ito r Concentration (Mi 11im olar) Veloci tyk x 103 1nhibi tor Concentration (Mi 11imolar) Velocity** x 10* Arsenate Concentration Arsenate Concentration 0. 00 M 0.013 M 0.00 1.78 0.00 9.00 0.125 1.76 0.30 1.72 5.46 8.60 0.40 1.72 0.55 1.66 10.9 7.50 0.60 1.60 0.71 1.54 16.4 6.50 0.80 1.52 0.90 1.46 28.1 5.10 1.025 1.42 1.0 1.32 33.6 5.00 1 .20 1.25 1.60 1.16 39.5 4.40 2.0 0 1.12 3.00 1.10 45.1 4.2 0 4.4 0 1.12 4.97 1.12 a Each reaction vessel contained 0.90 mg. protein per ml. and was 0.025 M in sodium b a rb ita l b u ffe r, pH 7*85. b V elocity is expressed as the change in absorbance at 310 nm. per mi nute. 33 Figure 5• Plot of 1/V versus trim ethylacetyl phosphate concentration for reaction of 3.56 x 10"^ M cinnamaldehyde with glyceraldehyde 3~ph°sphate dehydrogenase at 25° in the absence o f arsenate. Velocity is expressed as the change in absorbance at 310 nm. per minute at an enzyme concentration of 0.90 m g./m l. (1/v) x 10 CM k 5 2 3 (TMAP) x 103 , M 35 Figure 6. Plot o f 1/V versus trim ethylacetyl phosphate concentration fo r reaction of 3*56 x 10 ^ M cinnamaldehyde with glyceraldehyde 3“Phosphate dehydrogenase at 25° in the presence of 0.0132 M arsenate. Velocity is expressed as the change in absorbance at 310 nm. per minute at an enzyme concentration of 0.90 mg./ml. 37 *4 " The e ffe c t of varying the concentration of added NAD was determined employing cinnamaldehyde as substrate. V ariatio n of added NAD at a constant cinnamaldehyde concentration o f 2 .0 x 10 J M in -3 the absence and presence of 3-1 x 10 J M trim ethylacetyl phosphate suggests in h ib itio n is noncompetitive with respect to NAD+ as well as -5 with respect to cinnamaldehyde. Km is calculated to be 6 x 10 M. -5 + A Km value of 1.3 x 10 M has been reported fo r NAD at pH 8.6 and 26° when glyceraldehyde 3- phosphate is the other substrate (33) and 2.28 x 10 M with benzaldehyde as a substrate (28). The acyl phosphatase reaction of cinnamoyl phosphate w ith glycer aldehyde 3_phosphate dehydrogenase was investigated, but cinnamoyl phosphate was found to undergo spontaneous hydrolysis at rates roughly equivalent to the enzyme-catalyzed reaction. By substracting the change in optical density due to spontaneous hydrolysis from that of the enzyme-catalyzed reaction under sim ilar reaction conditions, in it ia l v e lo c itie s are obtained which allow calcu latio n o f a Km value fo r cinnamoyl phosphate of 7*98 x 10 ^ M. A s im ila r approach shows lin e ar in h ib itio n at high concentrations of trim ethylacetyl phosphate. To allow more accurate determination of enzymatic catalysis the (3-(2-fury 1 )acryloyl phosphate, which is e le c tro n ic a lly and s tru c tu r a lly very s im ila r to cinnamoyl phosphate, has been shown (57) to be re la tiv e ly stable under the reaction conditions employed. Figure 7 shows a plot o f 1/V versus 1 / (S) for the glyceraldehyde 3-phosphate dehydrogenase-catalyzed acyl phosphatase reaction of /3 -(2-furyl )acryloyl phosphate in the absence and presence o f in h ib i- 38 Table 4 . Acyl Phosphatase A c tiv ity of Glyceraldehyde 3“Phosphate Dehydrogenase w ith /3 -(2 -F u ry l)a c ry lo y l Phosphate and the E ffect o f a Trimethyl acetyl Phosphate In h ib itio n . FAPb Concentration x 104 (M) Veloci ty c x 103 FAPb Concentration x 104 (M) Veloci ty C x 103 TMAPb Concentration 0.00 M L TMAP Concentration 2 .0 x 10“3 m 1 .81 0.72 1.81 0.43 2.01 0.815 2.01 0.515 2.58 1.09 2.58 0.595 3.01 1.195 3.01 0.70 3.61 1.375 3.61 0.80 4.51 1 .535 4.51 0.925 6.02 J .825 6.02 1.165 9.03 2.235 9.03 1 .425 14.44 3.065 14.44 2.335 a Each reaction vessel contained 0.92 mg. protein per ml. and was 0.3 M in T ris b u ffe r, pH 7 .8 5 . b Abbreviations: FAP = 0 - (2 -fu r y l)a c r y lo y l phosphate; TMAP = trim eth ylacetyl phosphate. c Velocity is expressed as the change in absorbance at 360 nm. per minute. 39 Figure 7* Plot of 1/V versus 1 / (S) fo r the reaction of /3 -( 2 - fu ry 1)acryloyl phosphate w ith glyceraldehyde 3 _phosphate dehydrogenase at 25° in the absence ( o ) and presence ( ® ) of _3 2 .0 x 10 M trim ethyl acetyl phosphate. Velocity is expressed as the change in absorbance at 360 nm. per minute at an enzyme concentration of 0.92 mg./ml. (1 /v ) T T ro I o 2.0 1.5 1.0 0.5 (1/S) x 10“3 , M -fr O tio n by trim ethyl acetyl phosphate. In the absence o f trim eth ylacetyl - 3 phosphate, Km has a value o f 1.29 x 10 J M. Trim ethylacetyl phosphate is a good in h ib ito r fo r the reaction of ^ - ( 2 - f u r y l )acrylo yl phosphate with the enzyme, and i t can be seen that in h ib itio n is com petitive by the intersection on the v e rtic a l a x is . The competitive nature of trim ethylacetyl phosphate in h ib itio n is confirmed in Figure 8 in a plot of l/V versus in h ib ito r concentration at constant substrate -4 concentrations of 3-5 and 5- 0x10 M where the plots are c le a rly lin e a r and intersect above the horizontal axis. From the point of in te rs ectio n , a value o f Kj for the reaction is seen to be approxi- _ a mately 4.5 x 10 M. All the acyl phosphatase reactions were run in the absence of arsenate. I f arsenate is included in the (3-(2 -fu ry 1 )- acryloyl phosphate reaction at a concentration of 0.013 M no detectable rates are observed. Presumably, deacylation of the acyl-enzyme intermediate is so accelerated with regard to acylation th at no appreciable acyl-enzyme concentration can accumulate. /3 -(2 -F u ry l) aery 1 aldehyde must form the same th io l-e s te r enzyme intermediate as the phosphate. Figure 9 shows that /S - ( 2 - f u r y l) - aery 1aldehyde acts as an excellent substrate possessing a Km value of -3 1.59 x 10 M in the absence of any in h ib ito rs or arsenate. In contrast to cinnamaldehyde, however, trim ethylacetyl phosphate exhibits a very weak noncompetitive in h ib itio n (Figures 9 and 1 0), and a plo t of l/V versus in h ib ito r concentration is lin e a r at concentrations up -2 to 4 x 10 M. The data at constant /3 -(2 -fu ry l )acryl aldehyde -4 -4 concentrations of 1.0 x 10 M and 3-33 x 10 M extrap o late to 42 Table 5. Trim ethylacetyl Phosphate In h ib itio n o f the Acyl Phosphatase A c tiv ity of Glyceraldehyde 3 “Phosphate Dehydrogenase at 25° w ith (3 -(2-F u ry 1 )acryloyl Phosphate.3 b TMAP Concentration (Mi 11imolar) V e lo c ity 0 x 10 TMAP*5 Concentration (Mi 11imol a r) .. , ■ . c V elocity x 103 FAP Concentration*5 0.5 m M FAP Concentration*3 0.35 m M 0.0 3.13 0.0 1 .825 2 .0 2.805 2 .0 1 .38 4 .0 2.49 4 .0 1.145 6 .0 2.11 6 .0 0.98 8.0 2.015 8.0 0.89 10.0 1.82 10.0 0.765 12.0 1.735 12.0 0.655 14.0 1.653 14.0 0.69 16.0 1.49 16.0 0.565 18.0 1.325 18.0 0.513 2 0 .0 1.26 20 .0 0.495 a Each reaction vessel contained 0.92 mg. protein per ml. and was 0.3 M in T ris b u ffe r, pH 7-85. b Abbreviations: TMAP = trim e th y la c e ty l phosphate; FAP = 2 - fu ry l)a c ry lo y l phosphate. c Velocity is expressed as the change in absorbance at 380 nm. per mi nute. 43 Figure 8. Plot of l/V versus trim ethylacetyl phosphate concen- _ _y i tra tio n fo r reaction of 5-0 x 10 M (o ) or 3.5 x 10 M ( © ) /3 -( 2 - fu r y l Jacryloyl phosphate with glyceraldehyde 3- phosphate o dehydrogenase at 25 . Velocity is expressed as the change in absorbance at 360 nm. per minute at an enzyme concentration of 0.92 m g./m l. o 18 6 15 9 12 0 3 (TMAP) x 103, M -a- Table 6 . Reaction of /3-(2-Furyl)acrylaldehyde with Glyceraldehyde 3~Phosphate o a Dehydrogenase at 25 and the Effects of Trimethylacetyl Phosphate and Cyclic AMP. Substrate Concentration x 104 , M Velocity x 103 A^360^m’ n* Substrate Concentration x 10*, M Velocity x 103 ^ 360^mi n* Substrate Concentration x 10 , M Velocity x 103 , /min. 360 No Inhib itor Trimethyl acetyl Concentration Phosphate 5.0 m M Cyclic AM P 20 m M Concentration 0.844 0.70 1.00 0.69 1.43 0.825 1.00 0.80 1.54 1.025 1.67 0.969 1.11 0.925 1.67 1.125 2.00 1.21 1.43 1.175 2.00 1.25 2.22 1.16 1.67 1.35 2.50 1.50 2.50 1.40 2.00 1.70 2.86 1.85 2.86 1.49 2.50 1.85 3.33 1.95 3.33 1.89 3-33 2.35 5.00 2.90 5.00 2.31 5.00 3.325 6.67 3.75 6.67 3-015 10.0 5.575 10.0 4.45 10.0 3.66 a Reaction vessel contains 0.90 mg. protein per ml. and 0.15 M in sodium pyrophosphate, pH 8.64. U6 Figure 9. Plot of l/V versus 1 / (S) fo r reaction of (3-(2-fury 1 ) - acrylaldehyde with glyceraldehyde 3_phosphate dehydrogenase at 25° in the absence of inhib itors ( o ) , with 5 -0 x 10“3 M _2 trim ethylacetyl phosphate ( © ) , or w ith 2.0 x 10 M c yc lic AMP ( • ) . Velocity is expressed as the change in absorbance at 360 nm. per minute at an enzyme concentration of 0.9 mg./ml. 47 CM O 8 4 6 10 2 0 (1 /S ) x 10"3 , M 48 Table 7« T rim ethylacetyl Phosphate In h ib itio n o f the Reaction o f - (2-Fury I ) acrylaldehyde w ith Glyceraldehyde 3 “Phosphate O 3 Dehydrogenase at 25 • b TMAP Concentration (Mi 11im olar) V e lo c ity 0 x 103 TMAP*5 Concentration (Mi 11im olar) V e lo c ity 0 x 103 FAA Concentr< 1 .0 x 10"** at ion M FAA Concentration 3-33 x 10“^ M 0 1 .25 0 3.04 5 1 .19 5 2.8 6 10 1 .13 10 2.62 15 1.04 15 2.50 20 0.912 20 2.33 25 0.95 25 2.23 30 0.837 30 2.08 40 0.80 40 1.86 50 0.687 50 1.71 a Each reaction vessel contained 0.9 mg. p ro te in per m l. and was 0.15 M in sodium pyrophosphate b u ffe r , pH 8 .6 4 . b Abbreviations: TMAP = trim e th y la c e ty l phosphate; FAA = 0 - ( 2 - f u r y l ) acrylaldehyde. c V e lo c ity is expressed as the change in absorbance at 360 nm. per mi n u te . 49 Figure 10. Plot of l/V versus trim eth ylacetyl phosphate concentration fo r reaction o f 1.0 x lO- ** M (© ) and 3-33 x 10”^ M (o ) yS- ( 2 -f u r y l) acrylaldehyde with glyceraldehyde 3-phosphate dehydrogenase at 25°. Velocity is expressed as the change in absorbance at 360 nm. per minute at an enzyme concentration of 0.9 m g./m l. > (TMAP), Mi 11imolar 51 intercept on the horizontal axis, the point o f in te rs ectio n indicating -2 a K. binding constant o f 6.2 x 10 M w ith noncompetitive in h ib itio n . C yclic AMP acts as a noncompetitive in h ib ito r (Figure 9) fo r the reaction o f /3-(2 -fu ry l)a c ry la ld e h y d e w ith the enzyme. Figure 9 shows a Lineweaver-Burk double-reciprocal plot illu s tr a tin g this in h ib itio n , -2 giving a K. value of 2.12 x 10 M, and a p lo t of l/V versus cyclic AMP concentration in Figure 11 is lin e a r at constant substrate concentration. 52 Table 8. Cyclic AM P Inh ib itio n o f the Reaction of 1.0 x 10"^ M /3-{2-Fury 1 )acrylal dehyde with Glyceraldehyde 3"Phosphate o a Dehydrogenase at 25 . Cyclic AMP Concentration Mi 11imolar Velocity x 10^ A^gj/mi nute 0.0 4.95 1.0 4.50 3.0 4.02 4.0 3-56 5.0 3.43 7.0 3.11 8.0 2.95 9.0 2.78 12.0 2.28 13-0 2.43 a Each reaction vessel contained 0.9 mg. protein per ml. and was 0.15 M in sodium pyrophosphate b uffer, pH 8.64. Figure 11. Plot of l /V versus c y c lic AMP concentration for - 3 reaction of 1.0 x 10 M /3 -{2 -fu ry l )acry laldehyde with glyceraldehyde 3"Phosphate dehydrogenase at 2 5 °. Velocity is expressed as the change in absorbance at 360 nm. per minute at an enzyme concentration of 0 .9 mg./ml . (cAMP) x 103 , M 55 DISCUSSION Reaction of equimolar concentrations o f cinnamaldehyde and glyceraldehyde 3-phosphate dehydrogenase when arsenate is not present produces an increase in absorbance at 310 nm. u n til a constant value is reached. The constancy of absorbancy indicates that the a c y l- enzyme is reasonably stable under these conditions. The e le c tr o n i c a lly s im ila r /3 -(2 -fu ry l )acryloyl-enzyme is also reasonably stab le in the absence of arsenate or phosphate (57) • With cinnamaldehyde con cen tratio n greater than enzyme concentration a slow lin ear increase in absorbance indicates a deacylation reaction regenerating ac tiv e enzyme. Without arsenate or other external acyl group acceptors, the i n i t i a l v e lo c ity fo r acyl-enzyme formation should be unaffected by slow hydrolysis of the acyl-enzyme. Thus the data fo r cinnamaldehyde in the absence of arsenate (Figures 4 and 5) re la te s t r ic t ly to the oxidation reaction. The most lik e ly reaction scheme (Equation X I I ) involves reaction of the aldehyde substrate with the enzyme-NAD+ complex to form the hem ithioacetal, followed by oxidation to form the acyl-enzyme interm ediate, cinnamoyl-glyceraldehyde 3-phosphate dehydrogenase. This reaction scheme is id en tical to that determined fo r series of a lip h a tic and aromatic aldehydes (2 7 , 28). 56 U /H C H = C H -ct. *■ ESHfNAb*) OH C H = C .H ~ C - S E - fN A D * ) XI I . OH \ u /^cw=c.H-cf * NADH * The concentration of added NAD+ is considerably greater than its + _ c K . The K fo r NAD in the reaction with cinnamaldehyde (6 x 10 M) m m ' is not very d iffe re n t from that determined fo r acyl at ion of f3-( 2 - fu ryl)acry lo yl phosphate (3.5 x 10 ^ M) (57) or f ° r the enzyme catalyzed-oxidative arsenolysis of glyceraldehyde 3 - phosphate (9 x 10”* ’ M) (33). in a ll reactions described in this work the concentration of coenzyme is in substantial excess over the concentration of enzyme, Ue.. approaching the conditions fo r steady state k in e tic s . Therefore, Km values fo r reactions of aldehydes and acyl phosphates are those at + saturating conditions of NAD . Cinnamaldehyde and rS -(2 -fu ry 1Jacrylaldehyde and th e ir corres ponding acyl phosphates are good substrates for the enzyme with th e ir Km values lis te d in Table 9 . These Km values fo r the substrates under study are roughly intermediate in comparison to the natural substrate glyceraldehyde 3-phosphate and a series of a lip h a tic or aromatic aldehydes (27, 2 8 ). Further mention of K values in relatio n to m observed kinetics w ill be given la te r. In the reactions of a lip h a tic and aromatic aldehydes, the in it ia l 57 Table 9. Values fo r Various Substrates of Glyceraldehyde 0 3-Phosphate Dehydrogenase. Substrate K , Molar m’ Ci nnamaldehyde 5.19 x 10"3 0.013 M Arsenate 1.64 x 10”3 Cinnamoyl Phosphate 7.98 x 10"3 /3 -(2 -F u r y l) acryl aldehyde 1.59 x 10-3 0 -{2 -F u ry l)a c ry lo y l phosphate 1.29 x 10“3 b , -b Glyceraldehyde 3-pnosphate 1.3 x 10 c Benzaldehyde 5 .2 x JO-2 d Butyraldehyde o 00 X o 1 a Buffers and reaction conditions vary as described in Results or appropriate reference. b Reference 33* c Reference 28. d Reference 27. i I I ' I I I 58 velocity measurements are not affected by deacylation, since arsenate, which would enhance deacylation, has no e ffe c t on v e lo c ity . Arsenate at concentrations less than 0.002 M does have a small rate accelerating influence in the reaction with cinnamaldehyde, but at higher concen trations i t inhib its the reaction. !