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Steric effects in the acid, amine, and glyceraldehyde-3-phosphate dehydrogenase catalyzed hydrolysis of acyl phosphates

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Steric effects in the acid, amine, and glyceraldehyde-3-phosphate dehydrogenase catalyzed hydrolysis of acyl phosphates

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Content STERIC EFFECTS IN THE ACID, AMINE, AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE CATALYZED HYDROLYSIS OF ACYL PHOSPHATES by David Richard Phillips A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry) January 1969 UNIVERSITY OF SOUTHERN CALIFORNIA T H E G R A D U A T E S C H O O L U N IV E R S IT Y PA R K LO S A N G E L E S , C A L IF O R N IA 9 0 0 0 7 This dissertation, written by David Richard Phillips under the direction of h i D i s s e r t a t i o n Com mittee, and approved by all its members, has been presented to and accepted by The Gradu ate School, in partial fulfillment of require ments f or the degree of D O C T O R OF P H I L O S O P H Y Dean Date January, 1969 DISSERTATION CO M M ITTEE CkaU ACKNOWLEDGMENTS I wish to thank the members of my committee for aid in preparation of the manuscript. Particular thanks is owed to Professor Thomas H. Fife, whose ideas, direction, and example have been invaluable during graduate school. Finally, I would like to thank my wife, Mary, for her patience and encouragement in my academic studies and my son, Jeffrey, whose very presence inspired rapid completion of this work. TABLE OE CONTENTS Chapter Page ACKNOWLEDGMENTS........................... ii LIST OF TABLES...................................... v LIST OF ILLUSTRATIONS.............................viii INTRODUCTION ...................................... 1 Reactions of Acyl Phosphates Hydrolytic Reactions Catalyzed Reactions Reactions of Glyceraldehyde-3-Phosphate Dehydrogenase OBJECTIVES OF STUDY................................. 13 EXPERIMENTAL ...................................... 14 Materials Reagents Enzyme Preparations Kinetic Measurements Nonenzymatic Rates Acyl Phosphatase Activity of Glyceral- dehyde-3-Phosphate Dehydrogenase Dehydrogenase Activity of Glyceraldehyde- 3-Phosphate Dehydrogenase H2OI8 Experiments Product Analysis RESULTS..............................................21 Acid Catalysis Amine Catalysis and Neutral Hydrolysis Glyceraldehyde-3-Phosphate Dehydrogenase Catalyzed Reactions DISCUSSION OF RESULTS ............................. 95 Acid Catalysis Monoanion and Dianion Reactions The Activity of Glyceraldehyde-3- Phosphate Dehydrogenase iii TABLE OP CONTENTS (CONTINUED) Chapter Page SUMMARY................... ....................... 125 REFERENCES CITED.................................128 Table I. II. III. IV. V. VI. VII . VIII. IX. LIST OF TABLES Page No. Rate Constants and Position of Bond Cleavage for Reactions of Acetyl Phosphate with Amines at 39° and Ionic Strength 0.6 (Ref. 17).................8 Analytical Results for Acyl Phosphates .... 15 Rate Constants for the Hydrolysis of Aliphatic Acyl Phosphates at 25° and Ionic Strength 4.80 M with LiCl..........22 Rate Constants (k ^^min "S f°r Hydrolysis of Acyl Phosphates in Various HCl Solutions with No Added Salt at 25°...........26 Rate Constants and b Values for Hydrolysis of Acyl Phosphates at 25° in HCl Solutions................................... 30 -1 Rate Constants (k ^^min ) for Hydrolysis of'Abyl Phosphate! at a Constant HCl Concentration of 2.97 M and at 25°......... 33 Rate Constants for Hydrolysis of Aliphatic Acyl Phosphates in 4.72 M HCl in H O and 4.72 M DCl in D20 at 25°.................... 38 Rate Constants for Hydrolysis of Para- Substituted Benzoyl Phosphates at 25° in 25% Dioxane-Water (v/v) with an HCl Concentration of 3.91 M.................. 39 Rate Constants for the Hydrolysis of Aliphatic Acyl Phosphates in 2.87 M HCl at Various Temperatures with Ionic Strength 4.80 M Made Up with LiCl, and the Activation Parameters for the Hydrolysis Reactions ....................... 42 v LIST OF TABLES (CONTINUED) Table Page No. X. Rate Constants for Hydrolysis of Aliphatic Acyl Phosphates in 25% Dioxane-Water (v/v) at 25° and in HCl Concentration of 3-91 M............ • .44 XI. Rate Constants for Hydrolysis of Aliphatic Acyl Phosphates in Perchloric Acid at 25°...................... 45 XII. Observed Rate Constants for Hydrolysis of Aliphatic Acyl Phosphates at Various pH Values at 60° and Ionic Strength 0.6 . . . . ........................ 46 XIII. Rate Constants for Spontaneous and Hydroxide Ion Catalyzed Hydrolysis of Acyl Phosphates at'60°....................54 XIV. Rate Constants for the Hydrolysis of Acyl Phosphates at Various Tempera tures and the Activation Parameters Calculated at 39°.............................55 XV. Monoanion and Dianion Hydrolysis of Trimethylacetyl and 3,3-Dimethyl- butyryl Phosphate When Subjected to Various Solvent Conditions .................. 58 XVI. Rate Constants for the Amine Catalyzed Hydrolysis of Aliphatic Acyl Phosphates at 60°........................................60 XVII. Second-Order Rate Constants for Pyridine and Imidazole Catalysis of Acyl Phos phates at 60°................................. 63 XVIII. The Acetyl Phosphatase Activity of Glyceraldehyde-3-P Dehydrogenase with Varying Enzyme and Constant Substrate Concentrations at 25°.............. 68 vi LIST OF TABLES (CONTINUED) Table Page No. XIX. The Glyceraldehyde-3-P Dehydrogenase Catalyzed Hydrolysis of Various Aliphatic Acyl Phosphates at 25°............71 XX. The Kinetic Constants for the Hydrolysis of Various Acyl Phosphates by Glyceraldehyde-3-P Dehydrogenase at 25°• 79 XXI, The Glyceraldehyde-3-P Dehydrogenase Catalyzed Hydrolysis of the Inter mediate Branched Acyl Phosphates in the Presence of Arsenate and Phosphate at 25° . ..................................... 80 XXII. The Inhibition of the Dehydrogenase Activity of Glyceraldehyde-3-P De hydrogenase by Isobutyryl and Tri- methylacetyl Phosphate at 25°..............85 XXIII. Inhibition of the Acetyl Phosphatase Activity of Glyceraldehyde-3-P De hydrogenase by Trimethylacetyl and 3,3-Dimethylbutyryl Phosphate at 25°. ... 88 XXIV. Arsenate, Phosphate, and Monomethyl Phosphate Catalysis for the De hydrogenase Activity of Glyceralde- hyde-3-P Dehydrogenase at 25°..............93 vii LIST OF ILLUSTRATIONS Figure Page No. 1. Effect of pH on the Rate of Hydrolysis of Acetyl Phosphate at 39° and Ionic Strength 0.6. (Ref. 50)................................. . 4 2. Plot of log k , . for Hydrolysis of 3,3-dimethy2otityryl, Isovaleryl, Acetyl, Isobutyryl, and Trimethyl- acetyl Phosphate vs. log HCl Concen tration at 25° and Ionic Strength 4.80 with LiCl..............................24 3. Plot of for Hydrolysis of 3.3-dimetnylbutyryl, Acetyl, and Trimethylacetyl Phosphate vs. HCl Concentration at 25° (ionic strength not held constant)......................... 27 4. Plot of log kQKgfj for Hydrolysis of 3.3-dimethylbutyryl, Acetyl, and Trimethylacetyl Phosphate in Various HCl Solutions vs. -HQ at 25°.............. 31 5. Plot of log for Hydrolysis of 3,3-dimethylbutyryl, Acetyl, and Trimethylacetyl Phosphate vs. LiCl Concentration at a Constant Concen tration of HCl of 2.97 M and at 25°. . . . 34 6. Plot of log kobs(j for Hydrolysis of para - Substituted Benzoyl Phosphates in 25$ Dioxane Water (v/v) and 3-91 M HCl at 25° vs.*0" > the Hammett Sub stituent Constant......................... 40 7. Plot of log kobsd at 60° for Hydrolysis of Acyl Phosphate vis. pH for Isobutyryl, Trimethylacetyl, and 3,3-dimethyl butyryl Phosphate......................... 50 viii LIST OP ILLUSTRATIONS (CONTINUED) Figure Page No. 8. Plot of k0bSd for Hydrolysis of Isobutyryl Phosphate at 600 and Ionic Strength 0.6 vs_. Total Imidazole Concentration ................... 65 9. The Plot of the Acetyl Phosphatase Activity of Glyceraldehyde-3-P Dehydrogenase vs. Enzyme Concentra tion at 25° ................................. 69 10. Lineweaver and Burk Plot of the Acetyl Phosphatase Activity of Glyceralde- hyde-3-P Dehydrogenase in the Absence of Inhibitor, and in the Presence of Trimethylacetyl Phosphate and 3,3- Dimethylbutyryl Phosphate at 2 5 ........... 73 11. Lineweaver and Burk Plots of the Propionyl, Butyryl, Isobutyryl, and Isovaleryl Phosphate Activities of Glyceraldehyde-3-P Dehydrogenase at 25°..........................................77 12. The Rate of Disappearance of Propionyl, Butyryl, Isovaleryl Phosphate measured by the Hydroxamic acid assay at 540 my, in the Presence of Glyceral- dehyde-3-P Dehydrogenase and 0.01 M Sodium Arsenate at 25°......................8l 13. The Dehydrogenase Activity of Glyceral- dehyde-3-P Dehydrogenase in the Presence of Varying Amounts of Isobutyryl Phosphate at Inhibitor at 25°. • 83 14. The Dehydrogenase Activity of Glyceral- dehyde-3-P Dehydrogenase in the Presence of Varying Amounts of Trimethylacetyl Phosphate as Inhibitor at 25°...............89 Ix LIST OP ILLUSTRATIONS (CONTINUED) Figure Page No. 15. The Effect of Arsenate, Phosphate, and Methyl Phosphate on the Dehydrogen ase Activity of Glyceraldehyde-3-P Dehydrogenase at 25°.........................91 16. Plots of (log k0kS(j + Hq) vs. (log CH + HQ) for hydrolysis of 3,3-dimethylbutyryl, Acetyl, and Trimethylacetyl Phosphate in Various HCl Solutions at 25°.............. 99 17. Plot of log k at 60° for Hydrolysis of Acyl Phosphate Monoanions and Dianions vs . o * ...................................... 107 18. Plots of log kp^r for Acyl Phosphate Monoanions ana Dianions vs. a* at 60° . . . .109 19. Plot of log ko from Table I vs. Taft's Steric Constants, E_ ........................121 S X INTRODUCTION Since Lipmann’s discovery that an acyl phosphate, acetyl phosphate, was an intermediate in pyruvate oxidation (55), the presence of this class of compounds has been shown or implied in many metabolic pathways • ( 3^ , 69,70 ,80 , 86). Because of this presence, elucidation of the cata lytic mechanisms involved has been pursued both in enzym atic (69,73) and in non-enzymatic (17,18,19,^9,50,53,71) systems. Accordingly, a study is now made which extends our knowledge concerning the mechanisms of catalysis in acyl phosphate reactions. Furthermore, an attempt is made to apply the new and pre-existing data to an enzymatic re action involving an acyl phosphate: that of the acyl phos phatase activity of glyceraldehyde-3-phosphate dehydrogen- 2 Reactions of Acyl Phosphates Hydrolytic Reactions Much impetus was given to the fundamental problem con cerning the mechanism of acyl phosphate hydrolysis through the development of the hydroxamic acid assay procedure by Lipmann and Tuttle (56). Using this technique, Koshland (50) determined the pH - rate profile for acetyl phosphate hydrolysis, reproduced in Figure 1. Since acetyl phosphate has a pK^ of 4.95 (58), four distinct reactions could be observed depending upon the ionization state of the acetyl phosphate: acid catalysis, monoanion hydrolysis, dianion hydrolysis, and specific base catalysis. Earlier studies (5) showed that C - O bond cleavage occurred in base, mak ing probable a nucleophilic attack at the carbonyl carbon by hydroxide ion, displacing trinegative inorganic phos phate. This would be analogous to the specific base cat alyzed hydrolysis of normal esters (7). The lack of mechan istic data, however, prohibited discussion of the other re actions at that time. In 1961, Di Sabato and Jenks (18), in a most elegant study, elucidated the mechanisms of the monoanion and dian ion hydrolytic reactions. Their findings were consistent with a monomolecular elimination for both species, Equations (1) and (2), similar to that of other phosphate reactions (16) . The hypothetical metaphosphate intermediate reacts H g<:p.0 R<V v o o o R-tCT O ■> R-di-OH + [PO, ( 1) P O l l2)\ irapidly with water to yield orthophosphate, Equation (3). j |The more facile reaction of the monoanion, as shown in ' iFigure 1, is then due to an internal proton transfer in- ; volving a six membered cyclic ring in the transition state. ! pa + h2o — > h2po4- (3) The dianion reaction is simply a scission of the P - 0 bond.' These mechanisms were determined from the following criter ia, all of which are consistent with unimolecular reactions: ; (1) entropies of activation close to zero, (2) rates unaf- : ] Ifected when the solvent is changed from water to deuterium | i oxide, (3) volumes of activation close to zero, (4) rates 'unaffected by ionic strength of the medium, (5) rates of 'hydrolysis not changed by the dielectric constant of the ; isolvent, (6) a rho value for substituted benzoyl phosphates ■in accord with the postulated mechanisms, (7) position of bond cleavage, (P - 0), and (8) the formation of pyrophos- iphate in the presence of orthophosphate. Figure 1. Effect of pH on the rate of hydrolysis of acetyl phosphate at 39° and ionic strength 0.6. (Ref. 50) 5 00 O V— X XL 64 48 32 46 O 0 2 4 6 8 10 12 pH 6 Catalyzed Reactions Acyl phosphates belong to a class of compounds possess ing an "energy rich" bond as the free energy of hydrolysis, A F°, is -10.5 kcal mole ^ (64). The term "energy rich" may also be interpreted as high group transfer potential, a classification more meaningful when discussing transfer re actions of acyl phosphates. It is possible to apply the group transfer potential to the transfer of either the phosphate or acyl moiety. For example, the acetyl phos phate intermediate originally observed by Lipmann (55) ultimately undergoes a reaction at the phosphorous center, transferring the phosphate to ADP by an acetate kinase (56). However, in protein synthesis, the acyl amino AMP is trans formed into acyl amino tRNA (70). Other examples can be seen in acetic (86)/ succinic (69), and fatty acid (86) thiokinases, where the acyl group is transferred from phos phate to sulphur, and in glyceraldehyde-3-P dehydrogenase, where the acyl phosphatase activity can best be described as a transfer of the acyl moiety from phosphate to water (34). Since these acyl transfer reactions are catalyzed by enzymes, and since acceptor functions such as phosphate, sulfhydryl, and hydroxy groups do not appear to react directly with acyl phosphates (17), it becomes immediately obvious that functional groups on the enzyme surface must be participating in these reactions. Amine functional groups are found and have been implicated in acyl phos phatase reactions (69,73). The mode of interaction of these groups with acyl phosphates in nonenzymatic systems is important in understanding interactions in enzymatic 7 reactions. This was recognized early by Lipmann (57) who demon strated the facile acylation of a primary amine, aniline, by acetyl phosphate. Park and Koshland (74), however, found that a tertiary amine, pyridine, catalyzed the re action yielding P - 0 bond cleavage. This reaction was later shown to also be nucleophilic (17). Di Sabato and Jenks (17) extended this series and the position of attack by amines was indeed dependent upon the type of amine used. The tertiary amines, triethylenediamine, pyridine, 4- methylpyridine, and triethylamine yielded P - 0 bond cleav age while the secondary and primary amines, imidazole, N-methylimidazole, aniline, glycylglycine, morpholine, and glycine all produced a C - 0 bond fission. Chemically, there is no basis for this difference since the pK of the a amine is not related to its position of attack but only to the velocity of the reaction, Table I. This difference in reactivity may be relevant to en zymatic reactions. For example, the imidazole ring of histidine appears to be a functional group involved in the succinic thiokinase reaction (69), which transfers phos phate from, the acyl phosphate to ADP. Likewise, imidazole is involved in the phosphatase activity of glyceraldehyde- 3-p dehydrogenase (73). However, in this case, the acyl group is transferred from phosphate to a number of acceptor groups. Since the pK does not influence the attack posi tion, other influences must be examined. Table I. Rate Constants and Position of Bond Cleavage for Reactions of Acetyl Phosphate with Amines at 39° and Ionic Strength 0.6'a(ref. 17) . Base pKa k2 -1 . -1 M mxn k2 M-1min_1 1 k3 M 2min 1 Bond cleaved Aniline 4.3 0.54 C - 0 Pyridine 4.5 0.046 0.0087 P - 0 N-Methylimidazole 7.2 1.8 0.0034 b C - 0 Glycylglycine 8.3 51.9 0.047 b C - 0 Morpholine 8.8 165 0.15 b C - 0 Glycine 9.8 876 0.08 . 0.19 C - 0 a rate constants calculated from the rate law; v = k'(AcP~)(amine) = = 2 + k^(AcP )(amine) + k^(AcP )(amine) b no detectable reaction j Reactions of Glyceraldehyde-3-P Dehydrogenase ! r . i ^ , Glyceraldehyde-3-P dehydrogenase (D-glyceraldehyde-3- iphosphate: NAD oxidoreductase (phosphorylating), iEC 1.2.1.12) was first characterized and isolated from a I yeast source (85). The reversible reaction was dependent, I 5 in stoichiometric amounts, upon glyceraldehyde-3-P, NAD , land inorganic phosphate according to Equation (4) (68) . The |mammalian enzyme from rabbit muscle was R-CHO + NAD + HP04 ^ R-C02P03 + NADH + H (4) 'later isolated and appeared to catalyze an identical reac tion (15). The reaction sequence in Equation (4) is re versible and has been implicated as a control point in glycolysis (87,26). The reaction occurs in two steps (77) and it is convenient to discuss each part separately: (1) aldehyde binding and oxidation to an acyl enzyme, and (2) deacylation of the acyl enzyme intermediate. Aldehyde Binding and Oxidation to an Acyl Enzyme - Kinetic analysis using initial rates and product inhibi tions has revealed the binding of substrates to be in ran- jdom order at mutually independent sites (26). The NAD is |very tightly bound to the enzyme (8*0 and has been shown to [induce a cooperative conformational change in the tetramer- lic protein (30) upon binding (13,47,59,65). The glyceral- dehyde-3-P, when bound, appears to react with a cysteine [residue, forming a hemithioacetal which is subsequently [oxidized by NAD+ to yield the thiolester as in Equation (5) (77) • 10 — NAD+ -NAD+ -NADH , Q ^ (5) HCR LS" SC-R -S C R & 6 It is possible to acylate the same cysteine (29) by using p-nitrophenyl acetate as a substrate with the NAD free ;enzyme (75). Acetaldehyde (31), propionaldehyde, butyral- dehyde (33) , and glyceraldehyde (77 ) all react with glycer- aldehyde-3-P dehydrogenase, will form a thioester in the presence of NAD+, and appear to continue normally in the reaction process. Deacylation of the Acyl Enzyme Intermediate - Deacyla- tion occurs in vivo by phosphorolysis forming an acyl phosphate, but may occur through hydrolysis or be induced by arsenate in vitro (83). The phosphorolysis is a revers ible reaction indicated by the observations that acetyl :phosphate will acylate the same cysteine residue that the |natural substrate acylates (29) and that the incorporation |of labeled inorganic phosphate into acetyl phosphate is jcatalyzed by glyceraldehyde-3-P dehydrogenase (34). The re versibility of the phosphorolytic cleavage of the acyl en zyme accounts for the acyl phosphatase activity of glycer aldehyde- 3-P dehydrogenase (34). Studies oh this activity are, therefore, pertinent for understanding the deacylation of the acyl enzyme. Deacylation is undoubtedly aided by a functional group ion the enzyme surface, accounting for the great catalytic 11 activity of the enzyme. Serine may be involved, since both j i j ithe esterase and dehydrogenase activities are inhibited by j ! I jdiisopropylflurophosphate (76), a reagent which blocks this; | jresidue (2). However, in view of the high concentration required for inhibition, this result is not believed to be conclusive (73). More convincing evidence points to a his tidine residue. The esterase (73) and dehydrogenase (42) [activities, which are rate limiting in deacylation, are af fected by a group with a pK between 7 and 8. Furthermore, !photooxidation of the enzyme, which destroys 4 to 5 histi- Idine residues per tetramer, will not affect acylation by p-nitrophenyl acetate, but will inhibit deacylation of the enzyme (27). Destruction of the histidine residues also affects the phosphatase activity (27). Two theories prevail concerning the mechanism of his tidine involvement. Olson and Park (73), on noting that an absorption peak appears at 240 my when acetyl phosphate is the substrate (32), proposed a nucleophilic mechanism as described in Equation (6). The basis for this mechanism is [that the spectral maximum at 240 my more closely resembles |a: N-acylimidazole than a thiolester. Thiolesters, however, o -SCR S H2O or' HAsQJ if \— 10 i — S' HN +Rca (6) I I !are known to have an absorption maximum close to this re- j I i jgion (235-240 my) (21). Behme and Cordes (3) have observed | ‘ ' ! I a retardation of the estrolytic activity when the solvent i i • j jis changed from water to deuterium oxide and have according-; !ly concluded that a general base mechanism is operative. j S i |Deuterium oxide is known, however, to have a conformational | ; I {effect on proteins (39) which could affect enzymatic activ- j j I iity. The mechanism of deacylation or, conversely, the | 1 ; imechanism of the acyl phosphatase activity, remains to be | elucidated. j Objectives of Study i . i |(1) The mechanism of acyl phosphate hydrolysis has previ- j lously been examined for the monoanion, dianion, and base I ! i ^catalyzed reactions (18). The present study examines the |mechanism in the acid region. '(2) Alkyl groups on acyl imidazoles have been shown to i structure water in strong acid (22). These effects are now! i ’ i tested on acyl phosphates. (3) The position of attack by primary, secondary, and tertiary amines cannot be explained by differences in the basicity of the nucleophile (17) . It was thought that these differences may arise from a steric effect which is tested through variations in the steric bulk of the sub strate . (4) Mechanistic information pertaining to chymotrypsin has been obtained by changing the aliphatic group of the sub- jstrate (67). A similar attempt is made on the acyl phos- i Iphatase activity of glyceraldehyde-3-P dehydrogenase. i !(5) Cooperativity in glyceraldehyde-3-P dehydrogenase has + been demonstrated in the binding of the co-factor, NAD (47). Since highly branched acyl phosphates should be poor substrates for this enzyme, it may, therefore, be possible to demonstrate cooperativity in substrate binding. ! ,14 i ; [ • I j 1 j EXPERIMENTAL | ■ l Materials I • - i Reagents - Dilithium acetyl phosphate used for non- I ; enzymatic studies was purchased from Calbiochem Corp. and was used without further purification. All the other aliphatic acyl phosphates were prepared by the method of iLipmann and Tuttle (56), and isolated as the disodium salts.; ; Infrared spectra of the compounds were characteristic of acyl phosphates (41). Thin layer chromatography using 60% isopropanol-water as the solvent showed the compounds to contain a trace of inorganic phosphate. The compounds were purified for elemental analysis through isolation as the disilver salts by adding AgNO^ dissolved in water to a soluH tion of the acyl phosphates. The initial yellow precipitate was removed and the white precipitate which formed upon further addition of AgNO^ was the pure acyl phosphate, was analyzed by Elek Microanalytical Labs, and yielded the re sults in Table II. Dilithium p-nitrobenzoyl phosphate was also prepared by the method of Lipmann and Tuttle (56) as modified by :Ramponi, et al (78). The remaining benzoyl phosphates were synthesized by the procedure of Avison (1) with the modifi cations of Di Sabato and Jenks (18). The barium salts were converted to the disodium salts by stirring in water with an excess of sodium sulfate, adding sufficient ethanol to precipitate inorganic sulfate and phosphate salts, and isolated through addition of acetone to this mixture. The !acyl phosphates were stored in a desiccator at -4°, and Table II. Analytical Results for Acyl Phosphates. Acyl Group Formula Calcd %C %P Found %C %P Isobutyryl C4H7°5PAg2 12.57 1.83 8.12 12.58 2.11 8.20 Isovaleryl C5H9°5PAg2 15.15 2.27 7 .84 15.38 2.47 7.62 Trimethylacetyl C5H9°5PAg2 15.15 2.27 7.84 15.16 2.33 7.77 3,3-Dimethylbutyryl C6Hll°5PAg2 17.57 2.68 7.57 17.66 2.67 7.38 ! 