< „, for ci nnamaldehyde is consider ably less (1.64 x 10’ 3 M) in the presence of arsenate. Reaction of (2-fury 1)acryloyl phosphate is completely undetectable in the presence of 0.013 M arsenate, eith er through acceleration of deacyla tio n to a rate where change in absorbance due to accumulation of acyl- enzyme is decreased to indetectable lim its , or through even stronger in h ib itio n than evidenced with cinnamaldehyde. Deacylation of the /® -(2-fu ry1) acryl oyl-enzyme, produced by addition of arsenate (57) or phosphate is fa s te r than acylation under sim ilar conditions in the absence of arsenate. Thus there is a strong p o s s ib ility that the /3- (2 -fu ry l) acryloyl-enzyme, being more susceptible to arsenolysis than the cinnamoyl-enzyme, does not accumulate to appreciable concen trations in the presence of arsenate. However, this e ffe c t is not s u ffic ie n t to explain the decrease in observed with cinnamaldehyde. Keleti (48) previously observed that in the oxidation of glyceralde hyde the apparent Michael is constants are influenced by the presence of arsenate. Arsenate probably lowers the apparent Km of cinnamalde hyde through an e ffe c t on binding or rate of acylation. Data suggest that in h ib itio n by arsenate is noncompetitive (double-reciprocal plots intersecting on the abscissa axis of Figure 4) with respect to the cinnamaldehyde substrate. Since arsenate and 59 phosphate are s im ila r in s tru c tu re , the arsenate probably binds to the normal phosphate binding s ite of the enzyme near the aldehyde binding s ite to exert its e ffe c t. Trim ethylacetyl phosphate is an in h ib ito r fo r the reaction of the enzyme w ith cinnamaldehyde and /3- (2-fu ryl)a crylald e h yd e substrates as was previously found to be the case fo r a lip h a tic (27) and aromatic substrates (28) and the natural substrate glyceraldehyde 3"Phosphate (67). The form o f in h ib itio n is noncompetitive in a ll cases. A plot of l/V versus in h ib ito r concentration fo r in h ib itio n o f the reaction w ith benzaldehyde is lin e a r . A lin e a r p lo t of l/V versus in h ib ito r concentration has also been found fo r trim eth ylacetyl phosphate in h ib itio n when a lip h a tic aldehydes are the substrates, but w ith glyceraldehyde 3-phosphate a sigmoidal p lot is obtained with a maximum in h ib itio n at the r e la tiv e ly low acyl phosphate concentration of 0.003 M. In the absence of arsenate, as seen in Figure 5 when trim e th y l acetyl phosphate concentration is varied at constant cinnamaldehyde concentration, a plo t of l/V versus trim eth ylacetyl phosphate concen tra tio n is d e fin ite ly sigmoidal, showing maximum e ffe c t o f in h ib ito r at low concentrations (0,002 M ). The residual dehydrogenase a c tiv ity remaining at higher acyl phosphate concentration is not unexpected. Values ranging from 0.8 to 3 .8 have been obtained fo r the number o f s ite s acylated by ^ - ( 2 - f u r y l) acryloyl phosphate (57)> and fo r cinnamaldehyde even lower ratios were usual (approximately 60% o f an equivalent o f the enzyme is being acylated in the i n i t i a l re a c tio n ). Experiments by Chance and Park (17) Indicate that w hile a ll monomers o f the tetram eric yeast glyceralde hyde 3"Phosphate dehydrogenase have identical primary structures, they are not a ll equivalent in th e ir a b ilit y to be acylated by acetyl phosphate. Sites which are unfavorable to acetyl phosphatase a c tiv ity also have a decreased dehydrogenase a c tiv ity . The dehydrogenase a c tiv ity remaining at maximal trim ethylacetyl phosphate in h ib itio n may therefore be due to monomers which are not readily subject to in h ib itio n . Malhotra and Bernhardt (57) present evidence that h a lf of the monomers of the muscle enzyme are highly active while h a lf are less a c tiv e . Since coo perativity is observed in the present in h ib itio n experiments with the muscle enzyme, i t appears that one or more of the monomers is highly a c tiv e , and at least one is less a ctive. Glyceraldehyde 3“phosphate dehydrogenase derived from rabbit muscle is a tetramer consisting o f identical subunits (38). A possible in terp retatio n of the sigmoidal l/V versus trim ethylacetyl phosphate concentration plots with glyceraldehyde 3~phosphate and cinnamaldehyde is that with these substrates binding of acyl phosphate to one subunit induces or s ta b iliz e s a conformation change which is unfavorable for maximum velocity and which enhances fu rth e r binding of acyl phosphate u n til a maximum concentration is reached. In the acyl phosphate in h ib itio n , the shape o f the l/V versus ( I) plot is dependent on the aldehyde substrate when trim ethylacetyl phosphate is the in h ib ito r, the p lo t being lin ear fo r benzaldehyde and the a lip h a tic substrates, and plots of l/V versus 1/(S) are also lin e a r with constant concentration o f in h ib ito r. Sigmoidal plots of V versus (S) and l/V versus 1 / (S) are not seen w ith any o f the substrates in th is study. Although present data do not conclusively estab lish why th ere is such a c le a r d is tin c tio n between the various aldehyde substrates w ith regard to the type o f l/V versus ( l ) plot th at is obtained, there does appear to be a c o rre la tio n between Km values and the shape of these p lo ts . The natural substrate g ly c e r aldehyde 3-phosphate has the sm allest and gives sigmoidal l/V versus trim e th y la c e ty l phosphate plots w ith both acyl phosphate in h ib ito rs and also w ith c y c lic AMP as the in h ib ito r (2 0 ). A lip h a tic and aromatic aldehydes, however, are not good substrates, being _2 approximately 10 M. Cinnamaldehyde has an interm ediate Km value and gives sigmoidal k in e tic s . In any case, the sigmoidal plots are d e fin it e ly dependent on the aldehyde substrate. The noncompetitive in h ib itio n observed with acyl phosphate in h i b ito rs favors the p o s s ib ility that binding o f substrate is not being d ir e c tly influenced by binding o f in h ib ito r and th a t near eq u ilib riu m conditions must p r e v a il. Trim ethylacetyl phosphate, highly branched in the acyl group, binds very strongly to the enzyme in comparison to other acyl phosphates. The fa c t th a t the shapes o f the plots o f l/V versus ( I ) are dependent on both aldehyde and in h ib ito r must then in d icate th at the presence o f both a tig h tly binding substrate and in h ib ito r is necessary in order to obtain a large conformation change. In the conformation change pictured in Equation X I I I the d iffe r e n t geometrical shapes represent d iffe re n t extremes o f conformation o f the enzyme. Interm ediate conformations may e x is t between the two extreme 62 cases. Tight binding in both the acyl phosphate and the aldehyde binding sites is required before form T can exist at high concentra tions. In the case of acyl phosphate in h ib itio n , the binding of the R substrate must be equally good with e ith e r R or T, but the in h ib ito r must bind more tig h tly to T and conformation R must be more favorable fo r velo c ity . Intermediate conformations must then exist between R and T to account fo r in h ib itio n without detectable cooperativity e ffe c ts . Numerous studies (19, 20, 36) have suggested on the basis of binding studies of NAD+ to the muscle enzyme that a number of confor mations may e x is t. A more detailed schematic of the reactions and conformation changes involved, as well as the conditions necessary fo r observed kin e tic s , is given in Figure 12. Arsenate has a pronounced e ffe c t on trim ethylacetyl phosphate in h ib itio n of the reaction with cinnamaldehyde. At an arsenate con centration of 0.013 M, the acyl phosphate is an in h ib ito r at concen trations greater than necessary fo r maximum in h ib itio n in the absence of arsenate, but a plot of l/V versus trim ethylacetyl phosphate is linear at in h ib ito r concentrations as high as 0.045 M. In contrast, 63 Figure 12. Scheme fo r the Reactions and Conformation Changes in the Reaction o f Glyceraldehyde 3“ Phosphate Dehydrogenase S + I A R K! J cat S + I + X c cat S -X -l ■* Acyl-enzyme S cat K, = K T K, < K. kcat > cat K = KR cat = k R 'cat 6k arsenate has l i t t l e or no e ffe c t on in h ib itio n of the reaction w ith benzaldehyde. I f binding o f the acyl phosphate is contributing to a conformation change, then i t is cle a r that with cinnamaldehyde as the substrate arsenate can a lte r this effect,although in h ib itio n is not overcome. Since deacylation is enhanced by arsenate, i t is possible that more rapid turnover o f the enzyme could reduce the effectiveness of low concentrations of in h ib ito r, but this is unlikely since 0.013 M arsenate actually has an in h ib ito ry e ffe c t on the observed in it ia l velocity in the absence o f acyl phosphate. Arsenate may have great influence on the conformation of the enzyme, thereby modifying acyl phosphate in h ib itio n . A p o s s ib ility is that in the presence of arsen ate only a re la tiv e ly small intermediate conformation change can be induced by binding of acyl phosphate to one subunit with coo perativity then not detectable. In terms of Figure 12, binding of arsenate to the enzyme is much stronger to the R or intermediate conformations. /3-(2-Furyl)acrylaldehyde and # -(2 -fu ry lJ a c ry lo y l phosphate are good substrates fo r the dehydrogenase and phosphatase reactions with Km values of 1.59 x 10“^ M and 1.29 x 10"^ M, respectively. As previously discussed, no detectable rates are observed for /<3-(2-furyl )acryloyl phosphate in the presence of arsenate. Trim ethyl- acetyl phosphate, however, is a f a i r l y e ffic ie n t in h ib ito r, showing competitive in h ib itio n in a 1/V versus I / (S) plot at constant in h ib ito r concentration as well as in a l/V versus ( I) plot at two d iffe re n t concentrations of substrate. The dissociation constant fo r binding o f trim ethyl acetyl phosphate from both plots is about 5 .0 m illim o la r. The com petitive p attern o f in h ib itio n suggests that trim eth ylacetyl phosphate binds a t the same s ite as the less highly branched acyl phosphate substrate -(2 -fu r y l)a c r y lo y l phosphate. On the other hand, trim eth ylacetyl phosphate in h ib itio n o f the corresponding aldehyde /3~(2— f u r y I ) aery 1aldehyde shows noncompetitive k in e tic s , in common w ith a ll other aldehydes studied. Plots of 1/V versus 1 / (S) a t constant in h ib ito r concentration and 1/V versus ( I ) plots at con s ta n t substrate concentrations c le a rly e s ta b lis h the contrasting in h ib itio n patterns fo r the two substrates. Noncompetitive in h ib itio n o f aldehydes in the dehydrogenase reaction and com petitive in h ib itio n o f acyl phosphates in the phosphatase reaction indicate that the aldehyde and acyl phosphate substrates bind at d iffe r e n t s ite s . Since these substrates almost c e rta in ly form a common interm ediate (18), they probably bind to adjoining s ite s on the enzyme as seen in Equa tio n XIV. The properties o f the /3 -(2 -fu ry l)a c ry lo y l-e n z y m e from reaction o f /^ -(2 -fu ry l)a c ry lo y l phosphate are well established and in accord w ith the spectral and k in e tic properties o f the acyl-enzyme derived from reaction with the corresponding aldehyde. The plots of 1/V versus ( l) fo r trim eth ylacetyl phosphate in h ib i tio n of £ - (2 -fu ry l)a c ry lo y l phosphate are lin e a r . The Km fo r this ~3 substrate (1.29 x 10 M) is even less than the Km fo r cinnamaldehyde which shows sigmoid p lo ts . This does not necessarily argue against the possible c o rre la tio n o f sigmoid k in e tic s w ith K ,^ o f the substrate as described above, since Is - (2 -fu ry l )acrylo yl phosphate binds a t a 66 XV. d iffe re n t s ite from the aldehyde. However, cooperativity as seen in sigmoid plots o f 1/V versus trim ethylacetyl phosphate concentration must have d iffe re n t effects on the phosphatase reaction system than fo r the dehydrogenase system. The changes in subunit conformation produced by the strong binding of trim ethylacetyl phosphate must have no e ffe c t on the i n it ia l velocity fo r reaction at other acyl phosphate binding site s on the tetram er. Observed in h ib itio n is e n tire ly due to competition between substrate and in h ib ito r fo r the same binding s ite on the enzyme. Trimethylacetyl phosphate in h ib itio n of the /® -(2 -fu ry l )acryl - aldehyde dehydrogenase system requires some discussion. Although the of 1 .59 x 10 3 M indicates that /&-(2-furylJacrylaldehyde is a quite good substrate, the 1/V versus trim ethylacetyl phosphate plots at constant substrate concentration are lin e a r. In h ib itio n is also weak, w ith a Kj of approximately 0.062 M, and lin e a rity