16 ; I fresh samples were prepared periodically. I Acetyl phosphate for all enzymatic assays was purchased ■ from Sigma Chemical Company. The barium salt of DL-glycer- | I I aldehyde-3-P was purchased from Calbiochem and was convert-j |ed to the free aldehyde and the quantity of D isomer deter- j ' + ; mined by the method of Kochman and Rutter (48) . NAD was j i I |also purchased from Calboichem. K&K Laboratories, Inc., I i ■ I ;supplied the sodium monomethyl phosphate, and its concen- j jtration was determined through total combustion by the King ! j | .phosphate test (44). Glass distilled water was employed :for all aqueous solutions. Dioxane was purified by the method of Fieser (24) and was stored frozen. Deuterium oxide (99.8%) was obtained from Bio-Rad Laboratories. Other chemicals used were re- ; i agent grade. Enzyme Preparations - Glyceraldehyde-3-P dehydrogenase/ rabbit muscle enzyme, was from Worthington Biochemical Cor poration, Code 8 GB. Enzyme preparations were made by spinning the crystaline suspension at 3° for 20 min. at 14,500 x g. The crystals were then dissolved in 0.025 M sodium barbital buffer, pH 7.20, which was also 0.005 M in EDTA, respun to remove any solid material, and stored at 0°. Enzyme concentrations were determined by measuring the ab sorbance at 280 my of a 1:30 dilution of this stock enzyme 2-1 solution. An extinction coefficient of 1.002 cm mg and \a molecular weight of 140,000, determined by Fox and iDandliker (25), were used for calculations of enzyme concent jtrations. The ratio of absorbance at 280 my to that at ! i [260 my for all enzyme preparations was 1.20 ± 0.05. This enzyme preparation was employed as the stock solution for i jdetermining acyl phosphatase activity and was utilized with- !in two hours after its preparation. ! The stock enzyme preparation for glyceraldehyde-3-P de- I hydrogenase activity consisted of a 1:500 dilution of the Iprevious solution in mM mercapto ethanol and mM EDTA, | pH 7.40. This enzyme preparation was stable for several j hours when stored at 0°. ! 1 I iKinetic Measurements Nonenzymatic Rates - The hydroxamic acid assay was ;used exclusively for the kinetic runs. The neutral tech nique as described by Di Sabato and Jenks (17) was used for; the aliphatic phosphates. For the substituted benzoyl l phosphates, the alkaline technique of Hestrin (35) as util-; ized by Di Sabato and Jenks (18) was employed. All rates were run in duplicate to at least 75% completion, with less; than 5% deviation between the two rate constants in all — o cases. Temperature was controlled to +0.1 by means of a ■constant temperature bath regulated with a Precision Scien tific thermoregulator. Each experiment was initiated by I j I adding the acyl phosphate to the pre-equilibrated acid sol- ■ -3 jution making it approximately 5 x 10 M in acyl phosphate.: Rates did not change when the acyl phosphate concentration ' iwas varied 50%. At appropriate time intervals, aliquots iwere removed and added to the hydroxylamine solution. The !resulting mixture was then stoppered and shaken. A Hamil- i ton syringe was used to remove the aliquots in the measure ment of the faster rates. Development time for complete |formation of the hydroxymate was experimentally determined j for each compound. At least nine points were employed for ( a rate determination, and infinity points were taken at ten; i jhalf-lives. Pseudo-first order rate constants (k0bsd) were (calculated with an Olivetti-Underwood Programma 101 using a !computer program designed to calculate a least squares devaluation of the slope and intercept of a plot of In((0Do - OD00)/(ODt - OD^)) vs. time. j Acyl Phosphatase Activity of Glyceraldehvde-3-P De hydrogenase - The hydroxamic acid assay of Lipmann and j Tuttle (57) was employed to measure the amount of remaining ! acyl phosphate. For each experiment, the enzyme was dilut ed in 0.025 M sodium barbital buffer, pH 7.0, and equili brated at 25.0° for 5 min. in a circulating water bath. i This stabilizes enzyme activity (23) . The acyl phosphate, ; also dissolved in 0.025 M sodium barbital buffer, pH 7.0, was then added to the enzyme solution to initiate the reac tion. In the inhibition studies, the inhibitor was added with the substrate. Aliquots were withdrawn at appropriate time intervals and introduced into the hydroxylamine re- iagent. After complete formation of hydroxamic acid from [ ; [remaining acyl phosphate and any acyl enzyme intermediate Ipresent, aliquots were developed through addition of FeCl_ jand the absorbance was measured at 540 my. Each rate, de termined from 10 points, was followed to approximately 15% ! completion and was linear over this time interval. A plot (of absorbance vs. time was used to calculate the velocity (of the reaction, measured in n moles acyl phosphate hydro lyzed per mg protein per sec. i Dehydrogenase Activity of Glvceraldehyde-3-P Dehy- 19 i ! 1 i i drocfenase - This activity was determined as reported by Velick (83). Each reaction was initiated by adding the Iglyceraldehyde-3-P to the enzyme solution which had been :pre-equilibrated for 5 min. in the buffer. For all inhibi tion studies, the inhibitor was included in the pre-incuba- | !tion process. The reaction was followed through observa- ; ‘ i tiDn of conversion of NAD to NADH by watching the increase I :in absorbance at 340 mU with a Beckman DU spectrophotometer i :equipped with a Gilford Model 2000 recording attachment. j An extrapolation of the rate to time zero was used to ob- i tain the initial rate of change of absorbance. The concen- ; tration of NADH was determined (83) using an extinction co- : 6 2 “ X efficient of 6.22 x 10 cm mole (37). Acyl phosphate concentrations were determined by the hydroxamic acid assay i employed previously. NAD+ concentrations were determined by weight. The concentrations of all substrates and rea gents are reported in the text. 18 H20__ Experiments To determine the position of bond cleavage in acid jcatalysis, 3,3-dimethylbutyryl phosphate was dissolved in j T O Q |4.69 M HCl with 1.03 atom % excess H20 at 25 At 10 jhalf-lives the reaction was stopped by cooling to 0° and was neutralized to pH 8 with NaOH. Inorganic phosphate was then precipitated as the barium salt by the addition of 100% ex cess equivalents of BaCl^- The mixture was then filtered land the precipitate was washed and dried. An identical procedure was followed for inorganic phosphate, which was ;the control for this experiment. That no organic material was precipitated in the 3,3-dimethylbutyryl phosphate ex- 20 periment was confirmed by the lack of any carboxyl or car bonyl peaks in the infrared spectrum and, indeed, a spec trum identical with that from the control experiment was obtained. Oxygen-18 analyses were carried out by West Coast Technical Service Inc., San Gabriel, California. Product Analysis Disodium 3,3-dimethylbutyryl phosphate (1.01 gm) was dissolved in 20 ml of 5.29 M HCl. After complete hydrol ysis (10 half-lives) an oil layer formed which was isolated and combined with the ether extracts of the aqueous phase. After removal of the ether, the residue was treated suc cessively in dry benzene with thionyl chloride and aniline. Upon evaporation of the solvent, 0.5 g of the crude anilide (65% yield) was obtained. After two crystallizations from o ethanol-water, the melting point was 130.5 to 132 . An authentic sample of 3,3-dimethylbutyranilide melted at 131 o - to 131.5° (36) and a mixture melting point revealed no de pression (mp 131 to 132°). The major reaction products from the acid hydrolysis of 3,3-dimethylbutyryl phosphate are, therefore, 3,3-dimethylbutyric acid and inorganic phosphate. RESULTS Acid Catalysis - Table III presents the rate constants [ I at various HCl concentrations for hydrolysis of five acyl jphosphates, ionic strength being held constant at 4.80 M at 125°. The logarithms of the rate constants are plotted in i Figure 2 vs. the logarithms of the concentration of HCl. jit can be seen that in the case of the 3,3-dimethylbutyryl !derivative, branching at the 8-carbon has given rise to faster rates of hydrolysis than seen with the other com pounds in the series. The plots are linear and the slopes ; approach unity as determined from a least squares program, although some deviation does occur with each compound. The i values of the slopes are: acetyl phosphate, 0.99: isobu- tyryl phosphate, 0.86; trimethylacetyl phosphate, 0.99; isovaleryl phosphate, 1.05; 3,3-dimethylbutyryl phosphate, 1.12. The effect of allowing ionic strength to vary with acid concentration is seen in Table IV and Figure 3. The i rate constant, k^, for the acid-catalyzed reaction at a specified ionic strength, is dependent upon the ionic strength of the medium with a very pronounced positive salt effect being observed. Equation (7) where b is a constant .f f oh" ar particular salt and y is the ionic strength appears to be valid (9), and was used to calculate the curved lines in Figure 3. kobsd " <V + <VH+)> 1 0 b ( 7 ) The rate constant for hydrolysis of the neutral species of i Table III- Rate Constants for the Hydrolysis of Aliphatic Acyl Phosphates at 25° and y = 4.80 M with LiCl. Acyl Group HCl M kobs^3 min kH -1 -1 1. mole min Acetyl 1.08 0.070 1.94 0.132 0.064 2.87 0.184 3.82 0.246 4.80 0.315 3,3-Dimethylbutyryl 1.08 0.088 1.94 0.192 0.117 2.87 0.289 3.82 0.412 4.80 0.522 to to Table III. (Continued) Acyl Group HCl M k"obsd min-- * - 1. mole ^min' Isovaleryl 1.94 0.124 0.069 2.87 0.181 3.82 0.253 4.80 0.318 Isobutyryl 1.94 0.078 0.032 2.87 0.105 3.82 0.137 4.80 0.169 Trimethylacetyl 1.08 0.010 1.94 0.017 0.0093 2.87 0.027 3.82 0.035 4.80 0.044 Figure 2. Plot of log ^ for hydrolysis of 3,3-di~ methylbutyryl O, isovaleryl Q , acetyl isobutyryl Cl, and trimethylacetyl phosphate © vs. log HCl concentration at 25.0° and j i = 4-80 M with LiCl. o 0 o 0 m O © o (l-Ujfjj J pS ( ? o VOo 7 © © © © f t <0 o * a o * € O Table IV. Various HCl Rate Constants Solutions with (k . _ min for Hydrolysis obsd No Added Salt at 25°. of Acyl Phosphates in HCl M Acetyl Trimethylacetyl 3,3-Dimethylbutyryl 1.36 0.030 0.0049 0.015 2.03 0.048 0.0066 0.032 3.53 0.120 0.015 0.151 4.80 0.315 0.044 0.522 5.29 0.409 0.068 0.855 Figure 3- Plot of k . , for hydrolysis of 3,3-di- oJosd raethylbutyryl O, acetyl and trimethylacetyl phosphate © vs. HCl concentration at 25.0° (ionic strength not held constant). 1.0 0.9 0.7 0 .6 c E 0.5 ~o U) n O ^ 0.4 0.3 0. 1 4 3 5 0 1 2 HCl M acyl phosphates, kQ, is not significant, since kobsd ap proaches zero with decreasing acidity for all the compounds |studied. Values for the acid-catalyzed reaction rate con- S | | istants, k , and b were calculated from Equation (7) for HClj I H I ! | solutions and are given m Table V. Plots of log k , for hydrolysis reactions in acid obsd solutions with no added salt vs. -H , Hammett's acidity 'function, are linear as shown in Figure 4. However, the ‘slopes deviate greatly from unity with values of 0.77 for 1 acetyl phosphate, 0.76 for trimethylacetyl phosphate and 1.23 for 3,3-dimethylbutyryl phosphate. Since there is a large salt effect and since the slopes are not close to one, :mechanistic interpretations cannot be made from these plots. Rate constants for hydrolysis of the acyl phosphates at a constant HCl concentration of 2.97 M and with varying amounts of LiCl are presented in Table VI, The logarithms of k , are plotted vs. LiCl concentration in Figure 5. obsd — The b constants were also calculated in these HCl - LiCl solutions (0.13 M to 3.13 M LiCl). The lithium salt was : employed since lithium ion has nearly the same effect on ; 'the activity of water as hydrogen ion (79). For the HCl and HCl - LiCl solutions the calculated values of the con stants were quite similar. The values of b obtained for iacetyl phosphate, trimethylacetyl phosphate and 3,3-di- imethylbutyryl phosphate are given in Table V. | The acid-catalyzed hydrolysis of acetyl phosphate has been shown to take place predominantly with C - 0 bond ’ cleavage (74). 3,3-Dimethylbutyryl phosphate was analyzed ;by a similar procedure as outlined in the experimental Table V. Rate Constants and b Values for Hydrolysis of Acyl Phosphates at 25° in HCl Solutions. 