extending to 0.05 M in h ib ito r. Thus # -(2 -fu ry l)a c ry la ld e h y d e in h ib itio n appears anamalous in comparison to the sigmoidal in h ib itio n of glyceraldehyde 3_phosphate and cinnamaldehyde (whose K m value is even somewhat poorer). A possible explanation may be seen in the choice of reaction conditions. Glyceraldehyde 3~phosphate (67) and cinnamaldehyde were studied in 67 sodium b a rb ita l or t r i s b u ffe rs , but ^ -(2 -fu ry l)a c ry la ld e h y d e showed some form of u n id e n tifie d in te ra c tio n w ith these b u ffe rs , requiring a change to sodium pyrophosphate b u ffe r. Phosphate and pyrophosphate show a marked e ffe c t on the temperature-jump studies o f NAD+ binding to the enzyme (11, 23, 3 6 ). A conformation change o f the E(NAD+)^ species in t r i s b u ffe r is not observed in pyrophosphate, suggesting pyrophosphate s ta b iliz e s a s in g le conformation s ta te (3 6 ). Since phosphate is a substrate o f the c a t a ly t ic reactio n and pyrophosphate is a substrate analog, Bloch (11) has suggested an explanation in volving s p e c ific binding o f the anion to the enzyme subunits. A s im ila r e ffe c t could a c t on trim e th y la c e ty l phosphate binding to prevent sigmoidal k in e tic s from occurring in the reactio n o f £ - ( 2 - fu ry l)a c ry la ld e h y d e in pyrophosphate b u ffe r . Thus the lack o f sigmoid plots o f 1/V versus trim e th y la c e ty l phosphate concentration are pro bably a r e fle c tio n o f reactio n conditions rath er than an inherent anamoly o f the su b strate. The work o f Yang and Deal (80) has shown th a t various adenosine phosphate d erivativ es are in h ib ito rs fo r reactio n o f the yeast enzyme w ith the natural su b s tra te . ATP d issociates the enzyme but adenosine 3 1, 5 1 c y c lic monophosphate (c y c lic AMP) does n ot. The l a t t e r was 4* found to be com petitive w ith NAD , and i t was suggested th a t competi tiv e in h ib itio n was due to binding o f th e adenine nucleotide in the NAD+ s i t e . C yclic AMP shows noncompetitive in h ib itio n o f the reaction of /3 -(2 -fu ry l)a c r y la ld e h y d e w ith the enzyme. Apparently, a llo s te r ic •j" conformation changes in the subunit due to binding a t the NAD s ite can a ffe c t v e lo c itie s in much the same manner as acyl phosphate in h i b itio n . F ife and Szabo (29) have shown c y c lic AMP in h ib itio n o f the natural substrate glyceraldehyde 3- phosphate to be very s im ila r to the acyl phosphate/cinnamaldehyde/arsenate system. 1/V versus c y c lic AMP concentration plots at constant substrate concentrations are sigmoidal in the absence o f arsenate, but lin e a r and much weaker in the presence of arsenate. However, a plot of 1/V versus 1/(S ) a t constant c yc lic AMP concentration indicates in h ib itio n e ith e r com petitive or mixed with regard to glyceraldehyde 3-phosphate. With ( 2 -fu r y l) aery1 - aldehyde as substrate lin e a r plots of 1/V versus c y c lic AMP concentra tio n at constant aldehyde concentration are observed, although, again, th is could be a re fle c tio n of reaction conditions. Acyl phosphates and c y c lic AMP display great s im ila r ity in th e ir behavior. In view of the d ifferen ce in th e ir in h ib itio n patterns i t is u n lik e ly that acyl phosphates and c y c lic AMP are binding at the same s ite . That sigmoidal in h ib itio n plots are seen w ith both types o f in h ib ito rs , therefore suggests th at binding of in h ib ito rs at more than one s p e c ific s it e can produce these e ffe c ts . 69 Summation In the case of glyceraldehyde 3- phosphate as the substrate, low enzyme concentrations were employed and deacylation is rate determin ing. I t is now evident that the acylation process at high enzyme concentrations can, with the proper substrate, give sigmoidal plots of 1/V versus in h ib ito r concentration. I t is also c le a r, in view of the noncompetitive nature of the in h ib itio n and the d iffe re n t types of in h ib itio n plots which depend on aldehyde, in h ib ito r, and the presence or absence of arsenate, that the sigmoidal 1/V versus (I) plots must result because of a subtle in d irect interaction between s ite s . The glyceraldehyde 3-phosphate dehydrogenase reaction is very complex. There appear to be three substrate or in h ib ito r binding sites in close proxim ity. The acyl phosphate and aldehyde binding sites must be adjoining since they form a common intermediate. NAD+ participates in the oxidation reaction, and must also be in close proxim ity. Binding at a ll three sites by substrates or inhibitors are able to transmit a llo s te ric effects through conformational changes in the subunits. In addition, cooperative effects can be transmitted through binding at the NAD+ and acyl phosphate s ite s . Both binding, as in the case of NAD and acyl phosphate s ite s , and velo cities of reaction with the substrate can be influenced by the conformation changes. PART II INTRAMOLECULAR GENERAL BASE CATALYZED ALCOHOLYSIS OF AMIDES AND ESTERS 70 71 EXPERIMENTAL Materials and Syntheses All compounds and reagents were obtained commercially in reagent grade. Buffers were prepared using analytical grade materials and deionized water. Amine buffers were freshly d is tille d before use. Preparation of substituted phthalides. Phthalide (m.p. 73-7^°) was obtained from Matheson, Coleman and B e ll. 5-Nitrophthalide and 7-nitrophthalide were prepared through the appropriate n itr o -ortho- to lu ic acid by photochemical bromination using an adaptation of the method of Eli el and Rivard (24). A fter bromination in carbon te tra chloride, the solvent is removed on a rotary evaporator and hydrolysis in excess hydroxide solution replaces the °c-bromo group with a hydroxyl. Addition of acid catalyzes lactonization to the phthalide which precipitates out of solution. R ecrystallization is achieved from ether/hexane to yield pure samples of 5- nitrophthalide (m.p. 149-151°) or 7~nitrophthalide (m.p. 159"161°). Id e n tific a tio n of product is through infrared spectra and comparison with melting points from the lite ra tu re (73)• 6-Nitrophthalide is prepared by d irect n itra tio n in s u lfu ric acid as described by T iro u fle t (73)- Addition of water causes p re c ip ita tio n of the product, which is then recrystal.l ized from acetic acid and characterized by melting point (143°) and infrared spectra. Considerable d iffic u lty was encountered in the synthesis of 72 4 -n itro p h th a lid e . Photochemical bromination o f 2-m ethyl- 3 - n it r o - benzoic acid was unsuccessful even under extreme conditions with the methyl e s te r. The lit e r a t u r e synthesis (73) produces over 99% of the 6 -n itro p h th a lid e isomer. Since the melting points o f the two isomers d if f e r by only about 6°, and product elemental analysis or infrared spectra should be v ir tu a lly id e n tic a l, l i t t l e confidence can be placed in is*omer separation w ith such a small y ie ld . Reduction o f 3 -n itr o - phthalim ide by Zn/Cu in aqueous sodium hydroxide (34) also proved to be unsuccessful. 4-Aninophthalide was prepared by the method of Vene and T ir o u fle t (74) from the 3-acetamidophthalic anhydride to y ie ld 4-acetamidophthalide (m.p. 184-186°). Treatment with a le . KOH gives the 4-aminophthalide (m.p. 122-123°, HC1 s a lt decomposition 2 4 0 °). The diazonium fluoroborate p re c ip ita te s upon treatment o f the amino phthal ide w ith an equivalent of sodium borofluoride in concentrated HC1 in an ice bath and the slow addition of a solution o f sodium n i t r i t e . Addition of a large excess o f sodium n itrite /c o p p e r powder to an aqueous solution of the diazonium fluoroborate yield s the 4 -n itro p h th a lid e , which is re c ry s ta lliz e d from ethyl acetate/hexane (m.p. 135°). 5-Aminophthalide (m.p. 195-196°), 6-aminophthalide (m.p. 182°), and 7-aminophthalide (m.p. 122- 123° ) were prepared from pure samples of the appropriate n itro p h th a lid e by reduction w ith a 5% platinum catalyst on powdered charcoal under 60 psi hydrogen gas in ethyl acetate so lu tio n . C ry s ta lliz a tio n o f pure product is achieved from hexane. Preparation of the hydroxymethy1 esters. Triethyloxonium flu o ro borate (26) is synthesized by dropwise addition of epichlorohydrin in ether to gently refluxing boron tr iflu o r id e etherate in ether. The p re c ip ita te is filte r e d and dissolved in a c e to n itr ile . 2-Hydroxy- methylbenzoic acid is prepared by hydrolysis of the phthalide in aqueous base which is then neutralized with HC1 to pH 5.5. While holding the pH constant with molar sodium carbonate, a three-fold molar excess of the triethyloxonium fluoroborate solution in acetoni- t r i l e is rapidly added dropwise to the aqueous solution . Immediately a fte r the pH has s ta b iliz e d , the solution is extracted with ether, which is then dried with magnesium s u lfa te and evaporated under p a rtia l pressure. Elemental analysis by Elek M icroanalytical Labs (Table 10) of the ethyl 2-hydroxymethylbenzoate product shows some impurities are present. The liquid product is not susceptible to p u rific a tio n by solvents, and attempted vacuum d is t illa t io n causes lactonization to a re la tiv e ly pure sample of phthalide (Table 10). Hydroxyl protection groups such as dihydropyran can be used to allow d is t illa t io n of the tetrahydropyranyl ether, but removal o f the protecting group in d ilu te acid simultaneously cyclizes the product. Infrared analysis of the hydroxymethyl ester shows the absorption s h ifts from phthalide to be expected: a strong hydroxyl absorption peak a t 3-0 microns, and a s h ift of the lactone carbonyl absorption at 5.65 microns to the open chain ester carbonyl at 5-85 microns. Also, infrared analysis showed the presence of traces of phthalide, which must be the imputities indicated by elemental analysis. Since phthalide is the end product of c y c liza tio n in any case, and is c e rta in ly present only in small amounts, the ethyl 2-hydroxymethylbenzoate preparation was used fo r k in e tic determinations with reasonable confidence. Table 10 shows the structures and elemental analyses fo r a l l the esters and amides under study. The identical synthetic procedure was used fo r the preparation of ethyl 2-hydroxym ethyl-4-nitrobenzoate. As a solid th is product was re c ry s ta llize d from ether/hexane. The melting point of 62-64°, in fr a red analysis and elemental analysis (Table 10) a ll confirm its id e n tity and p u rity . Attempted synthesis o f the ethyl esters of 7 -n itro p h th a lid e and 4 -n itro p h th a lid e was unsuccessful, probably due to some form of s te r ic interference or rapid c y c liz a tio n in the aqueous so lu tio n . Synthesis o f the hydroxylmethyl benzamides. 2-Hydroxymethyl- 3-aminobenzamide (m.p. 7 4 -7 6 °), 2-hydroxymethyl-4-aminobenzamide (m.p. over 150°, decomposes slowly to phthalide at elevated tempera tu re s ), 2-hydroxymethyl-5-aminobenzamide (m.p. 135-136°), and 2-hydroxymethyl-6-aminobenzamide (m.p. 85- 87° ) were synthesized through treatment o f the corresponding n itro p h th a lid e in ethanol with ammonia gas in an ice s a lt bath at -1 0 °. The reduction vessel is sealed over night at room temperature. Solvent removal under p a rtia l pressure yields the hydroxymethyl nitrobenzamides, which are reduced by the platinum/charcoal c a ta ly s t in the procedure previously described, infrared analysis shows absorptions a t 3-05 microns and 6.05 microns and the absence o f any n itro group peaks at 6.6 or 7 .4 microns, as L A r> . Table 10. Analytical Analysis Results for the Ethyl Esters and Benzamides. Compound Structure Calculated Elemental Analysis Found Elemental Analysis % C % H % C ° / o H A Ethyl 2-hydroxymethyl' benzoate Phthalide (product of attempted d is tilla tio n ) Tetrahydropyranyl Ether B Ethyl 2-hydroxymethyl 4-nitrobenzoate C 2-Hydroxymethyl-3" ami nobenzami de C fe N 66.67 71.5 68.16 53.33 57.82 6.67 4.48 7-58 4.94 6.07 64.44 7.05 71.27 53.05 4.80 67.81 7-48 4.94 58.50 5.83 NH, Table 10 (Continued) Compound Structure Calculated Elemental Analysis Found Elemental Analysis % C % H % C % H D 2-Hydroxymethyl-4 - ami nobenzami de E 2-Hydroxymethyl-5 - ami nobenzami de F 2-Hydroxymethyl-6 - ami nobenzamide c - nh NH. CHjO H 57.82 6.07 57.82 6.07 57.82 6.07 58.11 6.04 58.08 6.04 57.63 6.12 77 predicted fo r these compounds. Elemental analysis shows the pu rity of the compounds, except fo r the 3-amino d e riv a tiv e which is s lig h tly contaminated with its phthalide. Rate Measurements and Spectra U ltra v io le t spectra of the amide and ester substrates and th e ir corresponding phthalides were obtained using a Cary 15 spectrophoto meter and 1 cm. quartz c e lls containing the appropriate buffer in both sample and reference compartments. Compounds were introduced as solutions in 10 to 30 *1. a c e to n itrile or methanol, and spectra taken immediately a fte r mixing. For a l l substrates, as the reaction pro- cedes th e ir u ltra v io le t spectra approach that o f the corresponding phthalide u n til the spectra are identical a t complete reaction. Typical comparison spectra fo r the in it ia l reactants and th e ir products are shown in Figure 13• The c y c liza tio n reactions were monitored by following changes in optical density due to appearance of the phthalide or disappearance of the hydroxymethyl ester or amide. The wavelengths chosen fo r monitoring the reaction fo r each substrate are given in Table 11, as well as the reaction observed in each case. _2 Stock solutions of substrate (1 x 10 M) were made up in anhy drous a c e to n itrile or methanol. In reactions using the conventional recording spectrophotometer, 50 m1. of the substrate stock solution is injected into the reaction cuvette containing 3 ml. b u ffe r, and the 78 Figure 13. Typical u ltr a v io le t spectra fo r the ester and amide substrates and th e ir products. A. Ethyl 2-hydroxymethyl- 4 - n itro ben zo ate/5-n itro p h th alid e system. B. 2-Hydroxymethyl- 6 - aminobenzamide/7-am inophthalide system. Phthalide spectra are drawn in dashed lin e s . Wavelength, nm. Extinction Coefficient x 10“**' o o — — • • • • O ' V O N J VJ1 N > C O o Va > o C D O o N 3 61 80 Table 11. Experimental Wavelengths Used to Observe the C yclization Reactions. Compound Reaction Followed Wavelength, nm, A Ethyl 2-hydroxymethyl benzoate B Ethyl 2-hydroxymethyl 4-ni trobenzoate C 2-Hydroxymethyl-3" ami nobenzami de D 2-Hydroxymethyl- 4 - aminobenzami de E 2-Hydroxymethyl-5 ” ami nobenzamide F 2-Hydroxymethyl- 6 - aminobenzami de G 2-Hydroxymethyl- 4 - ni trobenzami de Disappearance o f Ester Disappearance o f Ester Disappearance o f Anide 254 275 Appearance o f Phthalide 290 Appearance o f Phthalide 330 Appearance of Phthalide 315 Appearance o f Phthalide 315 285 81 reaction monitored at appropriate wavelength immediately a fte r mixing. Two to four ra te measurements were tabulated fo r each b u ffe r. In a few cases, p a rtic u la rly the lower pH reactions o f the 4-amino d eriva tiv e , a short lag period is observed. Temperature fo r a ll nonenzymatic reactions was controlled a t 30 i 0 .1 °, and the ionic strength of the buffers kept constant at 0.5 with KC1. Final concentrations were 1.67 x 10 M substrate and 1.67% in acetoni t r i le or methanol. Due to s o lu b ility problems, some rate measurements were carried out in 50% dioxane/water (v /v ). Reactions too rapid fo r the conventional recording spectrophoto meter were monitored using a Durrum-Gibson stopped-flow spectrophoto meter (Model D 110). The substrate is dissolved at desired concentra tion in an unbuffered solution at pH 3*0 (at which i t is generally stable fo r a few hours), and introduced into one of two identical drive syringes. The other syringe contains a high pH b u ffe r, such that on rapid mixing of equal volumes from the two syringes a reaction solution at the required pH was obtained. The drive syringes, mixing chamber, and cuvette were suspended in a water trough whose tempera ture was maintained at 30 t 0 .1 °. Optical density changes a fte r mixing were recorded on a Hewlett-Packard storage oscilloscope (Model 1207B), With each b u ffe r, four to six pairs of reactions with over lapping oscilloscope traces were tabulated. Reaction solution pH's were measured with a Radiometer pH meter Model 22 and G K 2303C combined electrodes standardized with M a llin - chrodt standard buffer solution s. pK_ values fo r buffers at 30° were 9 82 taken from lit e r a t u r e sources (50, 65) . C alculations An 01ivetti-Underwood Programma 101 programmed with a lin e a r regression program, was employed to c a lc u la te the least squares slopes and intercepts of lin e a r data. In a l l illu s tr a tio n s the lin e a r data are joined by a th eo retic a l le ast squares lin e . Pseudo-first-o rder rate constants were calcu lated w ith an IBM 360 computer using a program designed by Edwin Anderson fo r rigorous least squares adjustment of nonlinear data (76, 77)* 83 RESULTS Imidazole is a good catalyst for the cy c liza tio n reaction o f ethyl 2-hydroxymethylbenzoate (A) to phthalide at 30°, as seen in Table 12 where the observed pseudo-first-order constants are calculated fo r a series o f imidazole buffers o f increasing total imidazole concentra tion at constant pH values. Ionic strengths are held constant with KC1. The ra tio of Kg/(K g + a^) fo r each b u ffer, calculated from the known pK and the pH of the buffer at 30°, is used to determine the concentration of imidazole fre e base. Under pseudo-first-order condi tio n s, the concentration o f one reactant (imidazole concentrations range between 0.01 M and 0.5 M) is much greater than the concentration of the other (ester concentration is approximately 1.67 * 10"^ M). The advantages o f pseudo-first-order conditions are (1) experimental ease, (2) no need to know the substrate concentration, and (3) easy separation of the variables involved in the reaction. Observed rate constants are calculated with dimensions of reciprocal time (sec“ M , and are composite rate constants for b u ffer catalysis and spontaneous c y c liz a tio n . A plot of versus catalyzing b u ffer concentration is lin e a r and subject to Equation XVI (see Figure 14). kD, the rate XVI. kobs = kQ + kB (Buffer) constant fo r spontaneous c yc lizatio n at that pH value, is obtained by Table 12. Observed Pseudo-First-Order Rate Constants fo r the Imidazole-Catalyzed C y clizatio n o f Ethyl 2 -Hydroxymethyl benzoate to P h th a lid e .9 PH Imidazole Concentration (Molar) k , x 103 obs (s e c "l) 7-13 0.1 2.30 0.2 2.51 0.3 3.08 0 .4 3-50 0.5 4.12 7.28 0.1 3.04 0.2 3-78 0.3 4.41 0 .4 5.06 0.5 5.49 7.61 0.1 5.3^ 0.2 6.12 0.3 6.76 b 0 .4 7.22 pD = 8.03 0.1 2.07 0.2 2.25 0.3 2.42 0 .4 2.63 0.5 2.81 a C y clizatio n is monitored at 254 nm. at 30° and the buffers are kept at constant ionic strength o f 0.5 w ith KC1 . b Reaction run in D 20 to determine the deuterium k in e tic solvent isotope e ffe c t. pD = pH + 0.41 85 Figure 14. Plot of k0|jS versus the imidazole fre e base concen tra tio n fo r cyc liza tio n o f ethyl 2-hydroxymethylbenzoate to phthalide at 30° and at pH 7.13 ( o ) , pH 7-28 (<•>), and pH 7-61 (• ). (sec 86 ■ o x 0.1 0.2 0 .3 (Im idazole Free Base), M extrapolation of the plot to zero b u ffer concentration. The slope of the plot kg is the second-order rate constant fo r b u ffer catalysis with the dimensions M 'sec ' . Since kg is independent of hydronium or hydroxide ion concentration, plots at d iffe re n t pH values are p arallel only i f k0bs is plotted versus the catalyzing species of b u ffe r, in this case the fre e base o f imidazole. Thus, there is a firs t-o rd e r dependence on imidazole fre e base concentration in the reaction. The value of k |m, the pH-independent second-order rate constant fo r buffer . 0 - 1 - ] catalysis by imidazole fre e base, is 8.05 x 10 J M sec 1. Rate constants were also obtained in D^O as the solvent, the value of the H?0 DoO ra tio of rate constants (k, /k , ) being 3-^6. Im Im Other bases also act to catalyze intermolecular general base catalysis of this reaction and Table 13 gives typical data used to calculate the second-order rate constants tabulated in Table 14 for the lactonization reaction of ethyl 2-hydroxymethylbenzoate. Figure 15 shows a lin e ar plot o f the logrithms o f the second- order rate constants fo r buffer catalysis versus. pKg of the catalyzing base. This type o f relationship, called the Brjzfnsted catalysis law, is a lin e a r free energy relationship o f the c a ta ly tic e ffic ie n c y of a catalyst with its acid ity or b asicity (Equation X V Ii). The slopes of X V II. log kg = /@(pKa ) + Constant Such plots are called Br^fnsted co e ffic ie n ts (/3 ) and indicate the amount of proton transfer in the c r it ic a l tra n s itio n s ta te . For ethyl 88 Table 13. Observed Pseudo-First-O rder Rate Constants fo r B u ffe r- Catalyzed C y c liz a tio n o f Ethyl 2-Hydroxymethy1 benzoate. 3 B uffer PH Concentration ^obs (Molar) (sec ' ') Acetate 5 .6 0 0.2 5 .8 4 X 10"5 0.3 6 .4 4 CH,C00H 0.4 6 .4 4 J 0.5 7.20 Cacodyl ate 6.22 0.1 2.50 X 10"4 0.2 2 .7 7 ( ch3 ) 2As02 h 0.3 0.4 2.96 3.20 0.5 3 .6 0 Tri s 8.13 0.1 2 .20 X 10”2 0.2 2.30 NH_C (CH-OH) 0.3 2.36 2 2 3 0 .4 2.51 9 .3 8 2 .06 10"1 Borate 0.15 X 0.2 2 .30 h2b° 3 0.35 2.4 3 0.4 2.51 0.5 2 .6 9 N, N-Di methyl ethanol - 9.72 0.05 4 .7 9 X 10"1 ami ne 0.1 4 .8 8 (ch3 ) 2 nch2ch2oh 0.15 0.2 4 .9 9 5.02 Di ethyl ami ne 10.98 0.1 6.38 0.2 6.82 (ch3ch2) 2 nh 0.3 0.4 7.01 7 .2 0 0.5 8 .3 9 a C y c liz a tio n is monitored a t 254 nm. a t 30° and the b uffers kept a t constant ionic strength o f 0.5 w ith KC1. are 89 Table l4 . Rate Constants fo r Spontaneous and Genera] Base Catalysis of the C yclization of Ethyl 2-Hydroxymethylbenzoate. Buffer PH k , sec”^ 0 PKa k„, M’ 1 1 -1 sec Acetate 5.60 5.00 X 10-5 4.76 't.71 X O 1 Cacodyl ate 6.22 2.21 X 10_/f 6.33 4.70 X h T * Hydroxyl amine 6.44 2 .98 X 10-4 5.75 2.87 X 10-3 Imi dazole 7-13 1.72 X 10’ 3 7.00 8.05 X 10-3 b 7.28 2.50 X 10_3 7.61 4 .8 0 X 10“3 pD° = 8.03 1.87 X 10"3 7.54 2.52 X 10"3 T ris 8.13 2.08 X 10"2 7.93 1.71 X 10"2 Borate 9.38 1.79 X 10’ 1 9 .1 4 2.83 X 10"1 N,N-DImethyl- 9.72 4.42 X io - 1 9.13 2.53 X io ’ 1 ethanolamine DIethylami ne 10.98 6.16 10.89 10.00 HC1 0.00 3-36 X 10“3 0.38 1.59 X 1(r 3 0.58 9.29 X io -£ 1 .03 3-33 X 10“4 2.54 1.0 X 10-5 a C y clizatio n is monitored at 254 nm. at 30 and the buffers are kept at constant ionic strength of 0.5 with KC1, 0.67% acetoni t r i l e . b Second-order rate constants fo r bu ffer cata ly s is are averaged when more than one pH series is run with the same b u ffe r. c Reaction run in D2O as solven t. pD = pH + 0.41 d Acid catalysis is measured in unbuffered HC1 solutions w ith ionic strength kept constant at 0.5 with KC1. Figure 15. Br^nsted plot of log k versus the pK of the B 3 catalyzing base in the c yc lizatio n o f ethyl 2-hydroxymethyl- benzoate to phthalide at 30° in 1^0. -OH D ieth y1 ami ne Dimethyl ethanol ami ne Borate Tris Imidazole o Cacodylate Acetate PK , 92 2-hydroxymethylbenzoate has a value of 0,87, indicating considerable proton tra n sfer. Also of interest on Figure 15 is the fact that buffers of d iffe re n t types and charges ( I. e . amines, a lip h a tic acids, borate) form a lin e a r relationship. Hydroxyl amine gives an apparent buffer catalysis rate constant considerably above the Br^nsted lin e, but this apparent reaction contained an in terferin g reaction of hydroxyl amine with the phthalide product. The rate constants fo r spontaneous cyclizatio n (Table 14) of ethyl 2-hydroxymethylbenzoate are subject to both hydroxide and hydronium ion catalysis. These rate constants can be separated by calculations in pH ranges where other ions have no e ffe c t. For exam p le , Figure 16 shows the log kQ versus pH p ro file fo r the spontaneous reaction of ethyl 2-hydroxymethylbenzoate. The limbs of this p ro file ""6 **1 o intersect at 10 sec at 30 at a pH value of about 3*7 for the minimum rate of reaction, and the limbs have slopes of plus or minus one for hydroxide and hydronium ion catalysis, respectively. Second- order rate constants can be calculated from Equations XVI11-XIX. X V III. At pH 3 .7 kQ H = kD(OH") XIX. at pH 3-7 kH = ko (H30+) Thus, the spontaneous reaction it s e lf is a re la tiv e ly fa c ile process with kgj^ = ]Q k M”^sec“' and k^ = 3-55 x 10“3 M"lsec- ^ • Reaction in D 2O has a powerful e ffe c t on the rate o f spontaneous cyclizatio n catalyzed 93 Figure 16. Plot of log kQ versus pH p r o file fo r c y c liz a tio n of ethyl 2-hydroxymethylbenzoate ( o ) and ethyl 2-hydroxymethyl-k- ni trobenzoate ( © ) to phthalide and 5-n itro p h th al ide at 30° in H2O. i°g(k0 ) (sec- 1 ) i i i i 00 ON .p- M O M O M C T < o o o 95 by hydroxide ion, giving a r a tio o f 5-72. Hydroxide- catalyzed c y c liz a tio n on the Br^nsted p lo t (Figure 15) is roughly ten fo ld above the lin e fo r general base c a ta ly s is . The second-order ra te constants fo r general base b u ffe r c atalysis and f ir s t -o r d e r spontaneous c y c liz a tio n o f ethyl 2-hydroxymethyl-4 - nitrobenzoate to 5 -n itro p h th a lid e are given in Table 15. Figures 16 and 17 give the log kQ versus pH p r o file fo r hydroxide and hydronium ion-catalyzed spontaneous c y c liz a tio n and the log kg versus pKg Br^nsted p lo t. These figures show that the reaction o f ethyl 2- hydroxymethy1-4-n itro b en zo ate is s im ila r to th at o f the unsubstituted e s te r. Hydroxide c a ta ly s is is increased only f iv e -f o ld (kg^ = 5-01 x 4 -1 -1 10 M sec ) w hile acid catalysis is decreased by a lik e amount -if _ ] _] (k^ = 7*95 x 10 M sec ), and the Brpfnsted c o e ffic ie n t increases s lig h tly to 0 .9 7 . Im idazole-catalyzed c y c liz a tio n in D2O is decreased by a fa c to r o f 2.52 and the rate constant fo r spontaneous reaction is strongly lowered by a r a tio o f 6.90. 2-Hydroxymethyl-3-aminobenzamide (C ), 2-hydroxymethyl-4-amino- benzamide (D ), 2-hydroxym ethyl-5- aminobenzamide j(E), and 2-hydroxy- methyl-6-aminobenzamide (F) a ll undergo both b u ffe r-c a ta ly ze d and spontaneous c y c liz a tio n to th e ir respective p h thalid es. Figure 18 gives comparative log kQ-pH p ro file s fo r the spontaneous rate constants o f each o f these benzamides, whose in d ivid u al rate constants are lis te d in Tables 16-18. 