3 Acyl Group k x 10 H 1 mole-^ min-- * - b(HCl) b(LiCl) Acetyl 7.58 0.191 0.193 Trimethylacetyl 0.776 0.230 0.215 3,3-Dimethylbutyryl 4.07 0.302 0.289 31 Figure 4. Plot of log for hydrolysis of 3,3-dimethylbutyryl G, acetyl ©, and trimethyl acetyl phosphate © in various HCl solutions vs. -H at 25.0°. o lo9 kobsd(min1) w Rj L 0 * 0 0 0 o o f^> b uo r o Table VI. Rate Constants min f°r Hydrolysis of Acyl Phosphates at a Constant HCl Concentration of 2.97 M and at 25° with Variable Ionic Strength Made up with LiCl. LiCl M Acetyl Trimethylacetyl 3,3-Dimethylbutyryl 0.13 0.091 0.013 0.101 1.13 0.137 0.021 0.190 2.13 0.226 0.032 0.377 3.13 0.402 0.048 0.775 I j u> OJ 34 Figure 5. Plot of log for hydrolysis of 3,3-dimethylbutyryl O , acetyl ^ , and trimethyl acetyl phosphate © ;vs. LiCl concentration at a constant concentration of HCl of 2.97 M and at 25°* log' kobS(d (min'1 ) b t > o 36 I | t i section. The results, expressed in atom % excess oxygen-18 j I in Ba (P0.)„ above that normally occurring in H O, were j ! 3 4 2 2 ] 10.008 in the control and 0.000 for the complete hydrolysis j !of 3,3-dimethylbutyryl phosphate. It is readily apparent 18 I that in 4.69 M HCl, extensive incorporation of 0 into the jinorganic phosphate product has not taken place. Predomin- j jantly C - 0 bond cleavage must be occurring, as is also the ; i . : .case for acetyl phosphate. i In Table VII are given rate constants in D^O - DC1 j solutions. DCl in deuterium oxide increases the rates of hydrolysis compared to the rates obtained with an equal concentration of HCl in water. The ratios of k /k are be- : !} H tween 2.2 and 2.6 for all compounds studied. The rate constants for acid-catalyzed hydrolysis of I substituted benzoyl phosphates are given in Table VIII. As ; shown in Figure 6, a p value close to zero (-0.24) is ob tained when the logarithms of the rate constants are plot ted vs. c r , Hammett's substituent constants (28). Dioxane- ^ 0 (25% v/v) was chosen as the solvent for the reaction isince this was the best compromise between solubility of j j ' |the reactant and the reaction product. jo-Chlorobenzoic |acid, was insoluble in this solution and, therefore, the irate of hydrolysis of p-chlorobenzoyl phosphate could not ;be determined. It is assumed that the substituted benzoyl phosphates all have the same b value. ! In Table IX are reported rate constants for hydrolysis : of the aliphatic acyl phosphates in 2.87 M HCl and at vari ous temperatures. Values of AH* and AS* are also given 'in Table IX. It can be seen that the AS* for 3,3-dimeth- i I 37 i ; ! i ; j j ! ' ! jylbutyryl phosphate is 5.7 eu more positive than that for j !acetyl phosphate and 10.3 eu more positive than the AS* !for hydrolysis of trimethylacetyl phosphate. I ! Rate constants were also determined in 25% dioxane- | i H^O (v/v) at 25° and are given in Table X. Dioxane has a retarding effect on the rate in all cases but the effect is | i much greater for the 3,3-dimethylbutyryl derivative than j ifor acetyl or trimethylacetyl phosphate. \ : 1 i In Table XI rate constants are given for hydrolysis of the acyl phosphates in 4.20 M perchloric acid. It can be seen that the rates of hydrolysis of 3,3-dimethylbutyryl 'phosphate and trimethylacetyl phosphate are greater in perchloric acid than with an equal amount of HCl. The values of b were calculated in perchloric acid and were found to be 0.307 for trimethylacetyl phosphate and 0.389 for 3,3-dimethylbutyryl phosphate. Thus, b is greater in perchloric acid than hydrochloric acid, a result which y eliminates the possibility of nucleophilic attack by the anion since halogens are much better nucleophiles than perchlorate. The value of b could not be determined for I ; jacetyl phosphate in perchloric acid since that compound is iinsoluble in the solution. Amine Catalysis and Neutral Hydrolysis - Rate con stants for hydrolysis of the acyl phosphates at 60° and at I ■various pH values are given in Table XII. The pH-rate pro- ifiles for three branched acyl phosphates are presented in :Figure 7. The lines are theoretical and were calculated jfrom Equation (8) Table VII. Rate Constants for Hydrolysis of Aliphatic Acyl Phosphates in 4.72 M HCla in H20 and 4.72 M DCl in D20 at 25°. Acyl Group kH2° k v k A D H obsd obsd min ^ min“l Acetyl 0,284 0.627 2.21 3,3-Dimethylbutyryl 0.487 1.11 2.28 Isobutyryl 0.166 0.371 2.24 Trimethylacetyl 0.0413 0.106 2.56 a The rate constants in 4.72 M HCl were obtained by interpolation of the plots of log vs. HCl concentration. Table VIII. Rate Constants for Hydrolysis of Para-Substituted Benzoyl Phosphates at 25° in 25% Dioxane-H^O (v/v) with an HCl concentration of 3.91 M. Para-Substituent k . „ min ^ obsd OCEL 0.0200 CH3 0.0143 H 0.0194 N°2 0.0107 40 I I j j j Figure 6. Plot of log f°r hydrolysis of para-substituted benzoyl phosphates in 25% dioxane water (v/v) and 3.91 M HCl at 25.0° vs. a , the Hammett substituent constant. I —I • = d " 9 * 0 9 * 0 VO “1--------1 ------T - J _____L -O zo o zo- - O'Z- T 7 Table IX. Rate Constants for the Hydrolysis of Aliphatic Acyl Phosphates in 2.87 M HCl at Various Temperatures with u = 4.80 M made up with LiCl, and the Activation Parameters for the Hydrolysis Reactions. Acyl Group Temp. °C k , , AH* AS*a obsd / . _i kcal/mole eu m m Acetyl 3,3-Dimethylbutyryl 10 0.026 15 0.049 25 0.184 30 0.341 35 0.595 10 0.034 15 0.066 25 0.289 30 0.561 35 1.011 21.2 ± 0.1b -5.1 ± 0.5 23.2 ± 0.2 +0.6 ± 0.7 Table IX (Continued) Acyl Group m C ) Temp. C ■^obsd min~^" ah* kcal/mole AS*3 eu Trimethylacetyl 15 0.0080 25 0.027 21.2 ± 0.3 -9.7 ± 1.2 30 0.058 35 0.097 40 0.162 a Calculated at 25° employing values of (1. mole ^ sec ^). b Errors were calculated from the standard error of plots of log k vs. 1/T Table X. Rate Constants for Hydrolysis of Aliphatic Acyl Phosphates in 25% Dioxane-H^O (v/v) at 25° and an HCl Concentration of 3.91 M. Acyl Group obsd min-- * - Dioxane-H^O ^obsd min-l k /k dioxane H20 Acetyl 0.164 0.132 0.80 3,3-Dimethylbutyryl 0.213 0.146 0.69 Trimethylacetyl 0.0211 0.0191 0.91 a The rate constants in 3.91 M HCl were obtained by interpolation of the plots of log k . . vs. HCl concentration. ^ 3 obsd — Table XI. Rate Constants for Hydrolysis of Aliphatic Acyl Phosphates in o Perchloric Acid at 25 . Acyl Group hcio4 m • -1 k . nm m obsd 3,3-Dimethylbutyryl 0.964 0.0088 1.88 0.0362 2.91 0.144 4.20 0.738a Trimethylacetyl 4.20 0.0635b In comparison the rate constant in 4.20 M HCl, obtained by interpolation of a plot of log k ■ ■ . vs. HCl concentration, was 0.286 min-- * - obsd — The rate constant in 4.20 M HCl is 0.0263 min b Table XII. Observed Rate Constants for Hydrolysis of Aliphatic Acyl Phosphates at Various pH Values, 60° and y = 0.6. Acyl Group Buffer pH 60° k . n x 10 m m obsd Isobutyryl HCl (0.299 M) 159.4 HCl (0.101 M) 93.5 HCl 1.98 61.7 Formate 3.57 56.9 Acetate 5.02 36.3 Imidazole 5.83 20.4 Tris 8.83 14.5 KOH 10.60 16.3 KOH (0.0679 M) 82.2 Table XII (Continued) Acyl Group Buffer pH 60° in3 • -1 k , , x 10 ram obsd Trimethylacetyl HCl (0.497 M) 85.3 HCl (0.299 M) 65.3 HCl (0.101 M) 47.9 HCl 1.98 37.7 Formate 2.85 35.4 Formate 3.48 33.7 Acetate 4.61 28.1 . Acetate 5.02 22.8 Acetate 5.51 13.7 Imidazole 5.65 11.5 Imidazole 6.61 3.97 Imidazole 7.63 3.04 Tris 8.83 3.14 Table XII (Continued) Acyl Group Buffer pH 60° i m3 . -1 jc . x 10 m m obsd Trimethylacetyl KOH (0.0679 M) 10.2 KOH (0.170 M) 21.2 KOH (0.424 M) 51.9 KOH (0.501 M) 75.8 3,3-Dimethylbutyryl HCl (0.299 M) 188.6 HCl (0.101 M) 78.4 HCl 1.98 64.6 Formate 3.48 63.7 Acetate 4.61 46.5 Imidazole 5.65 15.7 Imidazole 6.61 8.98 Tris 8.83 8.78 KOH 10.66 8.68 Table XII (Continued) Acyl Group Buffer pH 60° i in3 ■ "I k , , x 10 m m obsd 3,3-Dimethylbutyryl KOH (0.0679 M) 15.6 KOH (0.170 M) 26.7 KOH (0.424 M) 48.8 KOH (0.501 M) 61.0 VD 50 Figure 7- Plot of log kobsd at 60° for hydrolysis of acyl phosphate vs, pH Isobutyryl phosphate; O Trimethylacetyl phosphate; Q 3,3-dimethylbutyryl phosphate. E x 13 -O O 90 80 r 70 6 0 50 40 30 20 — 0— - O ™ —G ____ J ____ Q pH 52 k obsd k (AcP ) + k dianion (AcP ) monoanxon (AcP) . - Total *0H (AcP )(OH ) (8) 1 employing the values of k monoanion' dianion' and the sec ond order rate constant for hydroxide ion catalysis, k , 0x1 for isobutyryl phosphate, trimethylacetyl phosphate, and 3,3-dimethylbutyryl phosphate respectively. These values 'compounds could not be determined by this method due to j :rapid hydrolysis of all the compounds below pH2- For this reason, theoretical lines were not continued below pH 3. The rate constants at various temperatures for tri- ; methylacetyl phosphate and 3,3-dimethylbutyryl phosphate are reported in Table XIV. Activation parameters are also o . : tabulated, calculated at 39.0 . For comparative purposes, ■ ;the literature values for acetyl phosphate are presented. i ] The effects of changing ionic strength and organic | I | jsolvent can be seen in Table XV. For monoanion and dianion . reactions of both trimethylacetyl and 3,3-dimethylbutyryl ' |phosphate the rate is slightly increased by changing the I ionic strength from 0.6 to 2.0 M. Similar effects are not- ;ed when the solvent is changed from water to 50% dioxane- Iwater. The rate constant for hydrolysis of the dianion of I trimethylacetyl phosphate is, however, doubled in 50% diox- ’ jane-H^O and a rate decrease is observed for the dianion of ; I in Table XIII. The pK values were determined by measuring! j 2 t I the pH of a half neutralized solution of the sodium salt. j ; o ■ The experimental pK^ values at 25 are 4.86, 5.02, and 5.11i o were used as pK^ of the compounds at 60.0 . pK^ for the 53 j3,3-dimethylbutyryl phosphate. The rate constants for |hydrolysis of the monoanion and dianion of trimethylacetyl |phosphate in deuterium oxide were also measured and are !also reported in Table XV. Comparing these values to those I ! !determined in H„0, D„0 solvent isotope effects, (k _/k__ ) | . 2 2 D 2O B. 2O [ I of 1.27 and 1.10 were obtained. j i The second order rate constants for pyridine catalysis,,' : ! ,calculated from the data in Table XVI, are presented in j j t 1 Table XVII and show a decrease for the more highly branched j 'compounds. No detectable reaction could be observed be- ; |tween imidazole or morpholine and trimethylacetyl or 3,3- dimethylbutyryl phosphate. There was no catalysis at pH 5.65, 6.12, or 7.50 by 0.5 M imidazole nor at pH 8.10 by 0.5 M morpholine. However, a pronounced imidazole catal- j ysis was observed in the case of isobutyryl phosphate at pH 5.62 where the monoanion would be at high concentration !although no catalysis was observed at pH 7.70 where little monoanion would be present. A plot of k , , vs. total im- obsa — ;idazole concentration from the data in Table XVI is shown ' !for isobutyryl phosphate in Figure 8. ^ O [ ! The pK^ of pyridine was determined at 60 and an ionic : | strength of 0.6 by half neutralization and was found to be ' 14.75. The pK^ of imidazole at 60° was found to be 6.58 by ! iextrapolation of values determined at a number of other j ;temperatures (67). i , I Glyceraldehyde-3-P Dehydrogenase Catalyzed Reactions - ! |The catalytic activity of glyceraldehyde-3-P dehydrogenase : iin the hydrolysis of aliphatic acyl phosphates can conven- ! t i jiently be divided into three classifications depending on Table XIII. Rate Constants for Spontaneous and Hydroxide Ion Catalyzed Hydrolysis of Acyl Phosphates at 60°. 