2-Hydroxymethy1-4-aminobenzamide (D) demonstrates k in e tic s analogous to the ethyl esters w ith the two limbs o f the log kQ versus pH p r o file in te rs ectin g at about 10 ^ sec ^ 96 Table 15* Rate Constants fo r Spontaneous and General Base Catalysis a of the C yclization of Ethyl 2-Hydroxymethyl-4-nitrobenzoate. Buffer PH kQ, sec"1 PKa kB, M-' -1 sec Acetate 5.54 2.46 X 10"4 4.76 1.57 X 10“4 Cacodyl ate 6.20 8.22 X IO"4 6.33 2.01 X 10"3 Imidazole 7.14 5-47 X 10"3 7.00 2.29 X 10"2 b 7.41 1.10 X 10“2 7-76 2.07 X IO-2 pDc = 8.02 9.09 X i o " 3 7-54 7.25 X 10-3 Tris 8.26 7.57 X ] 0"2 7.93 1.20 X 10"1 N,N-Dimethyl- 9.80 2.51 9.13 1.45 ethanolami ne Di ethyl ami ne 10.92 2.91 X i o 1 10.89 2.26 X 102 HC1d - 0.32 2.60 X 10‘ 3 0.00 7-95 X 10-4 0.38 3.40 X 10-4 0.58 2.21 X IO"4 1.03 8.37 X 10"5 a C yclization is monitored at 275 nm. at 30° and the buffers are kept at constant ionic strength of 0.5 with KC1, 0.67% acetoni t r i l e . b Second-order rate constants fo r bu ffer catalysis are averaged when more than one pH series is run with the same b u ffe r. c Reaction run in D 20 as solvent. pD = pH + 0.41 d Acid catalysis is measured in unbuffered HC1 solutions w ith ionic strength kept constant a t 0.5 w ith KC1. Figure . Br^nsted p lo t of log kg versus the pKg o f the c atalyzin g base in the c y c liz a tio n o f ethyl 2 -hydroxymethyl- 4-nitrobenzoate to 5-n itro p h th a lid e a t 30° in HgO. 98 -OHO b o / Diethyl ami ne 2 Dimethy 1 ethanol ami ne Borate 0 C O CD Tris Imidazole Cacodyl ate Acetate b 6 8 1 0 12 PK 99 Table 16. Rate Constants fo r Spontaneous C atalys is o f the C y c liza tio n o f 2-Hydroxymethyl- 3 ~aminobenzamide to 4-Ami nophthal id e .a B u ffer pH k , sec o -1 Formate 3 .2 0 5 .02 x o '4 3.60 2.36 x 0 _Zf A cetate **.95 1.65 x 0-3 pDb = 5.41 5 .6 x o~4 Imidazole 6.85 1.82 x 0-3 pDb = 7.45 4 .0 0 x o“4 T ris 8.50 1 .12 x o '3 N,N-Dimethylethanolami ne 9.28 9 .9 6 x o-4 Di ethyl ami ne 10.30 4.0 3 x 0-3 10.79 9.10 x O' 3 Cyclohexylami ne 11.50 3.95 x o-2 K0Hc 11.50 12.23 1.68 x 2.80 x C M — 1 1 o o HC1C 0.98 1.75 2.12 2.68 9.61 x 2.66 x 6.0 9 x 3.03 x °"-3 ° -4 ° - 4 0 4 a C y c liz a tio n is monitored at 290 nm. a t 30 and the b u ffers are kept at constant ionic strength o f 0.5 w ith KC1, 1.67% a c e t o n it r ile . 50% w ater/dioxane ( v /v ) . b Reaction run in D^O as s o lven t. pD = pH + 0 .4 l c HC1 and KOH solution s are unbuffered and kept a t constant ionic strength o f 0.5 w ith KC1. 100 Table 17. Rate Constants fo r Spontaneous and General Base C atalysis of the C yclization of 2-Hydroxymethyl-4-aminobenzamide to 5"Aminophthalide. Buffer3 PH kQ, sec"1 PKa kB, M -1 sec 1 Formate 3.23 3.75 3.41 x 10"4 2 Acetate 4.43 2 .3 0 X 10“5 4 .7 6 5.60 X IO' 4 b 4.60 6.0 x 10"5 3 Cacodyl ate 5.79 1.42 x 10J * 6.33 3.48 x 10“3 4 Imidazole 7.07 3.29 x 10"3 7.00 7.20 X IO"3 b -3 7.10 1 .75 x 10 J 7.51 9.85 x IO- 3 7.63 1.20 x 10“2 5 Tris 8.05 2.11 x 10"2 7-93 1.79 x IO"2 6 N,N-Dimethyl- 9.05 1.58 x , o - 2 9.13 5.05 X 10"2 b ethanol ami ne . -2 9.5 0 3-73 x 10 -2 9.69 6 .8 0 x 10 7 Cyclohexylami ne 10.28 2.3 0 X i o " 1 10.83 5.14 x 1 0 '1 8 Di ethyl ami ne 11.00 9-23 x 10-1 10.89 a C yclizatio n is monitored at 330 nm. at 30° and the buffers are kept at constant ionic strength of 0-5 with KC1, 1.67% acetoni t r i 1e. b Second-order rate constants fo r b u ffer cata ly s is are averaged when more than one pH series is run with the same b u ffe r. 101 Table 18. Rate Constants fo r Spontaneous and General Base Catalysis of Cyclization of 2-Hydroxymethyl-6-aminobenzamide to 7-Aninophthal ide. Buffer PH kQ» sec -1 pKg kg, M“ *sec- ' 1 Acetate 4.00 9.75 X 10 2 Imi dazole 7.16 6.97 X 10 7.45 6.25 X 10 7.^9 4.59 X 10 pD° = 7.99 5.52 X 10 3 Tris 7.84 8.17 X 10 8.32 2.15 X 10 4 Morpholi ne 8.43 1.27 X 10 5 N,N-Dimethyl- 9.05 1.16 X 10 ethanolamine 9.5 0 1.71 X 10 9.73 7.53 X 10 6 Ethylenediami ne 9.96 3.51 X 10 10.10 6.92 X 10 7 Cyclohexylami ne 10.83 4.87 X 10 10.87 2.99 X 10 8 Diethylamine 10.85 3.19 X 10 11.00 6.63 X 10 K0Hd 10.72 3.23 X io' 12.30 1.10 X 10 12.52 2.12 X 10 d HC1 0.35 4.91 X 10 0.68 2.18 X 10 1.35 4.04 X 10 1.75 1.53 X 10 2.51 3.66 X 10 -6 -> -6 -6 -6 ,-6 $ $ -k -2 r 2 > : ? -4 4.76 5.28 x 10-5 7.00 3-40 x 10-Zf b 7 .5 4 1 . 8 9 x 1 0 “^ 7.93 3.81 x 10“^ b 8.25 8.41 x 10-Zf 9.13 9.23 x 10“4 b 9.81 7-12 x 10“3 b 10.52 3.81 x 10"3 b 10.89 3*84 x 10”3 b a Cyclization is monitored at 315 nm. at 30° and the buffers are kept at constant ionic strength o f 0.5 w ith KC1, 1.67% acetonitri le . b Second-order rate constants fo r buffer catalysis are averaged when more than one pH series is run with the same b u ffer, c Reaction run in D2O as solvent. pD = pH + 0.41 d Acid and K0H catalysis is measured in unbuffered solutions with ionic strength kept constant at 0.5 w ith KC1. 102 Figure 18. Plot o f log kQ versus pH fo r spontaneous c y c liz a tio n o f 2-hydroxymethyl-3-aminobenzamide ( o ) , 2-hydroxymethyl-4 - ami nobenzami de ( © ) , and 2-hydroxymethyl-6-ami nobenzami de ( • ) at 30° in H^O. The 3- amino d e riv a tiv e is in 50% w ater/dioxane. log(kQ) (sec ') I I u n 1 “ I i ----------------- T V j O N J O' T 3 o c 00 to £01 104 at 30°• Hydroxide ion catalysis w ith a rate constant o f 1 x 10^ M"' sec” ^ is dram atically increased in comparison to that o f the unsubsti tuted 2-hydroxymethylbenzamide (3) due to what must be e lectro n ic e ffe c ts . Hydronium ion catalysis is affected to a much lesser degree (k^ = 1 x 1 0 ^ M ^sec” ' ) . As with the unsubstituted hydroxymethyl- benzamide (3 ), there exists a b r ie f plateau between pH 8 and 9, probably representing a change in ra te determining step. The gradual plateau observed w ith compound F is probably caused by some s te ric e ffe c t, but both arms are lin e a r and represent normal s p e c ific ion c a ta ly s is . Calculated rate constants fo r 2-hydroxymethyl-6-amino- benzamide are = 7*90 x 10”* M“*sec ^ and k^ = 1.01 x 10-2 M"'sec D 2O solvent isotope effects seem to be much weaker fo r the benzamides giving ratios of 1.80 for the general base-catalyzed reaction and 2.0 for hydroxide ion-catalyzed spontaneous la c to n iza tio n . The 4-aminophthalide product o f c y c liz a tio n of compound C is more insoluble in water than the other d e riv a tiv e s , a cloudy p re c ip ita te forming in the standard b u ffe rs . Reaction in 50% dioxane/water (v/v ) allows complete reaction showing a pH-independent spontaneous reaction _ 1 with a unimolecular rate constant o f 1.34 x 10 3 sec over a very broad pH orange of 3 .7 to 10, S p ecific acid and base cata ly s is can be seen at pH ranges outside th is plateau. For comparison purposes, some reactions o f compound D were run in id en tic al dioxane/water solvents and were shown to decelerate the hydroxide c a talysis about 3 to 4 fold w hile producing l i t t l e or no detectable e ffe c t in s p e c ific catalysis — 6 — 1 by hydronium ion. The 10 sec in tersectio n point fo r compound D is 105 unchanged in 50% dioxane/water b uffers. The pH-independent c y c lizatio n of 2-hydroxymethyl-3-aminobenzamide (C) procedes 2,82 times more slowly in 50% dioxane/D20 than in 50% dioxane/^O . A ctivation parameters are determined from a plot of log kQ versus the reciprocal of temperature of reaction, with temperature expressed in absolute degrees Kelvin. Equations XX are used to calculate the thermodynamic quantities fo r the c r itic a l tra n sitio n state o f the pH- independent cyclization of 2-hydroxymethyl-3-aminobenzamide to 4-amino- phthalide. Individual rate constants are given in Table 19 as well as the activation parameters. -Ea/RT = Slope of plot o f log vs.. 1/T ah* = Ea - RT XX. a F * = -2.3RT log(kr h/kT) AS* = ( A H * - A F * ) / T By measuring the absorbance at 290 mi. as a function o f pH in appropriate buffers immediately a fte r mixing to prevent appreciable cyc lizatio n (13 pH values from 2 .0 to 5 .4) at 23°, the pK of the O aromatic amino group was found to be 3*8. This value is in excellent agreement with the pK derived from the intersection of lines in the a log kQ-pH p ro file . 106 Table 19. Temperature Dependence and A ctivatio n Parameters fo r the 0 pH-1ndependent C yclizatio n o f 2-Hydroxymethyl-3-aminobenzamide. Temperature, °C k ( , sec-1 21.8 1 .766 x 10“3 30.3 3.413 x 10"3 39.5 7.495 x 10"3 50.1 1.537 x IO-2 58.9 2.043 x 10-2 Ea = 14.57 kcal/m ole * &H « 13.98 kcal/mole > II 20.89 kcal/m ole AS -2 3 .4 e .u . a Reaction in imidazole b u ffe rs , pH 7*22, and 50% dioxane/water (V /V ). 107 The hydroxymethylbenzamides are catalyzed by the fre e base form of the catalyzing b u ffer, analogous to the general base catalysis seen fo r the ethyl hydroxymethy1 benzoates. Typical data fo r the imidazole- catalyzed c y c lizatio n are given in Table 20, and Figure 19 shows the plots fo r catalysis by the imidazole free base at pH 7-16 and 7.49. As a consequence o f being on the rate plateau, the rates fo r these lines form a common lin e . Brpnsted plots (Figures 20 and 21) show marked deviations from those of the ethyl esters and unsubstituted benzamide (3 ). Brpnsted c o e ffic ien ts are found to be 0.47 for compounds 0 and E and 0.30 fo r compound F. Hydroxide ion rate constants lie above the Brjtfnsted lin e fo r general bases, in contrast to the ethyl esters. 2-Hydroxy- methyl-3 -aminobenzamide is not subject to s t a t is t ic a lly s ig n ific a n t bimolecular general base c a ta ly s is , the rate plateau apparently masking catalysis fo r the buffers employed and preventing construction of a Br^nsted p lo t. 2-Hydroxymethyl-5-aminobenzamide undergoes very slow reactions. At high pH values the hydrolysis of 6-aminophthalide in terferes with the c y c lizatio n reaction, e ffe c tiv e ly preventing the determination of an extended limb fo r hydroxide ion-catalyzed reaction in the log kQ- pH p r o file . At lower pH values, however, slow c y c liz a tio n of the ben zamide to its phthalide can be measured, and is found to be subject to buffer c a ta ly s is . The very slow reactions precluded extensive rate determinations, although some data fo r buffer and spontaneous catalysis are given in Table 21. The log kc -pH p ro file is very s im ila r to that 108 Table 20. Observed Pseudo-First-Order Rate Constants fo r the Imidazole-Catalyzed C yclizatio n o f 2-Hydroxymethyl-6-aminobenzamide to 7"Aninophthalide.a PH Imidazole Concentration (Molar) kobs * ,05 (sec“^) 7 .1 6 0.1 2.49 0 .2 4.7 6 0 .3 6.63 0 .4 8.40 7 M 0.1 3.06 0 .2 5.78 0 .3 8.58 0 .4 10.76 0 .5 13.37 7.49 0.1 2.89 0 .2 5.52 0 .3 8.66 0 .4 9.31 0 .5 13.58 PDb = 7.99 0.1 1.77 0 .2 3.55 0.3 4.83 0 .4 6.08 0 .5 7.^9 a C yclizatio n is monitored a t 315 nm. at 30 and the buffers are kept a t constant ionic strength of 0 .5 with KC1, 1.67% acetoni tr i le . b Reaction run in D_0 to determine the deuterium k in e tic solvent isotope e f f e c t . pD = pH + 0.41 109 Figure 19. Plot of k ^ g versus the imidazole fre e base concen tra tio n fo r c y c liza tio n o f 2-hydroxymethyl-6-aminobenzamide to 7-aminophthal ide at pH 7*16 ( o ) and pH 7*^9 ( © ) at 30° in H 2O and at pD 7.99 ( • ) in D^). 110 o Q ) if t U \ X 0.1 0.2 0.3 (Im idazole Free Base), M Figure 20. Brfinsted p lo t of log k_ versus the pK of the D 9 catalyzing base in the c y c liza tio n of 2-hydroxymethyl-4 - aminobenzamide to 5-aminophthalide at 30° in H^O. Numbers refer to the catalyzing bases lis te d in Table 17. 112 log (kg) (M- , sec- 1 ) - p - \ j J I O — ■ o — Io V jO o n vn o ' oo to O N 113 Table 21. Rate Constants fo r Spontaneous and General Base Catalysis of C y clizatio n of 2-Hydroxymethyl-5~aminobenzamide to 6— Aminophthalide.a Buffer PH l^o * ' sec .-1 PKa k„, mh sec-^ 1 Acetate 4 .5 8 5.17 X 10-6 PD° = 5.41 6 .0 X JO"6 Cacodyl ate 6.00 4 .0 X IQ '6 2 Imidazole 7.30 7-77 2.7 2 .0 X X 10"6 10"6 7.00 3 .0 X 10"5 b PDC = 7.72 1.8 X i o “ 6 7.54 1.62 x JO"5 3 Tris 8.13 3.7 X 10-6 7-93 3.17 x 10"5 4 Morpholi ne 8.62 1.35 X i o “ 5 8.25 1.28 x ]0~k 5 N, N-DImethy I - ethanol ami ne 9.15 2.82 X 10"5 9.13 1.31 x 10_if 7 Cyclohexylamine 10.20 1 . 11 X 10-4 10.52 5.77 x 10"4 a C yclization is monitored at 315 nm. at 30° and the buffers are kept at constant ionic strength o f 0.5 w ith KC1, 1.67% a c e to n itr ile . b Second-order rate constants fo r b u ffe r catalysis are averaged when more than one pH series is run w ith the same b u ffe r. c Reaction run in D2O as solvent. pD = pH + 0.41 Figure 21. Br^nsted plot of log kg versus the pKa of the catalyzing base in the cyclizatio n of 2-hydroxymethy1-6- ami nobenzami de to 7- aminophthal ide ( o ) and 2-hydroxymethy 1 - -5 -ami nobenzami de to 6-ami nophthal ide ( ® ) at 30° in H^O. Numbers refer to the catalyzing bases lis te d in Table 18. 115 X o vO C M 00 O O ‘•A O O CM vO o ( l_3asl_W) ‘ ( 9>l)6oi 116 of the 6-amino d erivative, with a hydroxide ion-catalyzed rate con stant of 5*6 x 10 ' M"'sec 'and a long pH-independent plateau at about 10 ^ sec ' . Table 22 l i s t the s ig n ific a n t c a ta ly tic data fo r a ll the compounds under discussion in this work, giving the rate constants fo r hydroxide and hydronium ion catalysis, D^O solvent isotope effe c ts , and Br^nsted co e ffic i ents. 117 Table 22. Comparison o f Rate Constants fo r C y clizatio n o f Esters and Ami des. Compound k0H M ^sec”' k h M *sec~^ kb k B k H k0H kD O H Ethyl benzoate 3 .0 x 10"2 (Reference 4) Ethyl 2-hydroxy- methylbenzoate 10** 3-35 x 10“3 0.87 3.46 5-72 Ethyl 2 -hydroxy- m ethyl-4-ni tr o - benzami de 5 .0 x 10** 7.95 x 10"** 0.97 2.52 6.90 Benzami de 1.15 x 10"5 (Reference 5) 2-Hydroxymethyl- benzamide 1.67 1.67 x lO"2 (Reference 3) 2-Hydroxymethyl- 3-ami nobenzami de 1 x 101 a 1.6 x 10"2 3 1 .0 x 10-1 a ,c „ b.c 2.82 2-Hydroxymethyl- 4-ami nobenzami de 1.0 x 10 3 3 .0 x 102 a 1.0 x 10"3 0 .3 0 2-Hydroxymethy1 - 5-ami nobenzami de 5 .6 x 10_1 0.47 1.85 1.0C 2-Hydroxymethyl- 6-ami nobenzami de 7.9 x 10"1 C M 1 O X o • 0.47 1.80 2.00 a Reaction in 50% dioxane/l^O. b Reaction in 50% dioxane/D^O. c pH-independent reaction. 118 DISCUSSION The study of intramolecular catalysis can lead to insight into analogous enzymatic reactions because of the s trik in g s im ila rity between an intramolecular reaction and an enzyme-catalyzed reaction proceeding through an enzyme-substrate complex. In the <c-chymotryps i n catalyzed hydrolysis of esters and amides the hydroxyl group of serine- 195 is i n i t i a l l y acylated in the reaction. Studies of intramolecular nucleophilic attack by alkoxide ions at the carbonyl group of esters and amides can give information concerning the a b ilit y of the serine hydroxyl group to p artic ip a te in intracomplex reactions. For example, the intramolecular cyc liza tio n of hydroxymethylbenzoates and benza mi des to th e ir corresponding phthalides provides an outstanding model of the <C-chymotrypsin-catalyzed reactions. In this section of the discussion, the mechanism o f these nonenzymatic c y c liza tio n reactions w ill be analyzed in d e ta il, followed in the next section by a consi deration of the implications fo r the enzyme-catalyzed reaction. The normal mechanism of both a lk a lin e and acid-catalyzed hydroly sis of esters and amides involves addition of hydroxyl ion or water to the neutral or protonated moiety, followed by elim ination of the leaving group. The hydroxide ion-catalyzed reaction (14) can be w ritten as Equation XXI, where hydroxide ion undergoes nucleophilic 119 attack upon the carbonyl to form a tetrah edral intermediate which then releases the leaving group to y ie ld the products of the reaction. O' XXI r_ £ _ x ^ R - c — x ■ > R y — C— OH + OH + "OH The typical acid-catalyzed reaction is quite s im ila r, involving nucleo p h ilic attack o f water on the protonated interm ediate. However, in the s p e c ific base-catalyzed c y c liz a tio n of esters and amides with neigh boring hydroxymethyl groups, th is mechanism would produce the fre e acid as product— the actual phthalide product indicating that apparent hydroxide ion catalysis must involve displacement of the leaving group through alcoholysis by the neighboring hydroxymethyl group (Equation x = o c h 2 c h 3 , nh2 Y = N02 , NH2 substituents on the 3) 4-> 5> or 6-posit ion The products of reaction of Compounds A through F (Table 10) were characterized by the id e n tity o f the u ltr a v io le t spectra taken at the end o f k in e tic runs w ith the spectra o f the corresponding phthalide. XXI I) o XXII + 'OH + 'X + HjO The phthalides were stable under experimental conditions in acidic and neutral conditions, but alkalin e solutions allow hydrolysis of the phthalide product to the 2-hydroxymethy1benzoic acid . In most cases, however, the rates o f cyclizatio n to form the phthalide were rapid in comparison to the rate of hydrolysis. Thus, hydrolysis of the phthal ide product does not s ig n ific a n tly in te rfe re with measurement of the rate constants fo r c yc liza tio n . As a case in point, consider the example of phthalide and ethyl 2-hydroxymethylbenzoate. The rate constant fo r alkaline-catalyzed hydrolysis o f phthalide (73) at 25° is known to be 1.95 x 10“* M *sec \ while the rate constant fo r specific base-catalyzed cyclization of the ethyl ester as determined in this 4 -1 -1 study is 10 M sec . Obviously with a rate difference of nearly 50,000 there is no interference between the rates o f these reactions. With 2-hydroxymethyl-4-nitrobenzamide, however, overlap does rise due to more rapid hydrolysis of the 5-nitroph thalide and much slower cyclizatio n of the benzamide. At pH 8.0 the firs t-o rd e r rate constant fo r cyclizatio n is 1.67 x 10"^ sec” *, only s lig h tly faster than the *6 ■ 1 rate fo r 5-nitroph thalide hydrolysis of 6.64 x 10 sec” . Thus in this case there is considerable interference, and in fa c t, the only reason any rates fo r cyclizatio n can be measured at a ll is due to the plateau observed fo r these benzamides at about pH values of 7 to 8. At pH values greater than 8 .0 no reaction is measurable since the 5-nitroph thalide hydrolyzes at a rate even greater than i t forms. The reaction of 2-hydroxymethy1-5-amino benzamide is s im ila r. Since the rate fo r a lk a lin e hydrolysis of 6-aminophthalide (6.7 M *min *) is 121 fa s te r than fo r the 5~ or 7_aminophthal1des, and since the rate o f c y c liza tio n is slower than fo r the k- or 6-aminobenzamides, no c y c liz a tion reaction can be measured. Comparisons o f rate constants fo r hydrolysis and c y c liz a tio n reactions of the other rin g-substituted derivatives under study are between the two extremes described. The sp e cific base-catalyzed alcoholysis of a n il ides and esters (58) is known to resemble in a ll respects hydroxide ion-catalyzed hydrolysis. As b r ie f ly described in the results section, the lacton- izatio n of these compounds fo llo w the same general rate equations fo r s p e cific ion catalysis as hydrolysis o f ordinary acyl d e riv a tiv e s . The applicable rate equation is shown in Equation XXIII. Under pseudo- fir s t-o r d e r conditions o f constant a c id ity and using a glass electrode to determine a c t iv it y o f the hydrogen ion (aH) , Equation XXIV can be derived from Equation XXIII. Kw is the known autoprotolysis constant fo r water and kj is the rate constant fo r spontaneous c y c liza tio n which is in sensitive to a^. From Equation XXIV it follows that these over the eas ily accessible experimental range from 0.1 M HC1 to 0.1 M KOH. In the pH range where k^jan « koH ^/a^ i t follows that XXI11. d(RCOX) dt (RCOX) XXIV. k “ k l + k0 H ~ + k»aH o constants may be determined separately since a^ and f^ /a ^ vary by 10 12 122 log kQ = log + anc* w^en ^HaH ^ ^OH^w^H t *ian ^°9 *^o = log k^ - pH. For a l l the compounds under study plots of log kQ versus pH at e ith e r pH range are lin e ar w ith slopes o f +1.0 fo r hydroxide ion catalysis and -1 .0 fo r hydronium ion c a ta ly s is . These slopes indicate the apparent order o f the reaction in hydrogen or hydroxyl ion. The spontaneous cyc liza tio n o f ethyl 2-hydroxymethylbenzoate is a r e la tiv e ly fa c ile process, subject to intramolecular nucleophilic attack by the hydroxymethyl group. The pK_ of the hydroxymethyl group a is undoubtedly comparable to that of ethanol. The ionized species is therefore s u ffic ie n tly basic to displace ethoxide. I t is of obvious interest to compare sp ecific base-catalyzed alcoholysis with hydroxide ion-catalyzed hydrolysis. Ethyl esters normally require high pH and elevated temperatures fo r rapid hydrolysis. Bender (4) has determined the rate constant fo r hydroxide ion-catalyzed hydrolysis o f ethyl benzoate to be 3.0 x 10 ^ M“'sec ^ at 25° in w ater. Therefore, kg^ fo r ethyl 2-hydroxymethylbenzoate is approximately 10^ g reater. This rate enhancement compares favorably with the enhancement seen fo r cyc liza tio n of 2-hydroxymethy1benzamide (3). At 25° the second-order rate constant fo r hydroxide ion-catalyzed hydrolysis of benzamide (5) is 1.15 x 10 M sec , again approximately 103 less favorable than the apparent hydroxide ion catalysis of the c y c liza tio n of 2-hydroxy- methy1benzam i de. Comparison of the unimolecular rate constant fo r the intramolecu la r reaction to the bimolecular rate constant fo r the corresponding interm olecular reaction proceeding by the same mechanism gives a ra tio w ith units of m o la rity . This e ffe c tiv e m o la rity can be taken to be the concentration o f c a ta ly s t in the bim olecular reaction necessary to give a p se u d o -first-o rd e r ra te constant o f the magnitude obtained in the intram olecular reac tio n . Koshland (53) has calcu lated th a t fo r such a p a ir of reactions, an e ffe c tiv e m o la rity o f only about 5“50 M can be a n tic ip a te d from proxim ity e ffe c ts alone. Such values are ■ a q commonly observed (5 6 ). In many cases, however, ratio s o f 10 to 10 are observed (42, 43) which g re a tly exceed expected magnitudes. Kosh land (72) suggests th at exceedingly s e n s itiv e o rie n ta tio n e ffe c ts may account fo r ra te enhancements in a neighboring group reaction in excess of any due to proxim ity e ffe c ts alone. Bimolecular o rie n ta tio n rate 3 5 enhancement factors may in fa c t range from 10 to 10 on the basis of various intram olecular and interm olecular e s te r ific a tio n and la c to n i- za tio n rate enhancements. Apparent hydroxide ion c a ta ly s is o f the c y c liz a tio n o f 2-hydroxy- methylbenzamide and ethyl 2-hydroxymethylbenzoate to p h th alid e is 10^ more favorable than hydroxide ion-catalyzed hydrolysis o f benzamide or ethyl benzoate. Likewise, neighboring hydroxymethyl groups have been studied as intram olecular nucleophiles in the decomposition o f carba mate esters (4 3 ). These very e f fic ie n t reactions proceed through atta c k o f the oxygen anions w ith e ffe c tiv e m o la ritie s fo r the neigh- 5 boring group o f 10 in comparison to the analogous bim olecular reactio n s. Correspondingly huge e ffe c tiv e m o la ritie s have been found fo r phenoxide (42) and carboxylate (15) anion nucleophiles. In the cited examples above, the apparent hydroxide ion catalysis is due to preequilibrium io n izatio n o f the neighboring group. However, the esters and amides in th is study are subject to general base c a talysis, indicating proton tra n sfer is not complete in the c r itic a l tra n s itio n s ta te . Br^nsted c o e ffic ie n ts of 0 .3 _0.5 fo r general base catalyzed c y c lizatio n of the substituted hydroxymethylbenzamides indicate proton tran sfer is not complete, and a deuterium solvent k in e tic isotope e ffe c t fo r apparent hydroxide ion-catalyzed c y c liza tio n ( k | ^ / k * * ^ 2. 2) indicates that 0-H and Q-D bonds are being stretched in the tra n s itio n s ta te . For the ethyl esters a Brtfnsted c o e ffic ie n t o f 0.87 fo r ethyl 2-hydroxymethylbenzoate and very large deuterium solvent isotope effects indicate that proton transfer is appreciable, but must s t i l l be incomplete fo r general base catalysis to be observed. I t can be concluded that a neighboring hydroxymethyl group is a power ful intramolecular nucleophile in alcoholysis reactions even when proton transfer is only p a r t ia l. Hydronium ion sp e cific acid catalysis is observed at low pH ranges fo r the esters and amides in th is study. The bimolecular rate con stant for sp e cific acid catalysis o f the lactonization of ethyl 2-hydroxymethylbenzoate to phthalide a t 30° was found to be 3*35 x 10“3 M *$ec“ *. This compares very favorably with the acid-catalyzed lac tonization o f 2-hydroxymethylbenzoic acid, whose rate constant a t 30° can be extrapolated from the temperature dependence o f rate constants lis te d by Bunnett and Hauser (16) to be 6.6 x 10”^ M"'sec“*. Therefore 125 the probable mechanism fo r acid -catalyzed alcoholysis o f the ethyl esters is the usual one fo r acid -catalyzed alcoholysis involving attack o f the a lc o h o lic hydroxymethyl group on the protonated carbonyl. In th is oxonium ion-catalyzed reaction, water is the conjugate base of the c atalyzin g acid and possibly functions to detach the proton from the 0-H o f the hydroxymethyl group. Equation XXV is a possible s tru c tu re o f the tra n s itio n s ta te fo r la c to n iz a tio n of 2-hydroxymethyl- benzoic a c id . Presumably, replacing the 0-H group on the carboxyl carbon w ith ethoxide does not s ig n ific a n tly a ffe c t the c y c lic a tio n . A cid-catalyzed reactions fo r the hydroxymethylbenzamides are somewhat more rapid. This e ffe c t is in accord w ith the gen erally observed fa c t th at amides are more susceptible to acid c a ta ly s is than th e ir cor responding e s te rs . This e ffe c t is probably due to the fa c t th at NHj is a b e tte r leaving group than ethanol. General base cata ly s is is observed fo r c y c liz a tio n o f these benzamides and ethyl benzoates. In c y c liz a tio n reactions o f carbamate esters (4-3) having a neighboring hydroxymethyl group, b u ffe r c a ta ly s is is not observed. This is probably due to the fa c t th a t the carbonyl group o f the esters is deactivated by the adjoinin g nitrogen so that XXV 126 the tra n s itio n state fo r formation of a tetrahedral intermediate w ill be d i f f i c u lt to a tta in , necessitating nucleophilic attack by a fu lly developed negative charge. With the compounds in the present study, however, the acyl group is not as deactivated, and proton transfer is not complete in the c r it ic a l tra n s itio n state allowing general base catalysis. As described in the Results section, the p a ra lle l lines a t two pH values in a plo t of l< 0(,s versus fre e base concentration show that the fre e base form o f the bu ffer is the catalyzing species for the reaction. Thus, there is a fir s t-o r d e r dependence on buffer fre e base concentration in the reaction. The value of k i„ , the second-order rate inr constant for im idazole-catalyzed cy c liza tio n o f ethyl 2-hydroxymethyl- benzoate, is 8.05 x 10~^ M”^sec”^. At 25° the second-order rate constant for hydroxide ion-catalyzed hydrolysis of ethyl benzoate is *2 “ 1 — 1 3 .0 x 10 M sec” (4 ). Thus (in a formal sense) attack by a weak base (imidazole) and a properly oriented unionized hydroxyl group can be nearly as e ffic ie n t in displacing ethoxide as d ire c t intermolecular attack by the powerful hydroxide ion nucleophite. A s im ila r argument has been presented by Belke (3) fo r 2-hydroxymethy1benzamide. There have not previously been any studies o f general base-catalyzed trans ester ific a tio n reactions of esters. The finding o f such catalysis in the c y c liza tio n of the ethyl esters of 2-hydroxymethylbenzoic acids to th e ir phthalides is o f great significance in consideration o f the o^-chymotrypsin acylation step. Rate laws in d icate stoichiom etry o f a tra n s itio n s ta te but provide no inform ation concerning how the components of th at tra n s itio n s ta te are arranged s tr u c tu r a lly . There are several k in e tic a lly equivalent mechanisms fo r the general base c a ta ly s is observed in th is study. Equations XXVla through XXVld i ll u s t r a t e these mechanisms. XXVIa. General base c a ta ly s is o f formation o f the tetrah edral i nterm ediate. XXVIb. General base c a ta ly s is o f breakdown o f the tetrah edral i nterm ediate cHtoH o O + X' XXVIc. General a c id -s p e c ific base c a ta ly s is . 