3 - 1 3 - 1 Acyl Group k x 10 min k n. . x 10 min k„„ monoanion dianion OH 1. mole"-*- min" a a Acetyl 128.0 58.5 15.7 Isobutyryl 56.9 14.5 1.0 Isovaleryl 57.6 16.9 Trimethylacetyl 36.0 3.14 0.115 3,3-Dimethylbutyryl 63.7 8.78 0.10 Value calculated employing the activation energy reported in reference 4. Table XIV. Rate Constants for the Hydrolysis of Acyl Phosphates at Various Temperatures and the Activation Parameters Calculated at 39.0C Acyl Group m o „ Temp C ^obsd min ^ AH* kcal/mole AS* eu Monoanion: Trimethylacetyl 3,3-Dimethylbutyryl Acetyl 25 0.00055 60 0.0337 70 0.107 80 0.286 40 0.00617 50 0.0214 60 0.0637 65 0.111 70 0.184 60 0.128° 22.9 ± 0.3 23.4 ± 0.1 22.5 -4.4 ± 0.8 -2.0 ± 0.4 -3.6 ui Table XIV (Continued) Acyl Group o Temp C ^obsd min-1 AH* kcal/mole AS* eu Monoanion: Isobutyryl^ Isovaleryl Dianion: Trimethylacetyl 3,3-Dimethylbutyryl 60 60 55 60 65 70 75 80 50 60 65 70 0.0569 0.0576 0.00205 0.00314 0.00736 0.0107 0.0222 0.0425 0.00274 0.00878 0.0170 0.0304 27.7 ± 0.5 +5.4 ± 1.3 25.7 ± 0.1 +1.1 ± 0.5 Table XIV (Continued) Acyl Group ° Temp C \>bsd min"'*' AH* kcal/mole AS* eu Dianion: 0 3,3-Dimethylbutyryl 75 0.0508 Acetyl 60 O.O5850 ~r- „d 25 .4 +3.7d 0 Isobutyryl 60 0.0145 0 Isovaleryl 60 0.0169 a 0.05 M formate buffer, pH 3.48; ionic strength 0.6 with KCl b 0.05 M formate buffer, pH 3.57; ionic strength 0.6 with KCl c Value calculated from activation energy reported in reference 4 d Reference 4 e 0.05 M Tris buffer, pH 8.83; ionic strength 0.6 with KCl Ln -j Table XV. Hydrolysis of Trimethyacetyl Phosphate and 3,3-Dimethylbutyryl Phosphate When Subjected to Various Solvent Conditions at 60° in 0.05 Molar Buffer at Ionic Strength 0.60, Except Where Indicated. Acyl Group Buffer pH PD k , n min' obsd Trimethylacetyl Formate 3.30a 0.0438 Formate13 3.47 0.0390 c Formate 4.57 0.0490 Tris 7 .93a 0.00345 m • k Tris 8.82 0.00416 TrisC 8.96 0.00866 3,3-Dimethylbutyryl b Formate 3.47 0.0655 c Formate 4.57 0.0814 m ■ k Tris 8.82 0.0102 m . c Tris 8.96 0.00582 Table XV (Continued) a pD values were determined from pH meter readings employing the glass electrode correction formula of T. H. Fife and T. C. Bruice, J. Phys. Chem., 65:1079 (1961). b y = 2 .0 c 50% dioxane-H20 Table XVI. Rate Constants for the Amine Catalyzed Hydrolysis of ‘ ‘ Aliphatic Acyl Phosphates at 60° and y = 0.6 with KCl. I Acyl Group Amine M PH k x 10^ obsd . t min x Acetyl Pyridine: 0.05 5.47 70.6 0.20 5.27 80.7 0.35 5.20 90.9 0.50 5.18 98.5 0.05 8.98 61.7 0.20 8.89 70.6 0.35 8.98 75.9 0.50 8.98 79.0 Isobutyryl Pyridine: 0.05 5.47 30.2 0.20 5.27 30.9 0.35 5 .20 34.9 0.50 5.18 39.6 0.05 8.98 11.9 o\ o Table XVI (Continued) Acyl Group Amine M PH k j x 10 obsd . _i m m x Isobutyryl Pyridine: 0.20 8.89 12.9 0.35 8.98 13.8 0.50 8.98 14.6 Imidazole: 0.05 5.59 23.2 0.20 5 .64 28.9 0.35 5.63 31.0 0.50 5.62 34.6 Trimethylacetyl Pyridine: 0.05 5.47 16.5 0.20 5 .27 18.0 0.35 5.20 19.4 0.50 5.18 21.6 0.05 8.98 2.96 0.20 8.89 3.38 0.35 8.98 3.51 0.50 8.98 3.20 c r > Table XVI (Continued) Acyl Group Amine M PH k x 10 obsd . _n m m 3,3-Dimethylbutyryl Pyridine: 0.05 5.47 26.8 0.20 5.27 29.3 0.35 5.20 32.3 0.50 5 .18 35.2 0.05 8.98 8.73 0.20 8.89 8.88 0.35 8.98 9.00 0.50 8.98 9.37 cn to Table XVII. Second-Order Rate Constants for Pyridine Catalysis of Acyl Phosphates at 60.0° and y = 0.60. Acyl Group k, 1 mole-1- Pyridinea min-1 Imidazole Morpholine13 Monoanion: Acetyl Isobutyryl 0.163° 0.114° 1.68 Trimethylacetyl 0.0375° b 3,3-Dimethylacetyl 0.0590° b Dianion: Acetyl Isobutyryl 0.0412d 0.0060d b b Trimethylacetyl 0.00060a b b 3,3-Dimethylacetyl 0.00134d b b < T i Co Table XVII (Continued) a Rate constants determined at four concentration from 0.5 to 0.05 molar of the amine b No detectable reaction in 0.5 molar amine c In pyridine buffer, pH 5.22 H In 0.05 molar Tris buffer, pH 8.89 Figure 8. Plot of f°r hydrolysis of isobutyryl phosphate at 60° and y =0.6 vs. total imidazole concentration. kobsd (mirr1) 66 0.040 0.035 0.030 0.025 0.020 pH 7.70 0.015 0.010 0 0 . 1 0.3 0.2 0.4 0.5 [Im idazole] Tot(j| ! 67 I I 1 I ' i | I i the steric bulk of the substrate: ' (1) that of the non- j i |branched compound, acetyl phosphate, which follows second j I order kinetics; (2) that of the intermediate branched com- j i i |pounds, propionyl, butyryl, isobutyryl, and isovaleryl j 'phosphate, which obey normal M.ichaelis and Menten kinetics; j j ' ; 'and.(3) that of the highly branched compounds, trimethyl- | ■ . i I acetyl and 3,3-dimethylbutyryl phosphate,- which are not i substrates. i j The acetyl phosphatase activity of glyceraldehyde-3-P i [dehydrogenase vs. enzyme concentration is presented in Table XVIII and Figure 9. The plot is linear to an enzyme concentration of 1.5 mg per ml, an enzyme concentration which was routinely used for all acyl phosphatase studies. This high enzyme concentration facilitates the rapid hy drolysis of the more branched compounds and stabilizes the enzyme in the reaction vessel (23) . In Table XIX, the ini tial velocities of the glyceraldehyde-3-P dehydrogenase catalyzed hydrolysis of acetyl phosphate for varying initial substrate concentrations are presented, and are plotted in Figure 10. It appears that this plot represents a K^. too i ! large to measure but yielding kinetics which appear to be [second order. The second order rate constants were calcu lated by varying substrate, holding the enzyme concentra tion constant (Figure 10) and by varying enzyme, holding the substrate concentration constant (Figure 9). k, . for 3 -1-1 the former was found to be 1.4 x 10 M min where k. , 3 -1 -1 ; for Figure 9 was 2.3 x 10 M min * in good agreement. j ! The glyceraldehyde-3-P dehydrogenase catalyzed hydrol- iysis of the intermediate branched compounds, propionyl, 68 Table XVIII. The Acetyl Phosphatase Activity of Glyceraldehyde-3-P Dehydrogenase with Vary ing Enzyme and Constant Substrate Concentrations Enzyme mg/ml Velocity n moles/mg protein/sec 2.5 1.97 1.5 1.70 1.0 1.16 0.5 0.590 0.25 0.482 a Each reaction vessel was 6.06 mM in acetyl phosphate and 0.025 M in sodium barbital, pH 7.0. Figure 9. The plot of the acetyl phosphatase activity of glyceraldehyde-3-P dehydrogenase vs. enzyme concentration at 25°. Activity is express in n moles acetyl phosphate hydrolyzed per mg protein per sec. Velocity n moles / ml / sec m 3 M 3 C D 3 cQ tn CJI CJ1 Ul ro cn o L 71 Table XIX. The Glyceraldehyde-3-P Dehydrogenase Catalyzed Hydrolysis of Various Aliphatic Acyl Phosphates at 25°.3 Acyl Group Concentration Velocity mM n moles/mg protein/sec Acetyl Propionyl Butyryl 19.8 3 .64 15.4 2 .62 8.81 1.56 6.61 1.52 4.41 1.13 3.31 0.762 2.20 0.424 35.1 2.96 25.4 2.40 17.6 1.88 11.7 1.45 7;80 0.830 3.90 0.528 37.3 1.23 26.9 0.957 12.4 0.512 8.29 0.356 4.14 0.218 72 Table XIX (Continued) Acyl Group Concentration mM Velocity n moles/mg protein/sec Isobutyryl 42.3 0.522 30.5 0.477 23.5 0.420 18.8 0.365 14.1 0.336 9.39 0.240 7.05 0.200 4.70 0.162 Isovaleryl 42.9 0.491 31.0 0.451 21.4 0.359 14.3 0.246 9.52 0.210 4.76 0.132 a Each reaction vessel contained 1.5 mg protein per ml and was 0.025 M in sodium barbital, pH 7.0. 73 Figure 10. Lineweaver and Burk plot of the acetyl phosphatase activity of glyceraldehyde-3-P dehydro genase in the absence of inhibitor © and in the presence of 0.00329 M trimethylacetyl phosphateED and 0.00424 M 3>3-dimethylbutyryl phosphate^ at 25°- Velocity is expressed in n moles acetyl phos phate hydrolyzed per mg protein per sec. I/V nmoles /m g protein/sec. O — ro oj - £ > o P o P Oi 75 butyryl, isobutyryl, and isovaleryl phosphate is presented in Table XIX and appears to be of the classical Michaelis and Menten type. Double reciprocal plots according to Lineweaver and Burk (54), Figure 11, are linear giving reasonable values for and VMax as reported in Table XX. The effect of arsenate on this catalyzed hydrolysis of propionyl, butyryl, and isovaleryl phosphate is presented in Table XXI. Figure 12 shows that the data used to calcu late these rate constants are linear, even up to 70% of the reaction. Work on other acyl phosphates (65,66) indicates that the kinetics for the intermediate branched acyl phos phates should be very similar to that for chymotrypsin (43), following the reaction scheme in Equation (9), where P^ represents organic phosphate, k1, E + S t * ES * ES' + Px *3 ^ j L ES -___* EP «.T * E + - 3 ^4 P^ the organic acid, and ES' the acyl enzyme intermediate. The arsenate effect indicates (see Discussion) that the kinetic expression for V would reduce to Equation (10). MciX This expression was used to calculate the values for V = E k_ (10) Max o 3 k^ reported in Table XX. An attempt was made to show that these compounds were reacting at the active site by demonstrating inhibition of the dehydrogenase activity of glyceraldehyde-3-P dehydro genase. The inhibition by one of these compounds, iso- 76 butyryl phosphate, is shown in Figure 13 and Table XXII. The plot is sigmoidal, with maximal inhibition at approx imately 500 mM isobutyryl phosphate, making it difficult to determine the nature of inhibition, i.e., competitive, non competitive, etc. Although active site specificity cannot be demonstrated by this technique, the sigmoidal nature of inhibition may have interesting ramifications pertaining to the cooperativity of glyceraldehyde-3-P dehydrogenase. This point will be developed further in the discussion. The highly branched compounds, trimethylacetyl and 3,3-dimethylbutyryl phosphate, are not substrates. Even with an enzyme concentration of 7 mg per ml, no catalysis could be observed for either compound. It is possible that the enzyme could be acylating and not deacylating. This appears unlikely, however, since no catalysis could be ob served even in the presence of 15 mM sodium arsenate, a reagent which favors deacylation. Both compounds are in hibitors for the acetyl phosphatase activity of glyceral- dehyde-3-P dehydrogenase, illustrated in Figure 10 and Table XXIII. Further evidence that the highly branched compounds are bound to the enzyme can be seen in Figure 14 and Table XXII where trimethylacetyl phosphate inhibits the dehydrogenase activity. It is apparent that this curve is sigmoidal, resembling the inhibition by isobutyryl phos phate, Figure 13. Maximal inhibition is at a lower concen tration, 3 mM, than that for isobutyryl phosphate. The concentration dependence of the dehydrogenase ac tivity of glyceraldehyde-3-P dehydrogenase on arsenate and phosphate is shown in Figure 15 and Table XXIV. The two 77 Figure 11. Lineweaver and Burk plots of the propionyl [7j * butyryl 0 , isobutyryl Q , and isovaleryl A phosphate activities of glyceraldehyde-3-P dehydrogenase at 25°■ Velocity is expressed in n moles acyl phosphate hydrolyzed per mg protein per sec. I/V x 10 n moles / mg protein / sec O • ro w cn & l o In O 0 01 b cn ro O ro cn c m o Table XX. The Kinetic Constants for the Hydrolysis of Various Acyl O 3 phosphates by Glyceraldehyde-3-P Dehydrogenase at 25.0 . Acyl Group ^Max n moles/mg protein/sec > M k3 sec”l Propionyl 6.3 0.046 0.89 Butyryl 2.2 0.039 0.30 Isobutyryl 0.70 0.016 0.097 Isovaleryl 0.67 0.020 0.093 a Each reaction vessel contained 1.5 mg protein per ml and was 0.025 M in sodium barbital, pH 7.0. Table XXI. The Glyceraldehyde-3-P Dehydrogenase Catalyzed Hydrolysis of the Intermediate Branched Acyl Phosphates in the Presence of Arsenate and o a Phosphate at 25.0 . Acyl Group Substrate mM Velocity n moles/mg protein/sec V Vb o arsenate vc phosphate Propionyl 17.6 1.9 4.3 --- Butyryl 18.7 0.72 2.7 --- Isovaleryl 10.2 0.20 0.39 0.17 a Each reaction vessel contained 1.5 mg protein per ml and was 0.025 M in sodium barbital, pH 7.0. b In the presence of 0.01 M sodium arsenate. c In the presence of 0.001 M sodium phosphate. 81 Figure 12. The rate of disappearance of propionyl Q , butyryl 0 , isovaleryl Q phosphate measured by the hydroxamic acid assay at 5^0 my., in the presence of glyceraldehyde-3-P dehydrogenase and 0.01 M sodium arsenate at 25°. Initial substrate concentrations were as reported in Table XXI. Absorbance 0.12 o.i I o.io 0.09 O 0.08 S 0.07 o . o s 0.05 0.04 0.03 0.02 O.Oi 0 1 0 20 30 40 50 6 Q 70 S O S O 1 0 0 N O Time (min) 83 Figure 13. The dehydrogenase activity of glyceral- dehyde-3-P dehydrogenase, measured as n moles NADH formed per mg protein per sec in the presence of varying amounts of isobutyryl phosphate as inhibitor at 25°. Initial concentrations are as follows: 5.64 x 10 NAD+, 3.82 x 10 glyceraldehyde-3-P, -4 2.0 x 10 mg/ml glyceraldehyde-3-P dehydrogenase. Isobutyryl phosphate l/V x I02 nm oles/m g protein / sec. ro ro cn ro + T 8 85 Table XXII. The Inhibition of the Dehydrogenase Activity of Glyceraldehyde-3-P Dehydrogenase by o a Isobutyryl and Trimethylacetyl Phosphate at 25 . Acyl Group Concentration Velocity mM Isobutyryl ^ 0.0 7.84 14.9 29.9 39.2 44.8 54.8 78.4 89.5 110 133 149 172 209 268 n moles/mg protein/sec 723 496 288 148 117 107 64.2 54.9 31.6 22.8 14.7 15.3 17.4 10.9 9.08 86 Table XXII (Continued) Acyl Group Concentration mM Velocity n moles/mg protein/sec Q Trimethylacetyl 0.0 765 0.195 364 0.392 196 0.978 84.7 1.95 58.2 3.92 51.2 a Each reaction vessel was 0.807 mM in EDTA and 0.025 M in sodium barbital, pH 7.0. —4 b Contained 2.00 x 10 mg protein per ml and was 0.382 mM in glyceraldehyde-3-P, and 0.564 mM in NAD+. c Contained 3.16 x 10 mg protein per ml and was 0.0686 mM in glyceraldehyde-3-P, and 0.256 mM in NAD+. 87 reagents appear to be equally effective, with maximal ac tivity at 3 mM. Methyl phosphate is also included on the graph for comparative purposes. Table XXIII. Inhibition of the Acetyl Phosphatase Activity of Glyceraldehyde- 3-P Dehydrogenase by Trimethylacetyl and 3,3-Dimethylbutyryl Phosphate at 25°.3 Acyl Group Acetyl Phosphate Concentration mM Velocity n moles/mg protein/sec Trimethylacetyl 8.2 0.336 11.9 0.424 16.4 0.620 Q 3,3-Dimethylbutyryl 3.43 0.364 5.14 0.541 7.42 0.742 10.3 1.14 a Each reaction vessel contained 1.5 mg protein per ml and was 0.025 M in | sodium barbital, pH 7.0. b 3.29 mM in trimethylacetyl phosphate c 4.24 mM in 3,3-dimethylbutyryl phosphate 89 Figure 14. The dehydrogenase activity of glyceral- dehyde-3-P dehydrogenase, measured as n moles NADH formed per mg protein per sec in the presence of varying amounts of trimethylacetyl phosphate as inhibitor at 25°. Initial concentrations are as -4 + -5 follows: 2.56 x 10 M NAD , 6.86 x 10 M glyceral- -4 dehyde-3-P, 3.16 x 10 mg/ml glyceraldehyde-3-P dehydrogenase. I / V x IQ '" nmoles /mg p ro tein /sec, 2.0 1 .5 1.0 0.5 0 4.0 3.0 3.5 2.0 i.O 1 .5 0 0.5 irimethylacetyl phosphate mM 91 Figure 15. The effect of arsenate O, phosphate O/ and methyl phosphate 0, on the dehydrogenase activ ity of glyceraldehyde-3-P dehydrogenase at 25°. Velocity is measured in n moles NADH formed per mg protein per sec. Initial concentrations are as _4 + _4 follows: 2.48 x 10 M NAD , 2.85 x 10 M glyceral- -4 dehyde-3-P, 3.38 x 10 mg/ml. Velocity . nmoles /mg protein / sec. CO o o o 73 o CD Z3 o o' 3 ss* o r o o o O o o o 00 o o o 04 00 26 Table XXIV. Arsenate, Phosphate, and Monomethyl Phosphate Catalysis for the o a Dehydrogenase Activity of Glyceraldehyde-3-P Dehydrogenase at 25 . Catalyst Concentration mM Velocity n moles/mg protein/sec Arsenate 0.00 39.6 0.098 340 0.197 276 0.490 413 0.983 537 4.90 666 Phosphate 0.327 296 0.973 518 1.61 562 3 .18 729 Monomethyl Phosphate 3.50 75.2 7.00 118 Table XXIV (Continued) Each reaction vessel contained 3.38 x 10 mg protein per ml and was 0.285 mM in glyceraldehyde-3-P, 0.248 mM in NAD+, 0.807 mM in EDTA, and 0.025 M in sodium barbital buffer, pH 7.0. 95 DISCUSSION OF RESULTS Acid Catalysis For all the compounds studied, the reaction in acid can be described in terms of an A-2 mechanism involving at tack of solvent on the protonated acyl phosphate as shown in Equation (11). Water very likely attacks as a nucleophile at the carbonyl carbon as indicated by the lack of sensitivity to electron- substituted benzoyl phosphates. The finding of C - 0 bond cleavage shows that attack of water is at the carbonyl car bon and not at phosphorous, and the D^O solvent isotope effects (k /k = 2.2 - 2.6) are consistent with the occur- D H rence of a pre-equilibrium protonation step. The small value observed for substituted benzoyl phosphate hydrolysis can be explained by invoking compensating effects; increas ed electron withdrawal in the benzoyl group will decrease the equilibrium concentration of protonated intermediate but will increase the ease of attack of water at the car bonyl carbon. Partial cancellation of these effects should R-C-O-lf-OH OH O O L i 4 - I ! I I H O H O ii i ii ic effects (p = -0.24) in the acid catalyzed hydrolysis of 96 give a p close to zero* An A-l reaction in which an acylium ion was formed, however, would be expected to show correlation with (72) with a highly negative value of p as in the acid catalyzed hydrolysis of substituted 2,6-di- methylbenzoate esters (4). At high ionic strength the rates of hydrolysis of the a -branched compounds, trimethylacetyl phosphate and iso- butyryl phosphate, are slower than the rate for acetyl phosphate, but the rates for the ^-branched compounds, 3.3-dimethylbutyryl phosphate and isovaleryl phosphate, are faster than that for acetyl phosphate. Increased steric bulk at the reaction center should not increase the equili brium concentration of protonated intermediate, and the rate of nucleophilic attack by water should be decreased. A change in mechanism to A-l for the 3,3-dimethylbutyryl derivative might account for the abnormal steric order of reactivity, although it would naturally be expected that the mechanism would be the same for all the compounds stud ied since the acylium ion that would be produced in an A-l reaction should be a highly unstable intermediate. How ever, the data are in accord with an A-l mechanism for 3.3-dimethylbutyryl phosphate and, therefore, this must be regarded as a possibility. The rate differences at high acidity are at least par tially due to substrate influenced ionic strength effects as seen from the larger value of b reported in Table V for 3.3-dimethylbutyryl phosphate than for acetyl or trimethyl acetyl phosphate. All of the compounds are subject to a marked positive salt effect, and at constant HCl concentra- 97 tion, increasing concentrations of LiCl have a greater effect on the hydrolysis of the 3,3-dimethylbutyryl deriva tive than on the hydrolysis of acetyl or trimethylacetyl phosphate. In addition to' salt effects, however, other factors are influencing the observed relative-rate ratios. The values of k reported in Table V also show differences that H would not be expected if increased bulk in the acyl group were simply influencing the reaction by steric hindrance to approach of an attacking water molecule. 3,3-Dimethyl- butyryl phosphate has a k that is only 1.9 times less than that for acetyl and which is 5.2 times greater than that for trimethyl acetyl- On the basis of steric hindrance the 3,3-dimethylbutyryl acyl group should produce a large re duction in rate compared to acetyl and should also give a slower rate than trimethylacetyl since its steric effects constant, E , is more negative (-1.74 compared to -1.54) s (Si). The rate constants for hydroxide ion catalyzed hy drolysis of these compounds show pronounced retardations owing to increased steric hindrance; the relative-rate ratios being, acetyl 1.0, trimethylacetyl 0.0073, and 3,3- dimethylbutyryl 0.0064. The mechanism for the hydroxide ion catalyzed reaction undoubtedly involves nucleophilic attack by hydroxide ion at the carbonyl group of the dian ionic species. Use of Bunnett's linear free energy rela tionships (10) partially clarifies the abnormal order of reactivity in acid. These relationships can serve as an indication of the involvement of water in the reaction, but do not necessarily yield conclusive evidence for the mechar>- 98 ism involved. Problems encountered in this regard have been discussed previously (63). The degree of water in volvement, < j > , is obtained from' the slope of (log + H ). From known reactions, Bunnett suggested that for wa- o ter not involved in the rate limiting step, c j > is less than zero? for water involved as a nucleophile, < j ) is 0.22 to 0.56; and for water involved as a proton transfer agent, c j ) is greater than 0.58. As seen in Figure 16, linear slopes are obtained with values of +0.31 for acetyl phosphate and -0.33 for 3,3-dimethylbutyryl phosphate, while trimethyl acetyl phosphate yields a curved line with a slope approach^ ing zero as the acid concentration is increased. From Bunnett's empirical criteria, therefore, water should act as a nucleophile in the hydrolysis of the conjugate acid of acetyl phosphate, should not be involved in the hydrolysis of 3,3-dimethylbutyryl phosphate, and may or may not be in volved in the hydrolysis of trimethylacetyl phosphate. In the latter case the curved plot could indicate a mechanism change as acid concentration is increased. Plots (not shown) of log ^otoscj + H0 vs* lo9 aH20 sl°Pes' that would be interpreted in the same manner (9), w being +1.8 with acetyl phosphate and -2.3 in the case of 3,3-dimethyl butyryl phosphate. Interpretation of the observed acid effects in terms of either the Zucker-Hammett hypothesis (62,88) or the Bunnett modifications (9,10) is obviously difficult, and neither of these treatments can be employed with great confidence as criteria of mechanism. A concerted reaction, as shown in Equation (12) in which the reaction becomes progressively more unimolecular 99 Figure 16. Plots of (log ^ + HQ) vs« (log C__ + H ) for hydrolysis of 3,3-dimethylbutyryl H o o , acetyl O , and trimethylacetyl phosphate © in various HCl solutions at 25.0°. 1 °s kobsd + U) o ro ui O ui 9 O H , [\3 o 100 101 9 R-C- I I I I I *+Q H H with Increased branching in the acyl group, would be in ac cord with the observed acid effects. The facilitation in rate produced by the highly branched acyl group might then be due to relief of strain upon stretching the C - 0 bond in the transition state. If this were occurring, however, it might reasonably be expected that trimethylacetyl phos phate would hydrolyze more rapidly than 3 *3-dimethylbutyryl phosphate which is not the case. It should be pointed out 18 that it is not known whether 0 exchange between water and the carbonyl oxygen of acyl phosphates takes place in the acid-catalyzed reactions and, therefore, evidence is lack ing as to whether tetrahedral intermediates might occur. If the mechanism is the same for all the compounds, then the Bunnett values may simply indicate that there is much less change in hydration upon going from the ground state to the transition state for 3s3-dimethylbutyryl phos phate than for acetyl phdlphate, and that for trimethyl acetyl phosphate, this change is also not as pronounced. The rate expression for an A-2 acyl-^phosphate hydroly sis reaction is shown in Equation (13) where f and f are s * M Q - 3 9-^°h OH (12) 102 the activity coefficients for substrate k f k = r n __ _ s obsd K + a-a f. (13) SH H H20 * and transition state and n is the formal order with re spect to water (62). Ions that salt out the substrate more than the transition state will raise Its activity co efficient and thereby increase the rate (60,61) if, of course, the activity coefficient effects can overcome the adverse effects of high salt concentration on the activity of water. These considerations have previously been ap plied to anhydride solvolysis where acid effects are also complicated (10). The increased rates and b values for 3,3-dimethylbutyryl phosphate In perchloric acid compared to HC1 are surprising since perchlorate generally salts in to a greater extent than chloride ion (12,60), but it was found that in the A-2 hydrolysis of acetic anhydride, per chloric acid at concentrations above 1.5 M was a better catalyst than HC1 although the reverse was true in the case of trimethylacetIc anhydride (12). The faster rate for the 3s3-dimethylbutyryl deriva tive in 2.87 M HC1 with an ionic strength of 4.80 is due solely to a more favorable AS* which might again indicate that with 3,3-dimethylbutyryl phosphate there is much less difference in hydration between the ground state and the transition state than with acetyl phosphate or trimethyl- 103 acetyl phosphate. It is perhaps surprising that the AS* values are in general as positive as was found. However, Kugel and Halmann (52) obtained a AS* of -6.0 eu for the acid-catalyzed hydrolysis of ethyl phosphate, a compound which hydrolyzes by an A-2 mechanism. Therefore, AS* is not expected to be highly negative, and the high ionic strength may make It even more positive. Steric hindrance to solvation of the transition state would not be expected to lead to the rate facilitation seen with 3s3-dimethylbutyryl phosphate. Therefore, if there is a smaller hydration change upon going from ground state to transition state with the 3,3-dimethylbutyryl acyl group it must be because of increased ground state hydration as is also probable in the case of N-3,3-dimethylbutyryl imidaz- olium ion (22). The rates of acid-catalyzed hydrolysis measured in 25$ dioxane-water are consistent.with solvent ordering in the ground state for 3s3-dimethylbutyryl phos phate since its rate was decreased much more than that of the two other compounds studied. This could be due to preferential solvation of the highly branched acyl group by the dioxane component of the solvent. It would, thus, appear that the values of k for the H acyl phosphates, which show a smaller sensitivity to steric bulk In the acyl group and a faster rate for 3,3-dimethyl butyryl phosphate than expected for an A-2 reaction, and io4 the unusual differences brought about by increasing acid concentrations can be explained either by increasing uni- molecular character in the rate determining step as branch ing in the acyl group increases, or smaller differences in hydration between ground state and transition state with greater acyl group branching, possibly due to the ability of alkyl groups to structure water around them in the ground state. These effects are not mutually exclusive and it is possible that both types of effects are important. Monoanion and Dianion Reactions It has been shown (18) that the monoanion and the dianion of acetyl phosphate hydrolyze by the unimolecular mechanisms in Equations (1) and (2), respectively. The D 0 solvent isotope effect (k /k =1.27) for tri- 2 D20 H20 methylacetyl phosphate monoanion is in accord with an in ternal proton transfer that is either complete. or Is par tially rate determining (45). Solvent isotope effects of about unity might be expected if a zwitterionic species was involved as an intermediate since D^O would have compensat ing effects on the equilibrium concentrations of monoanion and the zwitterion. Di Sabato and Jenks (18) likewise de termined this ratio for acetyl phosphate monoanion and found it to be 0.94. Salt effects and solvent effects are also similar for these compounds. In general, increased 105 branching decreases the rate of both monoanion and dianion hydrolysis. This rate decrease could be due to either steric or electronic factors, or to a combination of both. It is unlikely that increased size of the acyl group would cause the observed rate retardations since the rate determining step for both monoanion and dianion reactions is a unimolecular decomposition (18). As seen in Table XIV, the entropies of activation for the branched compounds and acetyl phosphate are very similar, with AS* for the monoanion of trimethylacetyl phosphate and the dianion of 3,3-dimethylbutyryl phosphate being only slightly more neg ative than the corresponding values for acetyl phosphate. Steric hindrance-to solvation could lead to large rate re ductions with increasing size of the acyl group, but it can be concluded from the similar entropies of activation that this is not an important factor. Inductive effects in the aliphatic series are probably important in producing the rate retardations observed for the highly branched compounds. Di Sabato and Jenks (18) found that the dianion rate of substituted benzoyl phos phates is facilitated by electron withdrawing substituents (p = +1.2), while the hydrolysis of monoanions is relative ly insensitive to electron withdrawal (p = +0.2). When the logarithms of the rate constants at 60° for the aliphatic series are plotted vs_. Taft a* constants (81), a straight line relationship is obtained as shown in Figure 17. The 106 value of p* Is +2.1 for the monoanion hydrolysis reaction and +4.9 for the dianion reaction. Data obtained at 39° were also plotted in order to utilize the rates of other compounds. The points for benzoyl phosphate (18) were found not to fit on the plots. Chloroacetyl phosphate was prepared and hydrolyzed at a rate in agreement with the re ported slopes . For chloroacetyl phosphate, k is -1 monoanion 0.204 min and k is 0.190 min at 39.0° and ionic dianion strength 0.6. Elemental analysis on this compound was not possible due to its rapid partial hydrolysis in aqueous solutions during the isolation procedure. Support for the importance of inductive effects can be drawn from the studies with pyridine. When the second- order rate constants, k (monoanion) and k (dianion) for ir y ^ ty the reaction of pyridine with the monoanion and dianion re spectively of acyl phosphates, are plotted against Taft's a* constants reasonably straight lines are obtained with p* values of +2.3 and +6.1 as shown in Figure 18. Steric effects should only account for a small part of the differ ences in the second-order rate constants since pyridine has been shown to attack exclusively at phosphorous when it re acts with either the monoanion or the dianion of acetyl phosphate (50,74). Thus, two atoms, oxygen and the carbon yl carbon, separate the reaction center from the point of branching thereby reducing greatly the magnitude of any steric effect. This can be illustrated by the similar Taft 107 Figure 17. Plot of log k at 60° for hydrolysis of acyl phosphate monoanions Q , and dianions O , vs. log k0bsd (min-i) O i q 108 109 Figure 18. monoanions Plots of log k for acyl phosphate □ , and dianions o vs. at 60°. lo g kpyr (I moie” * min.“ i) 110 i.O 2.0 3.0 0 , 2 Ill Eg constants (8l) for the groups: n-C^H^, (-0.36); i"C5Hn s (-0.35);■t_-C^HpCH2CH2, (-0.34), although in this comparison replacement of the carbonyl carbon and the oxygen by two saturated carbon atoms may not be exact. For $-glycerol- phosphate (82) the C - 0 - P bond distance is similar to the C - C - C distance, but for acyl phosphates the C - 0 - P distance could be shorter because of resonance interac tion between oxygen and the carbonyl group. It is, there fore, possible that steric and inductive effects are both affecting the nucleophilic attack of pyridine, but the pre dominant effect appears to be inductive. The larger rate constants for 3a3-dimethylbutyryl phosphate as compared to trimethylacetyl phosphate is, of course, not in accord with a steric order of reactivity. The value of p* for the reaction of acyl phosphate di anions with pyridine, is larger than for the dianion hydro lytic reaction. This result is expected since electron withdrawing groups would not only make it easier for the carboxylate anion to leave, as is the case in the hydro lytic reaction, but would also make the attack of pyridine more facile. Also, p* for the pyridine catalyzed hydrol ysis of acyl phosphate dianions is much larger than the p* for the pyridine catalysis of acyl phosphate monoanion hy drolysis. This observation can be attributed to a proton transfer step taking place in the monoanion reaction with 112 pyridine, as in the hydrolytic reaction (18). If the car- boxylate anion were the leaving group for both pyridine catalyzed reactions, similar p* values should be observed. Thus, the probable transition state for the pyridine reac tion with the monoanion would appear as shown in Equation A pentacovalent intermediate could also be forming and the kinetic data do not eliminate this possibility. Pyridine has been shown not to catalyze the hydrolysis of phenyl phosphate or p-nitrophenyl phosphate (14). The facile pyridine reaction with the monoanion of acyl phos phates may be due to the ease of proton transfer with these compounds, and in addition, the low pK of the carboxylate anion leaving group, 4.7, compared to 9-9 for phenol and 7.1 for £-nitrophenol, should greatly facilitate the reac tion. These differences in pK undoubtedly strongly influ ence the pyridine reaction with the dianion of acyl phos phates since proton transfer does not, of course, take place in the dianion reaction. Reactions of acyl phosphates which occur at the car bonyl carbon center are subject to normal steric effects. Imidazole and morpholine, two amine bases which attack at the carbonyl of acetyl phosphate (17), show no observable (14). (14) 113 reaction with trimethylacetyl phosphate and 3,3-dimethyl butyryl phosphate. Hydroxide ion, a much smaller nucleo phile, will catalyze the hydrolysis of the dianionic spe cies of these two compounds, although at a reduced rate in comparison to acetyl phosphate, as has been previously dis cussed . An interesting observation is that electronic and/or steric influences at the carbonyl carbon will not change the position of attack of amines. Imidazole will not pref erentially attack at phosphorous even when relatively high electron density (trimethylacetyl phosphate) or large ster ic hindrance (3s3-dimethylbutyryl phosphate) occurs at the carbonyl carbon. Some enzymes which exhibit acyl phosphatase activity have histidine at their active sites. When succinyl phos phate is utilized by succinic thiokinase, the acyl phos phate transfers the phosphoryl group to the enzyme to form a phosphoryl enzyme (69). The phosphorylated group in the enzyme has been identified as 3-phosphohistidine (38,69). The chemical data indicate that a phosphoryl group cannot be transferred directly from an acyl phosphate to histi dine. Transfer must then be mediated through an intermedi ate carrier or else the enzyme somehow utilizes a mechanism different from that normally seen in the non-enzymatic re actions . 114 The Activity of Glyceraldehyde-3-Phosphate Dehydrogenase The acyl phosphates employed should acylate or hind to the active site of glyceraldehyde-3-P dehydrogenase. Such widely varying compounds as 3-(2-furyl) acryloyl phosphate, (65) acetyl phosphate (33), and 2-acetamido-4-nitrophenol (46) have all shown active site specificity. Trimethyl- acetyl phosphate and 3,3-dimethylbutyryl phosphate inhibit the acyl phosphatase activity (Figure 10). In addition, trimethylacetyl phosphate and Isobutyryl phosphate have been shown to inhibit glyceraldehyde-3-P dehydrogenase activity (Figures 13 and 14). It is, therefore, likely that all acyl phosphates in the aliphatic series react at the active site of glyceraldehyde-3-P dehydrogenase. The sigmoidal nature of the plots in Figures 13 and 14 indicate that the inhibition caused by two representative acyl phosphates on the dehydrogenase activity of glyceral- dehyde-3-P dehydrogenase may be cooperative. The enzyme is a tetramer consisting of identical subunits (30) . Cooper- ativity in binding of NAD+ to yeast glyceraldehyde-3-P de hydrogenase has been aptly demonstrated by the kinetic ex periments of Kirshner, et_ al_ (47), and recently substantiat ed by Chance and Park (13)* Similar experiments are not possible on the muscle enzyme since the NAD+ is more firmly bound (84). By the use of other techniques, however, sub unit interactions have been noted. Velick (84) has observed that one mole equivalent of p-mercuribenzoate per mole pro- 115 teln weakens the affinity of the protein for NAD+ at all the remaining binding sites. Listowsky, et_ al (59) report ed through the use of ORD that the major changes in con formation of the protein upon NAD+ binding occurred when one equivalent of the cofactor was bound to the enzyme. Using $-(2-furyl)-acryloyl phosphate as a substrate, Malhotra and Bernhard (65) have demonstrated some coopera tive subunit interactions. The present experiments illus trate that a substrate which possesses active site specifi city for glyceraldehyde-3-P dehydrogenase, i.e., an acyl phosphate, also exhibits cooperativity in reacting with the enzyme. The residual dehydrogenase activity remaining at high acyl phosphate concentration is not unexpected. Values ranging from 0.8 to 3.8 have been obtained for the number of sites acylated by acyl phosphates (65). The experiments of Chance and Park (13) indicate that while all monomers of the tetrameric glyceraldehyde-3-P dehydrogenase have iden tical primary structures, they are not all equivalent in their ability to be acylated by acetyl phosphate. The sites which are unfavorable to acetyl phosphatase activity, i.e., on the less active monomers, also have a decreased de hydrogenase activity (13)- The dehydrogenase activity re maining at maximal propionyl and trimethylacetyl phosphate inhibition may, therefore, be due to those monomers which are not subject to acylation. The Chance and Park (13) ex- 116 periments Indicate the presence of one active monomer in the tetramer while those of Malhotra and Bernhard (65) sug gest that half of the monomers are active while half are less active. Since cooperativity is observed in the present inhibition experiments, it appears that more than one of the monomers is active, and at least one is less active. The acetyl phosphatase activity of glyceraldehyde-3-P dehydrogenase appears to be a second-order reaction, first order in enzyme and first order in acetyl phosphate. This is convincingly demonstrated by the similarity in rate con stants obtained by varying both the substrate and enzyme. The second-order nature of the reaction implies that the acetyl phosphate is only weakly bound to the enzyme, and that acylation occurs immediately upon binding. This is further substantiated by the slight decrease in Km obtained with the increased branching as seen in Table XX, and by the observation that trimethylacetyl phosphate gives maximal in hibition at a much lower concentration than isobutyryl phos phate. Acetyl phosphate, which does not have a branched acyl group, would fit into this trend as a compound which is very weakly bound to glyceraldehyde-3-P dehydrogenase. These results indicate that the mode of attachment of acyl phosphates is not strictly an ionic one between a positive charge on the enzyme and the phosphate moiety, but may be aided by a hydrophobic region in juxtaposition to the 117 active site. The arsenate effects clearly indicate that rate deter mining deacylation is occurring with the intermediate branched compounds. A rate of acylation comparable to de acylation would have been detected in the presence of ar senate by curvature in the lines of Figure 12. One of the compounds, propionyl phosphate, decreased linearly even to 70% of the reaction. The functional groups of the amino acids cysteine and histidine have been implicated in the acyl phosphatase ac tivity of glyceraldehyde-3-P dehydrogenase. In the cata lytic process cleavage of the C - 0 bond (7^) of the acyl phosphate occurs and an acyl enzyme is formed (3^)* Thiols are poor nucleophiles towards acetyl phosphate (17)3 where as, imidazole Is an excellent catalyst (17)* Imidazole catalysis could not, however, be detected in the non-enzym- atic hydrolysis of trimethylacetyl phosphate and 3,3-di- methylbutyryl phosphate. These same compounds are also not hydrolyzed by glyceraldehyde-3-P-dehydrogenase even though they bind strongly. The fact that both the non-enzymatic and enzymatic reactions are being influenced alike by the same steric factors supports, but does not prove, a mechan ism in which histidine is the attacking group in the enzym atic acylation reaction in a manner similar to that sug gested by Olson and Park (73). 118 Modification of the essential cysteine through treat ment with iodine (20) or iodoacetate (5137^) will acceler ate the acyl phosphatase activity but inhibits arsenolysis of the acyl enzyme (51)* When the sulfhydryl is not block ed a thiol ester invariably forms (66), possibly by an N to S shift. Blocking the cysteine could cause the reaction to proceed through an abnormal pathway, presumably only in volving the histidine. An intermediate acyl histidine should hydrolyze readily if transfer of the acyl group to cysteine is not possible, thus explaining the enhancement in activity when the essential thiol group is modified. Since acylation should then be rate limiting the arsenate effect would also be inhibited, as observed since arsenate can only promote catalysis by acting at or before the rate determining step. Histidine is believed to be involved not only in the acylation of the enzyme, but also in the deacylation of the thiol ester intermediate (73). Either a nucleophilic mech anism for deacylation, in which the acyl group is transfer red to histidine with the acyl histidine subsequently ’ undergoing rapid hydrolysis, or a mechanism in which histi dine acts as a classical general base in deacylation, par tially abstracting a proton from an attacking water mole cule in the transition state, is possible. It is more probable that histidine serves as a general base in the de- 119 acylation process, as in the scheme postulated by Behme and Cordes (3) from studies of the esterolytic activity of the A . NAD free enzyme. This deacylation mechanism is also in dicated by the almost total lack of effect of monomethyl phosphate on the dehydrogenase activity, a reaction which exhibits rate limiting deacylation. In contrast, a marked enhancement is observed in the presence of arsenate or phosphate. Both arsenate and phosphate possess an ioniz- able proton at pH 8.4. Histidine can partially remove this proton thereby facilitating catalysis by allowing attack of a more basic species. Monomethyl phosphate, which is com pletely ionized at that pH, cannot participate in such a reaction. If a nucleophilic mechanism were operative, all three reagents should be effective. The acyl groups of the acyl phosphates possess varying inductive and steric properties. It was thought that a correlation of the order of reactivity to the Taft steric constants, Es (8l), might permit discrimination between the nucleophilic and general base mechanisms of deacylation. The inductive effects constants, o* in Equation (15) are nearly the same over the series studied. Thus, the differ ences in reactivity are mainly due to differences in log k^ = o* p* + <5Es (15) the steric parameter, E_. In Figure 19 is shown a plot of o log k_ vs. E_. Compounds with more negative E„ values, 3 — S fa 120 such as triraethylacetyl and 3,3-dimethylbutyryl phosphate could not, of course, be Included since they are not sub strates. The slope of the line, 5, reflecting the sus ceptibility of deacylation to steric effects, is 1.1 ± 0.4. Steric effects in nonenzymatic hydrolysis reactions of thiol esters have also been studied. The value of 6 for the imidazole catalyzed hydrolysis of esters of thiolphen- ol, a nucleophilic reaction, was found to be 1.161. Steric effects on the hydroxide ion catalysis of these same com pounds should approximate the steric effects of general base catalysis of thiolesters, as a hydroxide ion is the attacking species for both reactions. The 6 for this reac tion was 1.15^. Applying this type of nonenzymatic data to enzymatic systems has been done with some success in a study on the deacylation of chymotrypsin (67). In that study (67) 3 the clear differences in the steric effects for classical gen eral base and nucleophilic catalysis on the oxygen esters enabled the investigators to confirm the general base mech anism of deacylation. However, for the imidazole catalyzed hydrolysis of thiolesters, the difference in 5 between the two mechanisms is non-existent, making the 6 of 1.1 obtain ed from the glyceraldehyde-3-P dehydrogenase catalyzed hy- 1 D. McMahon and T.H. Fife, unpublished results. 121 Figure 19. Plot of log from Table XX vs. Taft’s steric constants, E . s 122 LO LO o OJ d i 0 1 CO 0 1 CO d i CO IU o V | 00{ 123 drolysis of acyl phosphates of little value in distinguish ing between the two mechanisms of deacylation. Since a general base mechanism, as in Equation (16), is indicated for the deacylation step by the poor catalysis observed for methyl phosphate and the D2O solvent isotope effect found for deacylation of the NAD+-free enzyme (3)s a fundamental question arises as to the reason for a general base mechanism rather than one involving nucleophilic at tack by histidine. Thiol esters are very susceptible to nucleophilic attack by nitrogen bases (8) so that on chemi cal grounds a nucleophilic mechanism might be thought more likely. However, formation of an acyl histidine by attack on the thiol ester should be readily reversible as in Equa tion (17) since it is known that acyl imidazoles will pref erentially transfer the acyl group to a thiol acceptor O HlkV s £ R (16) o s-£ -r SH (17) 124 rather than water (40). Reversibility should be especially facile if the imidazole and sulfhydryl groups remain in close proximity. Thus, the general base mechanism could become the preferred pathway since it would allow products to be formed at a fast rate. Although the evidence cited for attack by histidine at the carbonyl of the acyl phosphate substrate in the acyla tion step cannot be regarded as conclusive, none the less, if such attack occurs, then an argument similar to that ap plied above would explain the subsequent formation of the relatively stable thiol ester. 125 SUMMARY The rates of acid-catalyzed hydrolysis of a series of aliphatic acyl phosphates with varying steric bulk in the acyl group have been measured in water at 25°. At constant ionic strength, plots of log kobsd HCl concentra tion, with acid concentration increasing to 4.80 M, are linear with slopes deviating slightly from 1.0. At high ionic strength an abnormal order of reactivity is observed, the 3,3-dimethylbutyryl derivative hydrolyzing faster than the other compounds in the series. There is a large posi tive ionic strength effect with each compound and 3,3-di methylbutyryl phosphate is affected to the greatest extent. The second-order rate constant for the acid-catalyzed reac tion independent of ionic strength effects for 3,3-dimethyl butyryl phosphate is only slightly less than that for acetyl and much larger than that for trimethylacetyl phos phate, Plots of (log k , , + H ) vs. (log C + H ) have c ob s d o — H o linear slopes of + 0.31 for acetyl phosphate and -0.33 for 3,3-dimethylbutyryl phosphate, while trimethylacetyl phos phate yielded a curved line. Values of k _./k„ ~ are be- D2' - / 2 tween 2.2 and 2.6 for all compounds. The AS* for 3,3-di methylbutyryl phosphate (+0.6 eu) is more positive than that for acetyl phosphate (-5.1 eu) or trimethylacetyl phosphate (-9.7 eu). The rate constants for acid-catalyzed hydrolysis of substituted benzoyl phosphates in 25% dioxane-water at 25° show only a small sensitivity to electronic effects (p = -0.24) indicating an A-2 mechanism for acyl phosphate 126 hydrolysis. Thus, with the aliphatic acyl phosphates, there are very likely differences in the importance of bond breaking in the transition state or differences in ground state and transition state hydration. The rate constants for hydrolysis of the series of aliphatic acyl phosphates have also been determined at 60°. Complete pH-rate profiles for three of the derivatives, isobutyryl, trimethylacetyl, and 3,3-dimethylbutyryl phos phate, were obtained as well as the monoanion and dianion hydrolytic rate constants for isovaleryl phosphate. The values of AS* were uniformly close to zero, consistent with the postulated unimolecular mechanism of hydrolysis of acyl phosphates. A decrease in the rate of hydrolysis was observed for the monoanion and dianion reactions as steric bulk and electron donation in the acyl group, as measured by the Taft a* constants, was increased. Good correlation with p* was obtained with a p* of +2.1 for the monoanion reaction and +4.9 for the dianion reaction. Second-order rate constants for reaction of pyridine with the monoanions and dianions were also correlated with the a* constants. The p* for k (monoanion) was +2.3 and for k (dianion) pyr pyr was +6.1. Imidazole and morpholine did not catalyze the hydrolysis of trimethylacetyl phosphate or 3,3-dimethyl butyryl phosphate. The acyl phosphatase activity of glyceraldehyde-3- phosphate dehydrogenase has been examined using the various acyl phosphates at 25°. This activity can conveniently be divided into three categories depending on the steric bulk of the substrate: (1) that of the non branched compound, 127 acetyl phosphate, which follows second order kinetics; (2) that of the intermediate branched compounds, propionyl, butyryl, isobutyryl, and isovaleryl phosphate, which obey normal Michaelis and Menten kinetics; (3) that of the high ly branched compounds, trimethylacetyl and 3,3-dimethyl butyryl phosphate, which are not substrates. The rate de termining step for the intermediate branched compounds was shown to be deacylation of the acylenzyme intermediate since arsenate increased the rate of catalysis. Methyl phosphate was found to deacylate the enzyme poorly. This and other observations are consistent with a general base mechanism for deacylation of glyceraldehyde-3-P dehydrogen ase. The highlyjbranched compounds bind to the enzyme since they inhibit acetyl phosphatase activity. 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This dissertation has been microfilmed exactly as received ’ PHILLIPS, David Richard, 1942- STERIC EFFECTS IN THE ACID, AMINE, AND GLYCER ALDEHYDE - 3 -PHOSPHATE DEHYDRO GENASE CATALYZED HYDROLYSIS OF ACYL PHOSPHATES. University of Southern California, Ph.D., 1969 Biochem istry University Microfilms, Inc., Ann Arbor, Michigan

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Creator Phillips, David Richard, 1942- (author)

Core Title Steric effects in the acid, amine, and glyceraldehyde-3-phosphate dehydrogenase catalyzed hydrolysis of acyl phosphates

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1969-01

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), [Lurf], David (committee member), Allerton, Samuel E. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-707873

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