128 XXVld. Nucleophilic catalysis by the base. CHjOH + X CHjOK + Intermolecular nucleophilic catalysis (XXVld) can be eliminated as a possible mechanism on the basis o f several arguments. Consider- ation o f the re la tiv e pK values of the leaving groups strongly Q suggests that should nucleophilic attack occur i t would almost c e rta in ly be an unproductive reaction with the intermediate undergoing breakdown to s ta rtin g products. For example, imidazole with a pKg of 7 is a much b e tte r leaving group than ethoxide (pKg over 14). Should nucleophilic attack by imidazole occur to form a tetrahedral in te r mediate by Equation XXVI1, the imidazolium group is a t least 10^-fold more lik e ly to be the leaving group in any subsequent step than the XX VII. C - Q £ t + Im CHjOH £OH OEfc ♦ Im ethoxide group, regenerating the o rig in a l compound w ith no o verall reaction. In add ition , the Br^nsted plots fo r base catalysis are very lin e a r with the rate constants fo r catalysis by bases o f d iffe rin g types f it t i n g on the same lin e . The Br^nsted plot fo r nucleophilic displacements at the ester carbonyl of para-nitrophenvl acetate (44) 129 serves to emphasize the inadequate gross c o rre la tio n between rate and pK fo r nucleo philic c a ta ly s is on the basis o f d iffe r e n t types of d nucleophiles (fo r example, nitrogen nucleophiles such as amines or imidazoles versus, oxyanion nucleophiles such as a c e ta te ). In Br^nsted p lo ts , the lines fo r general base reactions, the lines representing p a rtic u la r base types, are not as g re a tly separated as observed in nucleo philic reactions, so th is e ffe c t can sometimes be employed as a diagnostic tool (1 4 ). In a d d itio n , n u cleo p h ilic reactions are insen s it iv e to any major deuterium solvent isotope e ffe c ts . Rate constants obtained in D_0 as solvent g ive kl?2®/k*?2® as 3*46 fo r c y c liz a tio n of 2 im Im ethyl 2-hydroxymethylbenzoate, and small but s t i l l s ig n ific a n t ratios are found fo r the base-catalyzed c y c liz a tio n o f the benzamides. Thus, in conclusion i t is w ith reasonable confidence th a t we can e lim in a te any form o f nucleo p h ilic mechanism (XXVld) fo r base c a ta ly s is in th is system. The deuterium solvent isotope e ffe c t can be used fu rth e r to distingu ish between s p e c ific and general-catalyzed processes whose rate equations are k in e t ic a lly in distinguishable (1 4 ). S p ecific base H D catalysis exh ibits a k /k r a tio less than one. Both general base and general acid c a ta ly s is , on the other hand, e x h ib it ra tio s g re a ter than two. In a general a c id -s p e c ific base mechanism (XXVIc) the e ffe c ts should cancel to produce a very small o r no D^O solvent isotope e ffe c t. Since D^O does act to s ig n ific a n tly decrease the ra te constants fo r base c a ta ly s is in th is study i t is probable th a t some form o f general base mechanism is being follow ed, and the general a c id -s p e c ific base 130 mechanism (XXVIc) can be eliminated as a p o s s ib ility . It is o f in te r est here to note that hydroxide ion c a ta ly s is possesses a strong D^O e ffe c t and lie s r e la tiv e ly close to the Br0nsted lin e , indicating hydroxide ion probably acts as a general base in its c a ta ly s is . The general base mechanisms suggested (XXVIa,b) d iffe r in th e ir s ite o f c a ta ly s is . Since the deuterium solvent isotope e ffe c t in d i cates that 0-H bonds are being stretched in the tran sitio n s ta te of the rate determining step, these two mechanisms would also involve d iffe re n t rate determining steps (43) as shown in Equation X X V III. XXVI11. \ XXVlb XXVI a CH, o II c \ o / CH, The problem of distinguishing the s ite o f catalysis by general bases is approached by introducing intram olecular bases on the phthalide ring in positions allowing only one possible s ite of c a ta ly s is . Mechanisms XXVIa and XXVlb can be represented in these compounds as Equations XXIXa and XXIXb. With 2-hydroxymethyl-3-aminobenzamide XXI Xa XXI Xb 131 (XXIXa) the amino group is in a position to act as a general base to ab s trac t a proton from the hydroxymethyl group fo r c a ta ly s is o f the formation o f the tetrah ed ral interm ediate. The 6-amino d e riv a tiv e (XXIXb), however, could act to favor the anionic form o f the t e t r a hedral interm ediate and catalyze decomposition o f the tetrah ed ral interm ediate to the am?nophthalide. I t should be noted that a large body o f research w ith imidate esters (61, 68, 70) suggests the existence o f a t least one tetrah edral interm ediate on the reaction pathway between the amide reactant and products. Buffer c a ta ly s is o f both formation and breakdown o f the interm ediates (61) was suggested to explain curvature o f a p lo t of bicarbonate c a ta ly s is o f the la c to n iza tio n o f 2-hydroxymethylbenzamide. No such curvature was detected in th is study, the c a ta ly s is being lin e a r between concentrations o f 0.005 M to 0.5 M to ta l b u ffe r. Possibly the e ffe c t seen by Schmir was a p e c u lia rity o f the bicarbonate b u ffe r , or re s tric te d to a r e la tiv e ly narrow pH range not investigated in these stu d ies. At any ra te , the concept o f c a ta ly s is a t e ith e r step o f the reaction is fa m ilia r and deserves con sid eration. The log kQ-pH p r o f ile fo r the 6-amino d e riv a tiv e (XXIXb) shows no c a ta ly s is which could be a ttrib u te d to the amino group. In fa c t, on comparison w ith the 4-amino d e riv a tiv e where the amino cannot play any d ire c t ro le in the reac tio n , the rates o f la c to n iz a tio n show a dramatic decrease. The ra te constant fo r apparent hydroxide ion c a ta ly s is is decreased over 1 0 0 -fo ld . Any mechanism involving general base c a ta ly s is o f the tetrah ed ral interm ediate can obviously be 132 elim inated on this basis* The reason fo r the large decrease in re a c tiv ity with the amino group in the 6-position is unclear. Both the 6- and 4-amino deriva tives should have s im ila r inductive effects since both are meta to the hydroxymethyl group and in e ith e r ortho or para position to the carbonyl. The log kQ-pH p ro file does not indicate any e ffe c t of an ionizable group such as an amino on la c to n izatio n . Probably, the e ffe c t is some form o f s te ric deceleration. A Stuart-B riegleb model of the tetrahedral intermediate (XXIXb) indicates there should be re la tiv e ly l i t t l e s te ric interference, but the hydroxymethylbenzamide is less subject to s te ric interference than the aminophthalide. Thus the 6-amino group may act by p a rtitio n in g breakdown o f the tetrahedral interm ediate to b etter favor back-reaction to the amide rather than c y c liza tio n to the phthalide. A theoretical k in e tic a lly equivalent a lte rn a tiv e to intramolecular general base catalysis fo r 2-hydroxymethyl-3-aminobenzamide would be a rate enhancement due to an e le c tro s ta tic e ffe c t in the zw itterio n pictured in Equation XXX. However, the pH-independent c y c liza tio n of 2-hydroxymethyl-3_aminobenzamide proceeds 2.82 times more slowly in 50% dioxane/D^O than 50% dioxane/H^O indicating that proton transfer takes place in the c r itic a l tra n s itio n s ta te . The zw itte rio n as the o XXX +nh3 133 a c tiv e species would be unaffected or even fa s te r in a D^O solvent The only remaining mechanism l e f t to account fo r general base- catalyzed c y c liz a tio n reactions is shown in XXiXa and XXVIa. The log kQ— pH p r o f ile fo r 2-hydroxymethyl-3-aminobenzamide strongly sup ports th is mechanism by e x h ib itin g a broad p la te a u . The pH-independent o f 2-hydroxymethylbenzamide and 2-hydroxymethyl-6-aminobenzamide. Due to the r e la tiv e ly rapid hydroxide io n -catalyzed re a c tio n , there is no plateau in the p r o f ile fo r c y c liz a tio n o f the 4-amino d e riv a tiv e , where th e amino sub stitu en t cannot act w ith e ith e r the hydroxymethyl group o r a possible tetrah ed ra l in te rm ed iate. The limbs o f the log kQ - pH “6 -l p r o f ile fo r 2-hydroxymethyl-4-aminobenzamide in te rs e c t a t 10 sec which must represent a maximum value fo r a pH-independent re ac tio n . Thus, the ra te constant fo r pH-tndependent c y c liz a tio n o f the 3-amino 3 intram olecu lar d e riv a tiv e must be a t le a s t 10 g re a te r than fo r c y c liz a tio n o f the 4 - amino d e r iv a tiv e . In conclusion, the mechanism o f general b u ffe r c a ta ly s is almost c e r ta in ly involves c a ta ly s is by the mechanism shown in Equation XXXI w ith im idazole as the general base. • a reactio n from pH 3*7-10 is approxim ately 10^ fa s te r than in the case XXXI o \ H N NH 13^ i t seems appropriate at th is point to discuss the rate plateau observed in the pH 8 to 9 region for cy c liza tio n of the lt-amino derivativ e. Belke (3) recently suggested there occurs a change in rate determining step at about pH 8 w ith formation of the tetrahedral intermediate of 2-hydroxymethylbenzamide being rate lim itin g a t more a lkalin e pH values. However, Schmir (61) has given strong evidence fo r in te rp re tatio n of a change in rate determining step in the opposite sense. From the e ffe c t o f pH on product ratios of the hydrolysis of the iminolactone 2-phenyliminotetrahydrofuran (22, 70) and the iminolactone (61) of the hydroxymethylbenzamide/phthalide system, i t can be concluded that lacto n izatio n involves rate determining formation of a tetrahedral intermediate a t pH less than seven, with tra n s itio n to rate determining breakdown of the intermediate at higher pH. The plateau in the log kc-pH p r o file fo r c y c liza tio n of 2-hydroxymethyl-k- aminobenzamide between pH 8 to 9 is the range o f this tra n s itio n . A sim ilar e ffe c t is seen fo r the log kQ-pH p ro files fo r unsubstituted 2-hydroxymethylbenzamide and 2-hydroxymethyl-4-nitrobenzamide. These pH regions for cyc liza tio n o f the 3“ and 6-amino derivatives are masked by neighboring group e ffe c ts . Equation XXXII describes the s im p lifie d o XXXII mechanism fo r lactonizatio n presented by Schmir (61). This mechanism which holds in the pH region 5 to 9 is consistent with the tra n s itio n 135 from hydroxide ion ca ta ly s is to pH independent pathways as pH is increased. I t is important to remember in the present study th at a change in rate determining step , should one occur, does not a ffe c t our m echanistic conclusions. The mechanism o f ra te determining formation o f interm ediate is shown to be almost c e rta in ly v a lid fo r the neutral pH ranges o f im idazole buffers in H^O and D^O solvents — in agreement w ith Schmir's conclusions. Imidazole c e rta in ly acts to c a ta ly ze the ra te lim itin g form ation o f tetrah ed ral interm ediate. Of course the mechanism o f hydroxide ion s p e c ific base c a ta ly s is a t higher a lk a lin e ranges may w ell d i f f e r . However, the present findings are ap p licable to the ^-chymotrypsin reactions which also occur a t neutral pH ranges. The la c to n iz a tio n o f ethyl esters presented in th is work d iffe rs from th at of the benzamides in that th ere is no evidence of any change in rate determining step in t h e ir log kQ-pH p r o file s . Fersht (10, 25) suggests th at the changeover from ra te determining formation to breakdown of the tetrah ed ral interm ediate during alcoholysis of amides occurs a t about 1-2 pH units below the pK o f the leaving group 3 amine. I f th is p rin c ip le can be applied to the alcoholysis or tra n s - e s t e r ific a t io n o f the ethyl e s te rs , the tra n s itio n regions would be a t pH values beyond the range o f th is work, since ethoxide has a high pK valu e. Thus, no change in rate determining step is necessarily O expected w ith in moderately a lk a lin e pH ranges, a r e fle c tio n o f d i f f e r ing pK values o f the amine and ethoxide leaving groups. 3 There have been no previous studies o f intram olecular alcoholysis reactions where catalys is by a general base is also intram olecular. The c y c liza tio n o f 2-hydroxymethyl-3 -aminobenzamide allows c a lcu latio n of the e ffe c tiv e m olarity o f a neighboring general base, essential information fo r the analysis o f °C-chymotrypsin rates in terms of individual mechanistic factors (8 ). S ig n ific a n t interm olecular b u ffer catalysis fo r the c y c liz a tio n of this d e riv a tiv e can not be detected due to the rapid broad pH-independent plateau in the log kQ versus pH p r o file . Evidently the rate enhancement due to intram olecular buffer catalysis by the neighboring amino group is so strong that intermolecu la r buffer catalysis simply cannot compete. Since we cannot compare interm olecular and intram olecular b u ffe r catalysis In the same compound comparisons must be made with other benzamides of the amino-substituent s e rie s . The apparent pK value o f the amino group in the 3 _aniino position corresponds to the point at which tangents to the plateau and descending limb intersect in a log kQ-pH p r o file . This value is v i r tu a lly identical to the spectrophotom etrically determined pKg of 3*8 fo r the amino group. By extrapolatin g the pKg o f th is 3 - amino group onto the BrjSnsted p lo t fo r interm olecular b u ffer catalysis of the other d e riv a tiv e s , a bim olecular rate constant can be c Icu late d . A compari son of the pH-independent rate o f c y c liz a tio n fo r 2-hydroxymethyl-3 - aminobenzamide where the amino group can act as a general base with the interm olecular rate constant fo r b u ffer catalysis by a group of the same pKg would allow the c a lc u la tio n of the e ffe c tiv e m olarity of th at group in the intram olecular compound. The best d e riv a tiv e fo r comparison is 2-hydroxym ethyl- 5 -amino- benzamide where-—since both the 3 - and 5-p o s itio n s are meta to the carbonyl and e ith e r ortho o r para to the hydroxymethyl— the inductive e ffe c ts should be approximately equal. The c y c liz a tio n o f 2-hydroxy- methyl-5-aminobenzamide is q u ite slow. In fa c t, determ ination o f the r a te constant fo r hydroxide ion c a ta ly s is is somewhat u ncertain due to in te rfe re n c e by 6-am inophthalide h yd ro lys is. At moderate pH values, however, c y c liz a tio n o f 2-hydroxymethyl-5-aminobenzamide can be meas ured, and b u ffe r c a ta ly s is detected. The p a r tia l BrjSnsted p lo t fo r th is d e riv a tiv e shows a slope very s im ila r to the o th er su b stituted hydroxymethylbenzamides ( /3 = 0 .4 7 ), but the rates o f b u ffe r c a ta ly s is are much lower than fo r the 4-amino d e r iv a tiv e . With the 5-amino compound the amino group para to the hydroxymethyl exerts a much s tro n ger electro n donating inductive e ffe c t on the hydroxymethyl than w ith the 4-amino compound. The resu ltin g pKa o f the hydroxymethyl group is ra is e d , apparently leading to much less e f f ic ie n t b u ffe r c a ta ly s is . I t seems apparent th a t the c y c liz a tio n reactio n fo r the hydroxymethyl- benzamides is highly dependent upon the e le c tro n ic s ta te o f the hydroxymethyl group and the ease of proton a b s tra c tio n . The e x t r a p o la t io n o f th e Br0nsted p l o t to an amine b u f f e r o f pK 3 . 7 y ie ld s a v e ry low s e c o n d -o rd e r b im o le c u la r r a t e c o n s ta n t o f a p p ro x im a te ly 4 x 10“^ M *sec By com parison o f th e b im o le c u la r r a t e c o n s ta n t f o r c a t a l y s i s o f 2 -h y d ro x y m e th y l-5 -a m in o b e n za m id e by an amine o f pK 3-7 (4 x 10- ^ M~^sec w it h th e r a t e c o n s ta n t f o r pH- in d ep en d en t c y c l i z a t i o n o f 2 -h y d ro x y m e th y l-3 -a m in o b e n za m id e (k | = 138 1.6 x 10“3 sec ^ in 50% dioxane/H2 0 ; approximately 5 x 10~3 sec”' in pure H^O), an e ffe c tiv e m olarity fo r intram olecular general base catalysis by amine can be estimated as 103 - It/* M. This is a large rate enhancement fo r general c a ta ly s is . A s im ila r c a lc u la tio n in a comparison with 2-hydroxymethyl-4-aminobenzamide yield s an e ffe c tiv e m olarity about two or three orders o f magnitude lower (15 M). This e ffe c t, again, must be a re fle c tio n of d iffe r in g inductive e ffe cts of the amino group in the k- and 5~position s. In a S t u a r t- B r ie g le b model o f 2-h yd ro xym eth yl-3-am in ob en zam id e, gen eral base a b s tr a c tio n o f th e p ro to n from th e hydroxym ethyl group by th e neig h b o rin g amino group cannot be co n certed w ith n u c le o p h ilic alcoho lysis o f th e c a rb o n y l. Some r o t a t io n o f th e a lk o x id e group a f t e r p ro to n a b s tr a c tio n is necessary f o r carbonyl a tta c k in th e space f i l l i n g model even under th e best s te re o c h e m ic a l o r ie n t a t io n o f th e groups. However, the simultaneous occurrence of the D 2O solvent 3 isotope e ffe c t and l£ r rate enhancement o f the pH-independent spontan eous c y c liz a tio n requires the existance of some form of concerted mechanism. An a c tiv a tio n energy analysis gives a large negative (-2 3 .4 e .u .) entropy of a c tiv a tio n , strongly supporting the assumption of considerable r ig id ity or ordering in the stereochemical positioning of the functional groups. Either o f two explanations can be presented, ( l ) A Stuart-B rieg leb s p a c e -fillin g model does not necessarily accurately represent the actual chemical molecule, where i t is possible that bond angles and lengths may be more f le x ib le . Thus, s tra in in the c r it ic a l tra n s itio n state greater than indicated by the model may 139 be possible to allow a concerted reaction. The large negative entropy of activation could be a result of this s tra in . (2) General base catalysis by the neighboring amino group may act in a classical manner, j_.e. through the involvement of a water molecule as seen in Equation X X X III. In this case no unusual s tra in in the Stuart-Briegleb model need be suggested. The loss in entropy due to involvement of a water molecule in the tra n s itio n state may easily be reflected in the entropy of activatio n . Either mechanism is possible since they are k in e tic a lly equivalent. Reaction of 2-hydroxymethyl -3 - arn i nobenzamide in 50% water/dioxane seems to be three to four times slower than reaction in pure 1^0 w ithin the pH range o f the rate plateau, possibly lending some credence to the theory that water plays a functional role in the second mechanism. Considerable caution must be placed in this conclusion, however, since the reaction in pure ^ 0 may be an a r tifa c t due to p recip ita tio n of the phthalide product. In any case, this system is the f i r s t known genuine example of bifunctional catalysis with a large negative entropy. o XXXI11 H 140 IMPLICATIONS FOR oc-CHYMOTRYPSIN CATALYZED REACTIONS The oc-chymotrypsin-catalyzed hydrolysis of esters and amides involves acylation o f serine-195» w ith release o f the alcohol or amide portion o f the substrate, followed by deacylation to generate the a c tiv e enzyme (9, 14). The generally accepted mechanism fo r the acylation step involves h is tid in e -5 7 functioning as a general base, p a r t ia lly abstracting a proton from the serine hydroxyl as i t attacks the carbonyl group o f the substrate. Nonenzymatic acyl tra n s fe r reactions have been widely studied in attempts to understand the chemistry leading to cata ly s is by chymo- trypsin and other esterase enzymes. However, there are very few unambiguous observations o f cata ly s is o f acyl tra n s fe r by nucleophiles. No conclusive examples o f nonenzymatic nucleophilic c a ta ly s is of acyl tra n s fe r by serine or cysteine derivatives have yet been reported, although there is some evidence they may be suited fo r the function under some conditions. For example, N-acetylserinamide ( l) has a nucleophilic r e a c tiv ity toward im idazole-catalyzed a c y latio n by energy rich acyl compounds some three orders o f magnitude higher than the re a c tiv ity of water or ethanol. Belke (3) concluded that im idazole-catalyzed c y c liz a tio n of 2-hydroxymethylbenzamide to phthalide is a good model fo r acylation of -chymotrypsin by amide substrates. The present findings with 141 corresponding esters such as ethyl 2-hydroxymethylbenzoate and the substituted hydroxymethylbenzamides now make derivatives of 2-hydroxy- methylbenzoic acid reasonable models fo r reaction o f «c-chymotrypsin with both ester and amide substrates. The reaction of Equation XXXI is very lik e ly closely analogous to the reaction o f «-chymotrypsin with ethyl esters and amides. Acylation, proceeding through an enzyme-substrate complex, can be considered quite analogous to an intramolecular reaction. In te r- molecular general base catalysis of intramolecular alcoholysis of esters and amides s t i l l , therefore, does not proceed by the < > c - chymotrypsin mechanism where a ll functional groups are intramolecular. The cyclization of 2-hydroxymethyl-3 -aminobenzamide provides the f ir s t study of an intramolecular alcoholysis reaction with catalysis by a general nitrogen base also intram olecular. Comparison of this mechanism with the chymotrypsin-catalyzed mechanism in Equation XXXIVi clearly shows that the two reactions proceed vis v ir tu a lly identical reaction pathways. Bender (8) has attempted to analyze the catalysis by ®c-chymo- trypsin in terms of individual mechanistic factors. I t is now possible to discuss the workings of the enzyme on a straightforward XXXIV o 142 chemical basis with each fa c to r based on more s o lid chemical analogy than could be achieved at the time o f Bender's analysis. The non enzymatic hydroxide ion-catalyzed hydrolysis and the *c~chymotrypsi n- catalyzed hydrolysis of N-acetyl-L-tryptophanamide (Equation XXXV) can now be compared in q u a n tita tiv e d e ta il. xxxv. i ii i c B ' r N H ' NHCOCHj The bimolecujar rate constant fo r hydroxide ion-catalyzed hydrolysis of the amide group of N-acetyl-L-tryptophanamide can be -4 -1 -1 estimated as 3 x 10 M sec . To compare th is reaction w ith the enzymatic process, the f i r s t step is conversion to an interm olecular general base-catalyzed reaction involving imidazole. Jencks and Carruiolo (45) have shown th a t this comparison indicates that 1 M -6 imidazole is equivalent to 1 .6 x 10 M hydroxide ion. Thus the hydroxide ion-catalyzed hydrolysis can be transformed into an in te r- molecular im idazole-catalyzed hydrolysis of ra te constant 4 .8 x 10*"^ M ^sec So f a r , Bender's analysis is probably c o rre c t. At this p o in t, however, the present work allows more q u a n tita tiv e determination o f the k in e tic factors responsible fo r the enzyme- catalyzed reaction. Bender (7, 8) argues th at the alcoholysis o f a carboxylic d e riv a tiv e proceeds at a ra te approximately 100-fold fa s te r than the hydrolysis o f the corresponding compound, thereby assigning o a rate enhancement o f only 10 for the change in ra te determining step 143 from hydrolysis to alcoholysis. The present work cle a rly shows that a neighboring hydroxymethyl group undergoing p a rtia l proton transfer 5 allow intramolecular alcoholysis to undergo a 10 rate enhancement over the hydroxide ion-catalyzed hydrolysis. The e ffe c tiv e m olarity o f an intramolecular general base catalyst in 2-hydroxymethyl-3-aminobenzamide has been calculated to be approxi- 3 4 mately l ( r tolO M in comparison to the intermolecular general base catalysis of 2-hydroxymethyl-5-aminobenzamide. This is well in excess of the range predicted for simple proximity effects by Koshland (51) and Bender (6). These two intramolecular effects are a d d itiv e in the bifunctional catalysis of 2-hydroxymethyl-3-aminobenzamide. Assuming results for this model system can be extended to the intermolecular general base- catalyzed hydrolysis of N-acetyl-L-tryptophanamide in the oc-chymo- trypsin enzyme-substrate complex, an intramolecular rate constant of i 4.8 x 10 sec can be calculated. The rate constant for the ec-chymotrypsin-catalyzed hydrolysis at pH 8, the pH of maximal rate in water at 25°, is 4 .4 x 10”^ M”*sec”^ (78)* The rate enhancements for enzyme-catalyzed reactions can thus easily be e n tire ly accounted for by neighboring group e ffe c ts , with no ambiguous approximations for general acid catalysis or substrate freezing required. In conclusion, only three factors have been shown necessary to account for the enzymatic process: (1) the general base catalysis by imidazole; (2) its intramolecular character and the concomitant increase in e ffe c tiv e concentration of the neighboring hydroxymethyl c a ta ly tic group; and (3) the intram olecular nature o f the general base c a ta ly s is . These e ffe c ts are summerized in Table 23 to show the e ffe c t of each fa c to r on the comparison o f enzymatic and nonenzymatic processes. 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USA 6 6 : ^ 5 , 1970. 73* T ir o u fle t, J ., Bui 1. Soc. S c i. Bretagne 26:7. 1951. 74. Vene, J ., and J. T ir o u fle t, Compt. Rend. 231:911. 1950. 75. Wenkert, E., D.B.R. Johnston, and K.G. Dave, J. Org. Chem. 2 9: 2534, 1964. 76. Wentworth, W.E., _J. Chem. Ed. 42:96, 1965. 77* Wentworth, W.E., J.. Chem. Ed. 42:162, 1965* 78. Wolf, J .P ., I I I , and C. Niemann, Biochemistry 2:493. 1963. 79. Wu, R ., and E. Racker, J.. B io l. Chem. 234:1020. 1959• 80. Yang, S .T ., and W.C. Deal, Jr., Biochemistry 8:2806. 1969. INFORMATION TO USERS This dissertation was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation o f techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page{s)'\ If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being ‘ photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 A Xerox Education Company 74-21,454 BENJAMIN, B ru c e Monte, 1948- ! S U B S T R A T E A N D INHIBITOR INT ER A C TIO NS O F i G L Y C E R A L D E H Y D E 3 -P H O S PH AT E D E H Y D R O G E N A S E | A N D I N T R A M O L E C U L A R G E N E R A L B A S E C A T A L Y Z E D i A L C O H O L Y S IS O F AM IDE S AN D E ST ER S. j U n iv e rs ity of Southern C a lifo rn ia , P h.D ., 1974 1 Chem istry, b io lo g ic a l i \ University Microfilms, A X E R O X Company, Ann Arbor, Michigan j 1 0 THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.

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Asset Metadata

Creator Benjamin, Bruce Monte, 1948- (author)

Core Title Substrate and inhibitor interactions of glyceraldehyde 3-phosphate dehydrogenase and intramolecular general base-catalyzed alcoholysis of amides and esters

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1974-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Fife, Thomas H. (committee chair), Harding, Boyd W. (committee member), Weber, William P. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-836035

Unique identifier UC11356555

Identifier 7421454.pdf (filename),usctheses-c18-836035 (legacy record id)

Legacy Identifier 7421454.pdf

Dmrecord 836035

Document Type Dissertation

Rights Benjamin, Bruce Monte

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA