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Steric effects in imidazole and ɑ-chymotrypsin catalyzed ester hydrolysis

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Steric effects in imidazole and ɑ-chymotrypsin catalyzed ester hydrolysis

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Content STERIC EFFECTS IN IMIDAZOLE AND Ct-C H Y M O TRYPSIN CATALYZED ESTER HYDROLYSIS by J u l i e B lock M ils tie n A D is s e r ta tio n P re s e n te d to th e FACULTY OF THE G RA DUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In P a r t i a l F u lf illm e n t o f th e R equirem ents f o r th e Degree DOCTOR OF PHILOSOPHY (B io c h e m istry ) J a n u a ry 1968 UNIVERSITY OF SOUTHERN CALIFORNIA T H E G RA DUATE SC H O O L U N IV ER SITY PARK LO S A N G E LE S. C A L IFO R N IA 0 0 0 0 7 This dissertation, written by under the direction of hJStH ... Dissertation Com mittee, and approved by all its members, has been presented to and accepted by the Graduate School, in partial fulfillment of requirements for the degree of J u l i e Block M lle tie n D O C T O R OF P H I L O S O P H Y D m * J a n u a r y , 1 9 6 8 DISSERTATION COMMITTEE ACKNOWLEDGMENTS I w ish to th a n k th e N a tio n a l I n s t i t u t e s o f H e a lth f o r f i n a n c i a l a id d u rin g my te n u re a s a s tu d e n t, th e U n iv e rs ity o f S o u th ern C alifoi> - n ia Computer S c ien ce s L a b o ra to ry and i t s s t a f f , th e members o f my com m itte e , and, e s p e c ia lly , my husband S h eldon, who made me c o n tin u e when I w anted to q u i t . ii TABLE OF CONTENTS Page A CK N O W LED G M EN TS i i LIST OF TABLES iv LIST OF ILLUSTRATIONS Vi INTRODUCTION: a-CHYMOTRYPSIN 1 The Cfr-Chymotrypsin R e a c tio n Model S tu d ie s C la s s ic a l G eneral B a sic C a ta ly s is v s . N u c le o p h llic C a ta ly s is Hie K in e tic s o f th e C&-ChymotrypBin R ea ctio n R eagents S u b s tr a te s B u ffe rs Enzyme K in e tic M easurem ents C a lc u la tio n s The Im id a z o le -C a ta ly z e d H y d ro ly sis o f E s te r s o f N -A cety lserin am id e The Im id a z o le -C a ta ly z e d H y d ro ly sis o f E s te r s o f p -N itro p h e n o l The D eacylatT on o f Acyl-Gfr-chym otrypsins The A c y la tio n o f O-ChymotrypBin by E s te r s o f p -N itro p h e n o l MATERIALS A N D M ETHODS 8 RESULTS 19 DISCUSSION OF RESULTS 81 * S te r ic E f f e c ts A c tiv a tio n P a ra m eters The Enzyme Mechanism Summary REFERENCES CITED 10k i l l LIST OF TABLES T a tle Page 1 . Tem perature Dependence o f pH o f Im id azo le B u f f e r s .................. 11 2 . E x tin c tio n C o e f f ic ie n ts o f p -N itro p h e n o l In V ario u s B u ffe rs ............................................................................................. 12 3 . P s e u d o -F irs t-O rd e r R ate C o n sta n ts f o r th e Im id a z o le - C a ta ly z e d H y d ro ly sis o f E s te r s o f N -A cety lserin am ld e . . 22 Second-O rder R ate C o n stan ts f o r Im id azo le C a ta ly s is and P s e u d o -F irs t-O rd e r R ate C o n stan ts f o r S pontaneous H y d ro ly sis o f N -A cety lserin am id e E s te r s . . . . . . . . . . 24 5 . T a ft A lip h a tic S t e r i c C o n s t a n t s ............................................................ 25 6 . A c tiv a tio n P aram eters f o r th e Im id a z o le -C a ta ly z e d H ydrolysiB o f N -A cety lserin am id e E s t e r s ..................................... 31 7 . P se u d o -F irs t-O rd e r R ate C o n stan ts f o r th e Im id a z o le - C ataly ze d H y d ro ly sis o f p -N itro p h e n y l E s te r s ........................ 33 8 . Second-O rder R ate C o n stan ts f o r th e Im id a z o le - C ataly ze d H y d ro ly sis o f p -N itro p h e n y l E s te r s ........................ 35 9 . A c tiv a tio n P aram eters f o r th e Im id a z o le -C a ta ly z e d H y d ro ly sis o f p -N itro p h e n y l E s t e r s ............................................... 38 10. R ate C o n stan ts f o r th e D e a c y la tio n o f Acyl-Ct- chym o try p sin s a t V ario u s pH V a l u e s ............................................... 40 11. V a ria tio n o f R ate o f D e a c y la tio n o f Acyl-Qt-chymo- tr y p s in e w ith T r is B u ffe r C o n c e n tra tio n s ................................. k2 12. R ate C o n sta n ts f o r th e D e a c y la tio n o f Acyl-C&-chymo~ tr y p s ln s a t V ario u s T em peratures ................................................... 47 1 3 . pH and T em perature Dependence o f th e R ates o f D e a c y la tio n o f Some A c y l-C t-c h y m o try p s in s ................................. 48 14. T em perature Dependence o f th e I o n iz a tio n C o n sta n ts in th e D e a c y la tio n o f Some A c y l-C t-c h y m o try p sin s................... 49 15. A c tiv a tio n P a ra m e te rs f o r th e D e a c y la tio n o f Acyl-Ct- ch ym otrypsins .................................................................................................. 50 lv Table Page 16 . T em perature Dependence o f th e B ate o f D e a c y la tio n o f A cyl-O t-chym otrypsins In T ria B u ffe r . . . . . . . . . 56 17. A c tiv a tio n P a ra m e te rs f o r th e D e a c y la tio n o f Acylr- as~chym otryps±ns, D eterm ined in T rls B u f f e r ........................... 57 18. P s e u d o -P lrs t-O rd e r B ate C o n sta n ts f o r th e A c y la tlo n to o f O -C hym otrypslns by p -N itro p h e n y l E s te r s In 2 5 . pH 5 .9 2 , P h o sp h ate B u ffe r; pH 6 .1 9 / P h o sp h ate B u ffe rj pH 7 P h o sp h ate B u ffe r; pH 7*58) P h o sp h ate B u ffe r; pH 7 -9 9 / P h o sp h ate B u ffe r; pH 7*99/ T r ls B u ffe r; pH 8 .1 2 , T r ls B u ffe r; pH 8 .9 0 , T r ls B u f f e r .............................. 60-73 26 . Second-O rder B ate C o n sta n ts f o r th e A c y la tlo n o f a-C h y m o try p sln by p -N itro p h e n y l E s t e r s ...................................... 76 2 7 . T h e o r e tic a l pK V alu e s, pH O ptim a, and pH -Independent B ate C o n sta n ts f o r th e A c y la tlo n o f O -Chym otrypsln by p— N itro p h e n y l E s t e r s ........................................................................... 80 2 8 . B ate C o n sta n ts E x tra p o la te d to 95° f o r th e D e a c y la tio n o f A c y l-o t-c h y m o try p sln s ........................................................................... 85 2 9 . Com parison o f B e la tiv e B ate B a tio s f o r th e A c y la tlo n and D e a c y la tio n o f O -Chym otrypsIn by p -N itro p h e n y l E s t e r s ............................................................................... 90 v LIST OF ILLUSTRATIONS Figure Page 1 . P lo t o f kobga f o r th e Im id a z o le -C a ta ly z e d H y d ro ly sis o f N -a c e ty l-O -b u ty ry ls e rin a m id e v s . th e T o ta l C o n c e n tra tio n o f I m i d a z o l e ................................................................. 20 2 . T a ft P lo t f o r th e Im id a z o le -C a ta ly z e d H y d ro ly sis o f E s te r s o f N -A cety lserin am id e a t 9 5 ° ............................................... 2 7 3 . T a ft P lo t f o r th e Spontaneous H y d ro ly sis o f E s te r s o f N -A cety lserin am id e a t 9 5 * ............................................................. 29 4 . T a ft P lo t f o r th e H y d ro ly sis o f p -N itro p h e n y l E s te r s a t 3 ° ° .................................” ........................................................... 57 5 . T a ft P l o t f o r th e D e a c y la tio n o f Acyl-05- chym o try p sin s a t 2 3 ° ............................................................................... ^ -5 6 . P lo t o f A H * v s . A S * / U n c o rre c te d / f o r th e D e a c y la tio n o f A c y l-C t-c h y m o try p sin s............................................... 52 7 . P lo t o f A H * v s . A S* / C o rre c te d / f o r th e D e a c y la tio n o f A c y l-c s-c h y m o try p s in s............................................... 5^ 8 . E ad ie P lo tB f o r th e A c y la tlo n o f Ct-Chymotrypsin hy p -N itro p h e n y l E s t e r s ........................................................................... 59 9 . Second-O rder P lo t f o r th e A c y la tlo n o f Ct-Chymotrypsin hy p -N itro p h e n y l B u t y r a t e ................................ "jk 10. pH-Rate P r o f i l e f o r th e A c y la tlo n o f a-C hym otrypsin by p -N itro p h e n y l A c e t a t e ...................................................................... Ql 11. T a ft P lo t f o r th e A c y la tlo n o f Ct-Chymotrypsin by p -N itro p h e n y l E s t e r s , 2 5 ° ..................................................................... 82 12. T a ft P lo t f o r th e D e a c y la tio n o f Acyl-Ct-chymo- t r y p s i n s , 9 5 ° ....................................................... 86 vi INTRODUCTION: C M 2H Y M Q TRYPSIN The Ct-Chym otrypsin R e a c tio n The mechanism o f a c tio n o f th e p r o te o ly t i c and e s t e r a t i c enzyme, O -ch y m o tiy p sin , h as r e c e iv e d much a t t e n t i o n . C a ta ly s is o f th iB enzyme a p p e a rs t o in v o lv e th e fo rm a tio n o f an acyl-enzym e w ith a l l s u b B tra te s s tu d ie d (1- 3 )* The acyl-enzym e p r o b a b l y c o n ta in s a c o v a le n t lin k a g e betw een th e a c y l group o f th e s u b s tr a te and th e s e r in e h y d ro x y l a t th e a c tiv e s i t e (1 4 — 6 ) . The a c y la tio n and d e a c y la tio n o f t h i s s e rin e a r e p ro b a b ly a id e d by an im id a zo le r e s id u e w hich h a s been shown to be p r e s e n t a t th e a c tiv e s i t e ( 7 ~ l l ) . The pH dependence o f th e r a t e con s t a n t s , show ing th e inv o lv em en t o f an io n iz a b le group w ith a pK o f 6 . 5- 7 .5 (12, 1 3 ), i s c o n s is te n t w ith th e involvem ent o f im id a z o le . Thus, th e aim o f many s tu d ie s in th e p a s t s e v e r a l y e a rs h a s been to d e te rm in e , th ro u g h th e use o f sm all o rg a n ic m odel compounds, i f Im id az o le can f u n c tio n a s a c a t a l y s t in e s t e r and am ide h y d r o ly s is , an d , i f so , how e f f e c t i v e l y . Model S tu d ie s Im id azo le h a s been o b serv ed to a c t a s a c a t a l y s t i n e s t e r hy d r o ly s is ( 6 , l 4 - l 8 ) . However, im id a zo le may f u n c tio n a s a c a t a l y s t by two d i f f e r e n t m ethods: c l a s s i c a l g e n e ra l b a s ic c a t a l y s i s (6 , 16, 1 7 ) , in w hich th e Im id azo le a b s tr a c t s a p ro to n from w a te r in th e t r a n s i t i o n 1 s t a t e j and n u c le o p h ilic c a t a l y s is (1 4 ,1 5 ,1 8 ), In -which Im id azo le fu n c t i o n s a s a n u c le o p h ile to form an a c y l-im id a z o le -which th e n b re a k s down t o p ro d u c ts . B ecause o f t h i s d u a l fu n c tio n o f Im id a z o le , many s tu d ie s have been made to d eterm in e which o f th e s e r o le s Im idazole p la y s in th e enzym atic mechanism. th e h y d r o ly s is o f N ,0 -d ia c e ty ls e rin a m id e in im id azo le b u f f e r s a s shown by A nderson, e t a l . (6 ) . T h is r e a c tio n a ls o s e rv e s a s a good m odel f o r th e d e a c e ty la tio n o f acetyl-Cfc-chymo t r y p s i n . The t r a n s i t i o n s t a t e f o r t h i s r e a c tio n in v o lv e s p ro to n t r a n s f e r to im id a z o le . th e h y d r o ly s is o f p - n itro p h e n y l a c e ta te in im id azo le b u f f e r s a s s tu d ie d by Bender and T u rn q u est ( l4 ) and by B ru ice and Schm ir (15)* In t h i s c ase th e t r a n s i t i o n s t a t e in v o lv e s a tta c k by im id azo le d i r e c t l y a t th e ca rb o n y l carb o n . A r e a c tio n In v o lv in g im id az o le a s a c l a s s i c a l g e n e ra l b ase i s y NH-C-CH C Ho-C-O-CHo-CHC-NH A r e a c tio n in v o lv in g im id azo le a s a n u c le o p h ilic c a t a l y s t i s 5 o * - c ^ - ° H O hN0: C la s s ic a l G en eral B asic C a ta ly s is v s . N u c le o p h ilic C a ta ly s is A g r e a t d e a l o f e f f o r t h a s been expended to d i f f e r e n t i a t e be tw een th e s e two ty p e s o f c a t a l y s i s . The b e s t ev id en ce f o r th e e s ta b lish m e n t o f n u c le o p h ilic c a t a l y s i s i s th e o b s e rv a tio n o f a c o v a le n t a c y l-n u c le o p h lle in te rm e d ia te . In e s t e r h y d r o ly s is in sim ple ch em ical sy stem s, t h i s m ethod 1b p r a c tic a b le . In f a c t , i n th e h y d r o ly s is o f g - n itr o p h e n y l a c e ta te a s c a ta ly z e d by im id a z o le , N -a c e ty llm id a z o le h as a c t u a l l y been is o l a t e d a s an in te rm e d ia te (1 9 ). I t i s a ls o p o s s ib le to o b serv e th e fo rm a tio n and breakdown o f N -a c e ty llm ld a z o le sp e c tro p h o to m e trie s H y a t 2k$ mi*, a w av elen g th a t w hich a c y lim id a z o le s ab so rb ( l ^ ) . However, i n th e en zy m atic r e a c tio n , th e s i t u a t i o n i s more c o m p lica te d . Dixon and N eurath (20) re p o rte d a r a p id in c re a s e in ab so rb an ce a t 2k$ mil on a c y la tlo n o f th e enzyme. The k i n e t i c s o f th e ap p earan ce and d isa p p e a ra n c e o f t h i s 2 mil ab so rb an ce seemed to a g re e w ith th o s e f o r th e enzym atic r e a c tio n . I t was l a t e r shown t h a t th e ab so rb an ce a t 2Vj mil was a s s o c ia te d w ith th e p h y s ic a l s t a t e o f th e p r o te in u nder th e ex p e rim e n ta l c o n d itio n s used (2 1 ) . i f A n o th er m ethod f o r d is tin g u is h in g betw een th e two r o le s o f im id azo le i s th e D*0 s o lv e n t is o to p e e f f e c t . I f c a r r ie d o u t i n DgO, a r e a c t i o n in v o lv in g p ro to n t r a n s f e r in th e t r a n s i t i o n s t a t e w i l l p ro b a b ly p ro c e e d a t a r a t e two t o th r e e tim e s slo w er th a n in w a te r (17/ 2 2 ) . Thus th e c l a s s i c a l g e n e ra l b a se mechanism would p r e d ic t a DjjO s o lv e n t is o to p e e f f e c t , w h ile th e n u c le o p h ilic m echanism w ould n o t (1 4 ,1 8 ). The enzyme r e a c tio n h a s been c a r r ie d o u t in D^O and an lB otope e f f e c t o f two t o th r e e h a s been o b serv ed (2 } ). However, a s p o in te d o u t by Jen ck s (2 4 ), th e p r e d ic te d v a lu e s a re o n ly e m p iric a l and a re s u b je c t to a g r e a t d e a l o f v a r i a t i o n , th u s making th e r e s u l t s d i f f i c u l t to i n t e r p r e t in th e ab sen ce o f o th e r s u b s ta n tia tin g d a ta , e s p e c ia lly when ap p l i e d to enzym atic r e a c tio n s . A t h i r d m ethod o f d is tin g u is h in g betw een c l a s s i c a l g e n e ra l b a s ic c a t a l y s i s and n u c le o p h ilic c a t a l y s is l i e s in th e p a t t e r n o f s t e r i c e f f e c t s (2 5 ). G eneral b ase c a t a l y s is a p p e a rs to be o n ly mode r a t e l y a f f e c te d by in c r e a s in g s t e r i c b u lk i n th e c a ta ly z in g b ase (2 6 ), w h ile n u c le o p h ilic c a t a l y s is o f e s t e r h y d r o ly s is by im id azo le 1b s tr o n g ly r e ta r d e d by s u b s t i t u t i o n o f la r g e g roups in th e 2- p o s itio n o f im id az o le (2 7 ) . There i s a ls o a d if f e r e n c e betw een th e two mechanisms when th e s t e r i c b u lk i s in th e s u b s tr a te r a t h e r th a n in th e c a ta ly z in g b a s e . In c re a s e d a lk y l group b ra n c h in g in th e a c y l group o f p - n i t r o p h e n y l e s t e r s ca u se s a marked d e c re a se in r a t e i n th e n u c le o p h ilic c a t a l y s i s o f th e s e e s t e r s (1 8 ). T h is i s p ro b a b ly due to in c re a s e d d i f f i c u l t y o f a tta c k by th e n u c le o p h ile a t th e e s t e r c a rb o n y l. In th e im id a zo le g e n e ra l b a se c a ta ly z e d h y d r o ly s is o f H -a c y l-im ld a z o le s , b ra n c h in g a t th e a -c a rb o n p ro d u ces l i t t l e e f f e c t on th e h y d ro ly s is r a t e , even c a u sin g a s l i g h t a c c e le r a tio n In th e r a t e I n some c a s e s , ■while "branching a t th e P -c a rb o n d e c re a s e s th e r a t e , b u t t o a sm a ll ex t e n t (1 8 ) . S e v e ra l s tu d ie s have b een made t o d ete rm in e th e e f f e c t on da— a c y la tlo n o f a c y l-c h y m o try p sin s o f changes In th e s t e r i c b u lk o f th e a c y l group (28- 5 2 ) . However, no stu d y o f an e x te n s iv e s e r i e s in c lu d in g b o th s t r a i g h t c h a in and b ran ch ed compounds u nder th e same e x p e rim e n ta l c o n d itio n s had been made. Thus i t v a s d ecid ed to stu d y s t e r i c e f f e c t s on th e r a t e s o f h y d r o ly s is and on th e a c t i v a t i o n p a ra m e te rs In th e f o l low ing r e a c tio n s : (a ) a c l a s s i c a l g e n e ra l b ase c a ta ly z e d r e a c tio n , th e h y d r o ly s is o f e B te rs o f N -a c e ty lse rin a m ld e In im id azo le b u ffe rs } (b) a n u c le o p h ile c a ta ly z e d r e a c tio n , th e h y d ro ly siB o f e s t e r s o f p - n i t r o - p h en o l In im id azo le b u f f e r s ; (c ) th e enzym atic r e a c tio n , th e a c y la tlo n and d e a c y la tio n o f e s t e r s o f p - n itr o p h e n o l. The K in e tic s o f th e Ct-Chymotrypsin R e a c tio n The k i n e t i c s o f th e r e a c tio n o f QS-chymotrypsin w ith p - n i t r o p h en y l a c e ta te have been w e ll s tu d ie d (5>-55)• W ith t h i s s u b s tr a te i t 1b p o s s ib le to o b serv e b o th th e a c y la tlo n and d e a c y la tio n o f th e enzyme by th e s u b s tr a te . The enzym atic r e a c tio n shows a n i n i t i a l " b u r s t” o f 5— n itro p h e n o l fo llo w e d b y a z e r o -o r d e r r e le a s e o f p ro d u c t. T h is behav i o r h a s been in t e r p r e t e d to in d ic a te th r e e s te p s in c lu d in g th e form a t i o n o f th e enzyme— s u b s tr a te com plex, th e rearran g em en t o f t h i s complex to form an acyl-enzym e, and th e breakdown o f th e acy 1-enzyme to p ro d u c ts 6 E + S f : \ ± ES — -» ES1 ■ ■ > E + P *, ( l ) k - i + P i •where E I s th e enzyme, S i s th e s u b s t r a te , p - n itr o p h e n y l a c e t a t e , ES i s th e e n z y m e -su b stra te com plex, ES* i s th e acy l-en zy m e, P*. i s p - n ltr o — p h e n o l, and P£ i s a c e ta te Io n . T h is fo rm u la tio n y ie ld s th e e q u a tio n ( 5 5 ^ 5 ) (ka + E3) (S) + ksK (s) W (3 )0 + K n - (S )c + K m + (2 ) where ( J ) m ^ k . , I s th e p s e u d o - f ir s t- o r d e r r a t e c o n s ta n t f o r th e b u r s t r e a c tio n , obsd x and (S )q i s th e i n i t i a l s u b s tr a te c o n c e n tr a tio n . W ith p - n itr o p h e n y l a c e t a t e , in th e p re se n c e o f an excesB o f s u b s t r a te , kg can be d e te rm in e d d i r e c t l y . I t i s th e z e ro -o rd e r v e lo c i t y d iv id e d by th e enzyme c o n c e n tra tio n (3 6 ) . I f (ka + k 3 ) (S )q » ksK , e q u a tio n (2 ) can be s im p lif ie d to m (kg + k 3 ) (S) k . = — 7 - r -------------£ . (lv) obsd (S) + K v ' 'o m I f t h i s i s th e c a s e , th e d a ta can be a n a ly z e d by an E ad ie p l o t (}7 ) j p l o t t i n g fcol3B(i V£* ^ o b s d /^ ^ o 8 1 1 1 4 K m ^ + k 3 ) from th e s lo p e and th e i n t e r c e p t , r e s p e c tiv e ly . H ien ke can be d eterm in e d sin c e k 3 i s known. I d e a l l y , in o rd e r t o d e term in e K^, th e s u b s tr a te concen t r a t i o n should be v a r ie d from 0 .1 K to ID K . m m F o r p— n itr o p h e n y l a c e t a t e a t low s u b s tr a te c o n c e n tr a tio n s , e q u a tio n (2 ) m ust be u se d . In t h i s c a se an E a d le p l o t o f (k otB(i “ * 3 ) y s . (k . , - k 3 ) /( S ) g iv e s K and ke d i r e c t l y from th e slo p e and th e — ' o b sd o m . i n t e r c e p t , r e s p e c t i v e l y . The s iz e o f th e b u r s t , a s g iv e n by F a l l e r (35 )> I 0 [A ( p - n itr o p h e n o l) ] b u r s t ** (S ), o (kfi + k 3 ) (S )o + k^Cm (E )0 - (5 ) Thus I t i s lim ite d b y th e I n i t i a l enzyme c o n c e n tr a tio n . U nder p se u d o - f i r s t - o r d e r c o n d itio n s , where (S )0/( K ) q m ust be a t l e a s t te n , th e s o lu b i l i t y o f th e B u b s tra te l i m i t s th e ran g e o f s u b s tr a te c o n c e n tr a tio n s w hich nan be u sed a t a n enzyme c o n c e n tr a tio n la r g e enough to show an o b s e rv a b le b u r s t . I n th e c a se o f p - n itr o p h e n y l a c e t a t e , F a l l e r (35) h a s fo u n d t h a t u n d er h i s e x p e rim e n ta l c o n d itio n s s u b s tr a te s o l u b i l i t y i s so low t h a t th e h ig h e s t a t t a i n a b l e s u b s tr a te c o n c e n tr a tio n i s alw ays l e s s th a n K^. F o r t h i s c a s e , an E a d ie p l o t o f e q u a tio n (2 ) w i l l g iv e an i n f i n i t e v a lu e o f K . T h is Im p lie s t h a t th e c o n c e n tr a tio n o f ES i s m so mmj-1 a s t o be e x p e rim e n ta lly u n d e te c ta b le , and th e r e a c tio n m ust be t r e a t e d a c c o rd in g t o B eco n d -o rd er k i n e t i c s . The s e c o n d -o rd e r r a t e c o n s ta n t i s th e r a t i o ka/Km (35>38) . MATERIALS AND M ETHODS R eagents Hie Im id azo le used was from. Eastm an Kodak Company (W hite L a b e l) . A cid c h lo r id e s were e i t h e r co m m ercially o b ta in e d (M ia th e son Coleman and B e ll) o r were p re p a re d from th e com m ercially o b ta in e d c a rb o x y lic a c id s (K & K L a b o ra to rie s ) by r e a c tio n w ith th lo n y l c h lo r id e , w ith th e excep t i o n o f t rim e th y l a c e t y l c h lo rid e (A ld ric h Chem ical Company). A cid an h y d rid e s w ere o b ta in e d from M atheson Coleman and B e ll e x c e p t f o r is o v a l e r i c an h y d rid e (K & K L a b o r a to r ie s ) . S e rin e m eth y l e s t e r hydro c h lo r id e was o b ta in e d from A ld ric h C hem ical Company. Dloxane was p u r i f i e d by th e method o f F ie s e r (59)• A c e t o n i t r il e was Eastm an Kodak S p ectro Qrade w hich was d i s t i l l e d tw ic e o y e r P^Og and once o v e r K 2 C O 3 . S u b s tr a te s N -A eety lserin am id e was p re p a re d from s e r in e m ethyl e s t e r hydro c h lo r id e by th e method o f R o th s te in (ho). N ,0 -D ia c e ty lse rln a m id e and N -a c e ty l-O -trim e th y la c e ty ls e rin a m id e w ere p re p a re d by a d d in g 0.0 5 eq o f th e a c id c h lo rid e dropw ise w ith s t i r r i n g t o 0.05 eq o f N -a c e ty l- serin am id e d is s o lv e d in 85 m l p y r id in e . A f te r r e f lu x in g th e m ix tu re a t room te m p e ra tu re f o r two h o u rs , th e s o lv e n t was e v a p o ra te d u n d er r e duced p r e s s u r e . The re s id u e was w ashed w ith e th e r and r e c r y s t a l l i z e d f i r s t from a c e t o n i t r i l e and th e n from a v e ry sm a ll amount o f 951 > e th a n o l. N ,0 -D ia c e ty lse rin a m id e had up 185- 187°; l i t . ( 6 ), op 157-159°* 8 A n a l. C alcd. f o r CTHj^lfeO*: C, 4 4 .6 7 ; H, 6 .4 3 ; N, 1 4 .8 9 . Found: C, 4 4 .7 7 ; H, 6 .4 8 ; N, 1 4 .7 5 . N -A c e ty l-O -trlm c th y la e e ty ls e rlnamlde h ad mp 191- 194*. A n a l. C alcd . f o r CioHialfeO*: C, 5 2 .1 6 ; H, 7 -8 8 j 1 2 .1 7 . Found: C, 5 1 .9 7 ; H, 8 .0 6 ; N, 1 2 .0 3 . The o th e r se rin a m id e s were p re p a re d In a s im ila r m anne r ex ce p t t h a t th e a c id a n h y d rid e s were u sed r a t h e r th a n th e a c id c h lo r id e s , and b ecau se o f th e d i f f i c u l t y e n c o u n te re d In r e c r y s t a l l i z a t i o n , th e r e s u lt i n g w h ite c r y s t a l s were in s te a d washed s e v e r a l tim e s w ith e th e r . N — A c e ty l-O -b u ty ry lse rin a m id e had mp 106-108°. A n a l. C alcd. f o r C®Hi*N2 0 4 : C, 4 9 .9 9 ] H, 7 .4 6 ; N, 1 2 . 9 6. Found: C, 4 9 . 72; H, 7 .4 1 ; N, 1 2 .6 3 . N-Acety1 -0 -1 s o b u ty ry ls e r In amide had mp I 56- I 58*. A n a l. C alcd . f o r CaHxaNaO,*: C, 4 9 .9 9 ; H, 7*46; N, 1 2 .9 6 . Found: C, 5 0 .1 4 ; H, 7 .6 1 ; N, 1 2 . 7 8 . N -A c e ty l-O -iso v a le ry lse rin a m id e h ad mp 127-130°* A n a l. C alcd. f o r CxoHiaHfiO*: C, 5 2 .1 6 ; H, 7 . 8 8; N, 1 2 .1 7 . Found: c , 5 1 -99; h, 7 . 98; N, 1 2 . 0 1 . S e v e ra l o f th e p - n itro p h e n y l e s t e r s were th e same a s p re v io u s ly s tu d ie d (1 8 ) . p— N1tro p h e n y 1 is o v a le r a te was p re p a re d by th e same m ethod and b o ile d a t 123° a t 1 .5 mm; n^2^*^, 1 .5 1 9 3 ; l i t . ( 4 l ) , bp 158- 160* a t 6 mm. p -N itro p h e n y l t r i e t h y l a c e t a t e was p re p a re d by ad d in g tr i e t h y L - a c e t y l c h lo rid e ( l 6 .6 g , 0 .1 eq) to a s o lu tio n o f p -n itr o p h e n o l ( l4 .2 g , 0 .1 eq ) i n 37 m l p y r id in e (0 .4 6 eq.). Hie m ix tu re was h e a te d f o r one h o u r, th e n poured w ith s t i r r i n g in to ic e w a te r. The w a te r s o lu tio n was w ashed w ith 5$ sodium c a rb o n a te s o lu tio n and th e n e x tr a c te d w ith e t h e r . The e th e r was f la s h e v a p o ra te d , and th e r e s id u a l l i q u i d was d i s t i l l e d . The p ro d u c t was a y e llo w l i q u i d t h a t b o ile d a t 139-140° a t 0 .8 mm; 22 5 njj , 1 .5 1 9 0 . The m a te r ia l s o l i d i f i e d when p la c e d in th e c o ld and 10 m elte d a t * 4 - 5 — *46.5"* A n a l. C alcd . f o r C, 6 5 . 58; H, 7*22; N, 5 .2 8 . Found: C, 6 5 .0 5 ; H, 7*25J N, 5-*46. p -N itro p h e n y l h ex an o ate 2 2 .5 was p re p a re d In a s im ila r m anner and had bp 155- 15$* a t 0 .8 mm; n^ * , 1 . 5178J H t . (4 1 ), bp 1714- 175* a t 6 mm. B u ffe rs Im id azo le b u f f e r s f o r th e h y d r o ly s is o f e s t e r s o f N - a c e ty ls e r in - amide w ere made up a t th r e e b u f f e r r a t i o s to 1 .0 M In KC1. S e r i a l d i l u t i o n s w ere made w ith 1 .0 M KC1. The pH o f each b u f f e r s o lu tio n was m easured on a R adiom eter Model 22 pH m eter a t v a rio u s te m p e ra tu re s In o rd e r to d eterm in e th e pH v a lu e s a t 95° by e x tr a p o la tio n . These pH v a lu e s a re g iv en I n T able 1. The v a lu e s f o r th e r a t i o s (K& /(K & + a ^ ) ) f o r each b u f f e r were d eterm in e d i n th e fo llo w in g way. U sing th e known v a lu e o f p K ^ a t 50% 7 .1 0 (*42), and th e pH o f each b u f f e r a t 50% th e r a t i o (Ka/(K R + a ^ ) was d eterm in ed f o r each b u f f e r a t 50*. T h is r a t i o was assum ed to be te m p e ra tu re In d e p e n d e n t, and was u sed a t each te m p e ra tu re f o r th e b u f f e r f o r w hich i t was c a lc u la te d . The r a t i o (Ka /(K & + a ^ ) ) f o r each b u f f e r i s a ls o g iv en i n T able 1 . The Im id azo le b u f f e r u sed i n th e stu d y o f th e h y d r o ly s is o f th e p - n itr o p h e n y l e s t e r s was made up a t a b u f f e r r a t i o o f 5 **495:1* The pH v a lu e s a t th e te m p e ra tu re s s tu d ie d w ere m easured d i r e c t l y on a Model 22 R adiom eter pH m e te r. The pKR v a lu e s a t th e s e te m p e ra tu re s w ere de term in e d d i r e c t l y , ta k in g th e v a lu e a t 50° a s 7*10 (*42). These de term in e d v a lu e s w ere a s fo llo w s : 2 0 ° , 7 -2 6 ; *40*, 6.9*4; 5 0 °, 6-73* The r a t i o (K& /(K & + a ^ ) ) was found t o be te m p e ra tu re In d e p e n d e n t, a s a s sumed above. 11 TABLE 1 TEMPERATURE DEPENDENCE OP pH OF IMIDAZOLE BUFFERS A N D (Ka/(K & + Hg) FOR EACH BUFFER A pproxim ate B u ffe r R a tio T em perature ( • c . ) pH pH a t 95* (KaAKa + aH)) 1 :1 30 6.92 5.72 0.751 4o 6.75 50 6.55 75 6 .0 1 4 :1 30 7.43 5 .9 7 0 .6 8 1 40 7.24 50 6 .9 8 75 6.45 5 :1 30 7.58 6 .3 0 0.398 4o 7-35 50 7.19 75 6 .6 3 12 P o tassiu m p h o sp h ate b u f f e r s v e re made up a t an Io n ic s tr e n g th o f 0.075 M, u sin g a nomogran (4 5 ). T ris-H C l b u f f e r s v e re made up a t an Io n ic s tr e n g th o f 0.075 M. P h o sp h ate b u f f e r s v e re u sed f o r pH 5 *88- 7 .9 9 IQ th e enzyme s tu d ie s , and T r ls b u f f e r s from 7 -9 9-8*90. E x tin c t i o n c o e f f ic ie n ts v e re d eterm in e d f o r th o s e b u f f e r s uBed In th e deacyla^- t l o n stu d y u n d er e x p e rim e n ta l c o n d itio n s a n d a re g iv en In T able 2 . TABLE 2 EXTINCTION COEFFICIENTS OF p-NITHOPHENOL OR p-NITROPHENOIATE ION IN VARIOUS BUFFERS8 , B u ffe r pH A « x 104 P hosphate 5 .8 8 330 0 .8 5 1 P h o sp h ate 6 .2 0 It 0.762 P hosphate 7 .2 8 400 1 .1 6 P hosphate 7*37 tt I .1 9 P hosphate 7 *66 ff 1.46 T r ls 8 .8 0 It 1 .6 7 T r ls (0 .1 M) 8 .8 0 It 1 .6 8 T r ls (0.2 M) 8 .8 0 tl 1 .7 1 T r ls (0 .5 M) 8 .8 0 t t 1.73 an = O.075 M, 25.0® , 1 .6 # a c e t o n i t r i l e . Enzyme Ct-Chymotrypsinji 5X c r y s t a l l i z e d , v a s o b ta in e d from W orth in g to n B iochem ical C o rp o ra tio n . The enzyme v a s made up to 2 6 .7 m g/ml, 10 ml a t a tim e , in pH 5*05 a c e ta te b u f f e r , p. = 0 .1 M. The o p e r a tio n a l n o r 13 m a lity o f th e enzyme s o lu tio n s v a s d eterm in ed by th e t i t r i m e t r i c p ro c e d u re o f Schonbaum, e t a l . (M+) a s 8 X 10”* 4 N. The n o rm a lity , N, a t 310 m y. v a s ,a °'9&9 Aa + A* - A3 . . N - T*oi7f ' ' v h e re Ag i s th e ab so rb an ce o f .t h e s u b s tr a te a lo n e , A* i s th e ab so rb an ce o f th e enzyme a lo n e , and A3 i s th e ab so rb an ce a f t e r a c y la tio n o f th e enzyme i s co m p lete. Enzyme p r e p a r a tio n s v e re fo u n d t o have 7 0 * 80$ a c t i v i t y b ase d on a m o le c u la r w eig h t o f 2^ ,800 f o r O -chym otrypsin. K in e tic M easurem ents H y d ro ly sis o f N -A cety lserln am id e E s te r s by Im id azo le The r a t e s o f th e im id a z o le -c a ta ly z e d h y d ro ly s is o f e s t e r s o f N -a c e ty lse rin a m id e v e re fo llo w e d b y th e hydroxam ate m ethod u sed by B ru lc e and Bruno (4 2 ). One m l o f th e a p p r o p ria te Im id azo le b u f f e r s o lu tio n , 0.015 M i n e s t e r , v a s p i p e t t e d in to each o f te n P y rex tu b e s . These v e re t i g h t l y c lo s e d v l t h T e flo n - lin e d screw caps and p la c e d in a Haake NBe 657^6 U ltr a - th e r m o s ta t, w hich m a in ta in e d th e te m p e ra tu re to + 0 .1 ° . Tubes w ere w ithdraw n a t i n t e r v a l s . The r e a c tio n v a s quenched i n i c e , and t o each tu b e v a s added two m l o f f r e s h ly p re p a re d b u ffe re d hydroxy 1 amlne s o lu tio n , c o n s is tin g o f one p a r t 28$ hydroxylam ine s o lu t i o n in v a t e r , one p a r t 14$ NaOH, and two p a r t s b u f f e r v h ic h v a s fo u r p a r t s 0 .1 M sodium a c e ta te and one p a r t 0 .1 M a c e t i c a c id . The tu b e s v e re th e n re p la c e d in th e b a th f o r fo rm a tio n o f hydrox&mic a c id . A f te r an a p p r o p ria te le n g th o f tim e th e y v e re p la c e d in i c e , and to each tu b e v a s added two ml o f v a t e r , one ml o f 3 N HC1, one ml o f 5$ 7 eC l3 in 14 0 .1 N HC1. The c o lo r was re a d a t 5^0 q i on a Z e is s FM Q I I sp e c tro p h o to m e te r . Each r a t e v a s m easured In d u p lic a te . D u p lic a te v a lu e s ag ree d to + 5$. Developm ent tim e v a s d eterm in e d f o r each r e a c tio n u n d er ex p e rim e n ta l c o n d itio n s . A t th e end o f each k i n e t i c ru n , th e s o lu tio n v a s checked f o r ^ - e lim in a tio n hy m easu rin g th e ab so rb an ce a t 2 4 l mp. / The amount o f e lim in a tio n v a s alw ays l e s s th a n 5$ and u s u a lly l e s s th a n 2 $, ta k in g a m o lar e x tin c tio n o f 5000 f o r th e d eh y d ro a la n in e d e r iv a tiv e (4 5 ). H y d ro ly sis o f p -N ltro p h e n y l E s te r s by Im id azo le The im id a z o le -c a ta ly z e d h y d r o ly s is o f th e p - n ltro p h e n y l e s t e r s v a s fo llo w e d a t 400 mp on a Z e is s PM Q I I sp e c tro p h o to m e te r In im id azo le b u f f e r s . C o n stan t te m p e ra tu re to + 0 .1 ° v a s m a in ta in e d w ith a p r e c i s io n S c i e n t i f i c L o-T em ptrol l^lf c i r c u l a t in g te m p e ra tu re b a th and a Z e is s c o n s ta n t te m p e ra tu re c e l l - h o ld e r . To 5 .0 ml o f th e a p p r o p ria te p r e - e q u ilib r a te d b u f f e r s o lu tio n v a s added e x a c tly 0 .0 5 ml o f th e e s t e r s o lu tio n in d io x an e, so t h a t th e r e a c tio n m ix tu re was a lv a y s 1 .6 4 $ d io x a n e . The m ix tu re was th e n s t i r r e d v ig o ro u s ly . F in a l c o n c e n tra tio n o f e s t e r in th e c u v e tte v a s ab o u t 10"* M. Each r a t e v a s m easured in t r i p l i c a t e . T r i p l i c a t e v a lu e s g e n e r a lly shoved an av erag e d e v ia tio n o f + 5$* The pH o f each s o lu tio n v a s m easured b e fo re and a f t e r each k i n e t i c ru n on a Model 22 R adiom eter pH m eter and v as found to rem ain c o n s ta n t. 15 D e a c y la tio n o f Acyl-CS-chymotrypBlnB The r a t e s o f d e a c y la tio n o f th e acyL-C t-chym otrypsins w ere ob ta in e d by fo llo w in g th e ap p earan ce o f p - n itr o p h e n o l on a Z e is s PM Q 11 o r G ilfo rd M odel 2000 re c o rd in g sp e c tro p h o to m e te r a t 350 mu o r o f p - n itro p h e n o la te io n a t ^00 mp.. C o n sta n t te m p e ra tu re (+ 0 .1 ° ) was main— ta ln e d d u rin g th e k i n e t i c ru n s by c i r c u l a t i n g w a te r from a Haake Model F c i r c u l a t in g b a th th ro u g h & Z e is s c o n sta n t te m p e ra tu re c e ll- h o ld e r o r from a P r e c is io n S c i e n t i f i c L o-T em ptrol 15 ^ c i r c u l a t i n g w a te r b a th aro u n d th e c e l l com partm ent o f th e Beckman D U component o f th e G ilfo rd . In a l l r a t e s m easured on th e G ilfo rd in s tru m e n t, te m p e ra tu re was moni to r e d c o n s ta n tly by m»*nB o f th e te m p e ra tu re probe s u p p lie d w ith th e in s tr u m e n t. E s t e r s to c k s o lu tio n s w ere made up in a c e t o n i t r i l e a t a con s t a n t c o n c e n tr a tio n o f J X 10- 3 f o r a l l e s t e r s so t h a t " s u b s tr a te a c t i v a t i o n '1 e f f e c t s (3^ , 3 8 ) were m inim al. A v a r i a t i o n o f s u b s tr a te con c e n tr a tio n tw o - t o f o u r f o ld gave no s i g n i f i c a n t change in th e r a t e c o n s ta n ts . The r e a c tio n c u v e tte c o n ta in e d 0 .0 5 ml each o f e s t e r and enzyme s o lu tio n s in a f i n a l volume o f 3*1 ®1 b e in g 1.6$ a c e t o n i t r i l e . The r a t i o o f (S )q/ ( e ) q was betw een th r e e and f o u r . A p p ro p ria te c o rre c t i o n s w ere made f o r sp o n tan eo u s h y d r o ly s is o f th e e s t e r w hich was neg l i g i b l e e x c e p t a t h ig h e r te m p e ra tu re and pH. R ates w ere m easured in t r i p l i c a t e a t each pH and te m p e ra tu re , and t r i p l i c a t e v a lu e s showed an a v erag e d e v ia tio n o f + 5$. pH was found t o rem ain c o n s ta n t th ro u g h o u t each ru n . 16 A c y la tlo n o f Ct-Chymotrypsin b y p -JH tro p h e n y l E a te rs The r a t e s o f a c y la tlo n o f O -chym otrypbIn by th e p -n ltro p h e n y l e s t e r s v e re fo llo v e d a t 330 o r 400 mji on a Z e is s P M C J XI spectrophotom e t e r eq uipped w ith a Brush m odel No. IU>-^6l^-00 h ig h g a in a m p lif ie r , a B rush model No. RI>-23 2 1 -0 0 /S-2020 o s c illo g r a p h , and a s to p p e d -flo v ap p a r a tu s a s d e s c rib e d by F ren c h , e t a l . (4 6 ). C o n stan t te m p e ra tu re to + 0 .1 * was m ain ta in e d by c i r c u l a t in g v a t e r from a Haake Model F c ir c u l a t i n g b a th o r a P r e c is io n S c i e n t i f i c L o-T em ptrol 154 c i r c u l a t in g v a te r b a th th ro u g h th e b r a s s th e rm o s ta t b lo c k o f th e s to p p e d -flo v a p p a ra tu s . S u b s tra te and enzyme s o lu tio n s v e re made up in p re v io u s ly de g a ssed b u f f e r so t h a t th e f i n a l (S)q/(E )q r a t i o a f t e r d i l u t i o n in th e m ixing chamber v a s g r e a te r th a n te n in o rd e r to m a in ta in p s e u d o - f i r s t - o r d e r c o n d itio n s . S u b s tra te and enzyme s o lu tio n s v e re each made up i n 4 . 68$ a c e t o n i t r i l e in o rd e r t o a v o id s c h lie r e n e f f e c t s on m ix in g . Thus th e f i n a l c o n c e n tra tio n o f a c e t o n i t r i l e in th e o b s e rv a tio n chamber v as 4 . 68$ . A p p ro p ria te c o r r e c tio n s v e re made f o r spo n tan eo u s h y d ro ly s is o f th e e s t e r s . C a lc u la tio n s The d e a c y la tio n r a t e c o n s ta n ts v e re c a lc u la te d from th e slo p e s o f th e s te a d y s t a t e p l o t s o f ab so rb an ce v s . tim e by c o n v e rtin g to p - n itro p h e n o l c o n c e n tra tio n u n i t s u sin g th e d eterm in ed e x tin c tio n co ef f i c i e n t s in T able 2 and d iv id in g by th e enzyme c o n c e n tra tio n . P s e u d o - f ir s t- o r d e r r a t e c o n s ta n ts f o r th e b u r s t r e a c tio n v e re d eterm in e d by th e m ethod o f F a l l e r (35)* From e q u a tio n ( l ) , th e r e s u l t a n t e x p re s s io n f o r th e r e le a s e o f p - n ltr o p h e n o l i s (p -n itro p h e n o l) * A t + B(1 - e”^ ) . 17 (7 ) A i e a c o n s ta n t v a lu e e q u a l to th e r a t e o f p ro d u c tio n o f p -n itr o p h e n o l a f t e r th e e x p o n e n tia l term i n (7 ) h a s decayed) t h a t i s , when th e b u r s t r a t e i s o v e r. A a ls o in c lu d e s n o n -en zy m atic h y d r o ly s is and i n s t r u m e n ta l d r i f t . When A t i s s u b tr a c te d from e q u a tio n (7 ) / th e rem ain in g curve i s d e s c rib e d by th e fo llo w in g e q u a tio n : B i s th e v a lu e o f th e curve on decay o f th e e x p o n e n tia l, o r ( p - n i t r o - p h e n o l)^ . In p r a c tic e th e v a lu e o f (OD)^ r a t h e r th a n B i s d e te rm in e d . A p l o t o f ab so rb an ce (which i s p r o p o r tio n a l to th e c o n c e n tra tio n o f p - n itr o p h e n o l r e le a s e d ) v s . tim e was made. The s t r a i g h t l i n e p o r tio n o f th e curve v a s e x tr a p o la te d to zero tim e . T h is v a lu e v a s ta k e n as O liv etti-U n d erw o o d Programma 101 w hich v as programmed to do r e g re s s io n an d c o r r e l a t i o n a n a ly s is . The o u tp u t o f i n t e r e s t c o n s is te d o f th e re g r e s s io n c o e f f i c i e n t ( r a t e c o n s ta n t) o f In [ (0D^ - OD^)/ (OD^ - OD^) ] v s . tim e , th e i n te r c e p t o f th e lo g f u n c tio n v s . tim e , th e c o r r e la tio n c o e f f i c i e n t , and th e s ta n d a rd e r r o r o f th e e s tim a te . rith m of th e r a te c o n sta n ts v s . E , th e T a ft B te ric e f f e c ts c o n sta n ts — s (^ 7 ), v e re c a lc u la te d on th e O liv e tti-U n d e rw o o d Programme 101 u s in g a s im ila r l i n e a r r e g r e s s io n program . The o u tp u t i n t h i s case c o n s is te d o f th e r e g r e s s io n c o e f f i c i e n t , th e i n t e r c e p t , th e c o r r e l a t i o n c o e f f i c i e n t , and th e s ta n d a rd e r r o r o f th e e s tim a te . (8 ) A ll p s e u d o -firs t-o rd e r r a te c o n sta n ts v e re determ ined on an A ll se c o n d -o rd e r r a t e c o n s ta n ts a s w e ll a s p l o t s o f th e lo g a - 18 A c tiv a tio n e n e rg ie s were c a lc u la te d , on a H oneyw ell 800 o r an O liv etti-U n d erw o o d P ro gramma 101 by means o f a program d esig n e d to c a r ry o u t l e a s t sq u a re s e v a lu a tio n o f th e lo g a rith m o f th e r a t e con s ta n t v s , l/T °K . The e r r o r s r e p o rte d In A H * and A S* w ere c a lc u la te d from th e s ta n d a rd e r r o r o f th e e s tim a te . RESULTS The Im id azo le— C a ta ly z e d H y d ro ly sis o f E s te r s o f K -A cety lserin am ld c I t was f i r s t n e c e s s a ry to d eterm in e t h a t th e b a s ic s p e c ie s o f Im id azo le was th e a c tiv e form o f th e c a t a l y s t f o r th e h y d r o ly s is o f each o f th e e s t e r s o f B -a c e ty ls e rin a m id e . U lIs was acco m p lish ed by ch an g in g th e r a t i o o f th e b a s ic form to th e a c id form o f Im id az o le a t 9 5 °, and th e n p l o t t i n g th e o b serv ed p s e u d o - f ir s t- o r d e r r a t e c o n s ta n ts v s . th e t o t a l im id az o le c o n c e n tra tio n . A t y p i c a l p l o t I s shown In F ig u re 1 . I t can be seen t h a t th e r a t e in c r e a s e s a s th e pH i s in c re a s e d ; th e re fo re ,, i t 1b th e b a s ic form o f im id az o le t h a t i s c a t a l a t - l c a l l y a c tiv e . Each compound was s tu d ie d a t two b u f f e r r a t i o s a t 95* ex c e p t f o r th e tr im e th y la c e ta te e s t e r . T his e s t e r was s tu d ie d a t o n ly one pH b ecau se o f th e extrem e slow ness o f th e r a t e s and th e sm a ll t o t a l O D c h a n g e.^ The iB O v a le ry l e s t e r showed c u rv a tu re a t th e h ig h pH s tu d ie d , b r a n c h in g in th e a c y l group seemed to g iv e s m a lle r t o t a l O D changes i n th e r e a c tio n . T h is was p resu m ab ly p a r t l y due to s t e r l c h in d ra n c e to th e a t t a c k o f hydroxylam ine a t th e c a rb o n y l. A lso , a t th e e le v a te d te m p e ra tu re s a t w hich th e stu d y was done, th e r e was some F eC l3 c o lo r even in th e b la n k s o lu tio n o f N -a c e ty ls e rin a m id e . The amount o f t h i s c o lo r In c re a s e d w ith th e lo n g e r developm ent tim e s r e q u ire d f o r th e more h ig h ly b ran ch ed compounds. T h is c o lo r was assum ed t o be cau sed by hydroxy lam ine a t t a c k a t th e amide carb o n s end was co r r e c te d f o r in a l l r e a c tio n s . 19 20 8.0 c E C O o X — X -Q pH 5.72 -\ 1.0 (Im). F ig . 1 . —P lo t o f fc0tjB ( j _ ^o r In d -d a z o le -c a ta ly z e d h y d r o ly s is o f K -a c e ty l-O -b u ty iy ls e rin a a iid e y b . th e t o t a l con c e n t r a t Ion o f im id a zo le (im + ImH+ ) a t 9 5 °. 21 and so th e s e d a ta a re n o t In c lu d e d . The p s e u d o - f ir s t- o r d e r r a t e con s t a n t s f o r compounds a t a l l te m p e ra tu re s s tu d ie d a re g iv en in T able 3 . To d eterm in e th e s e c o n d -o rd e r r a t e c o n s ta n ts f o r Im id azo le c a t a l y s i s , kotB(^ v a s p l o t t e d v s . (lm )g / w here ( l» ) B i s th e c o n c e n tra t i o n o f Im id azo le In th e b a se form . T h is I s d eterm in ed by e v a lu a tio n o f th e e x p re s s io n (lm )^(K a/(K a + a ^ ) ) w here (im )^ I s th e t o t a l Im id azo le c o n c e n tra tio n o f th e b u f f e r , and K& /(K a + a ^ ) was d eterm in ed a s p re v io u s ly d e s c rib e d (T able l ) . The B econd-order r a t e c o n s ta n ts a re th e s lo p e s o f th e r e g r e s s io n l i n e s . They a re th e av erag e v a lu e s In th e c a se s where th e r a t e s w ere o b serv ed a t two pH*s. They a re g iv e n In T able 4 . The in t e r c e p t s a t zero Im id azo le c o n c e n tra tio n co rresp o n d to t h a t p o r tio n o f th e o b serv ed r a t e due to sp o n tan eo u s h y d r o ly s is by w a te r and h y d ro x id e Io n , k . The v a lu e s o f k p re s e n te d In T able 4 a re B S th o s e d eterm in e d a t pH 6 .30 ( a t 93*)t e x c e p t f o r th e is o v a le r a te e s t e r , w hich was d eterm in ed a t pH s in c e i t co u ld n o t be d eterm in e d a t pH 6 .3 0 , a s m entioned above. The v a lu e b o f k a r e re p o rte d a t o n ly one pH w here p o s s ib le In o rd e r t o a v o id any d is c re p a n c ie s w hich may o c cu r I f th e h y d ro x id e Io n r a t e c o n s ti t u t e s a la r g e p o r tio n o f k . 8 The norm al s t e r i c o rd e r o f r e a c t i v i t y Im p lied t h a t th e d a ta co u ld be f i t by th e e q u a tio n lo g k 1 L k ’° = p* a* + B E . ( 9 ) 0 The c o n s ta n ts a* and a re th e T a ft a l i p h a t i c s u b s titu e n t p o la r and s t e r i c c o n s ta n ts , r e s p e c tiv e ly , f o r th e v a rio u s a c y l groups (4 7 ). 22 T A B U ! 3 PSEUDO-FIRST-ORDER RA3E OONSTAHTS FOR THE JKEDAZOIZ-CATAL3CZED HYDROLYSIS OF ESOXRS OF H-^AGETYLSERIHAMIDEa ' ’ 1 "1 ." ...................... — .............. .................... 1 I 1 L ' 1 ■ I I I I I .......................I . T em perature kob sd X 10* E s te r (° C .) (lm )t (mln- 1 ) A c e ta te 95 A c e ta te*1 G A c e ta te B u ty ra te B u ty ra te c iB o b u ty ra te Q I s o b u ty r a te I so v a le rate** T rim e th y la c e ta te 1 .0 1 4 .9 + 0.13 0.50 8.35 + 0.27 0.25 5.52 + 0.02 0.05 3.51 ± 0-05 1 .0 10.8 + 0.05 0.50 7 .t e + 0 .0 4 0.25 5 .5 6 + 0.01 0.05 3-92 ± 0-11 1 .0 6.32 + 0.25 0.50 4 .0 1 0.25 2 .2 6 + 0.09 0.05 0 .8 4 8 + 0.03 1 .0 8 .75 + 0.31 0.50 4 .9 4 + 0.14 0.25 3 .5 7 + 0.15 0.05 2 .0 1 + 0 .0 1 1 .0 3 .2 4 + 0.02 0.50 2.83 + 0.05 0.25 2.08 + 0.19 1 .0 6 .79 + o .o 4 0.50 5 .0 9 + 0 .2 4 0.25 3 .^ 1 + 0 .2 7 0.05 1.82 + 0.002 1 .0 3-93 + 0.13 0.50 2.06 + 0 .0 1 0.25 1 .4 8 + 0.001 1 .0 3.68 + 0.01 0.75 4 .2 0 + 0.05 0.50 3.29 + 0.12 1 .0 2.12 0.50 1-33 23 TABU 3— C ontinued E s te r T em perature ( #c . ) (lm )t k . . X 10a obsd (mSxTX) A c e ta te 85 1 .0 8 .5 2 + o .l 4 0.75 5 .6 5 + 0.31 0.50 4 .7 3 + 0.02 0.25 3.80 + 0.02 B u ty ra te 1 .0 4 .5 8 + 0.12 0.75 3 .2 4 + 0.10 0.50 2 .7 6 ± o . i 4 Is o b u ty ra te 1 .0 4 .9 8 + 0.12 0 .7 5 3.88 + 0.12 0.50 2 .7 9 ± 0 -3 ° I s o r a le r a te 1 .0 4 .2 2 + 0.22 0.75 2 .0 8 0.50 l .o 4 A c e ta te 75 1 .0 4.05 + 0.09 0.50 3*09 + 0.06 0.30 2.12 + 0.09 B u ty ra te 1 .0 2 .4 0 + 0.05 0.75 1 .7 1 + 0 .0 7 0.50 1 .8 4 + 0,003 I s o b u ty r a te 1 .0 4.03 + 0.05 0 .75 2 .5 7 + 0 .2 8 0.50 2 .0 0 + 0.17 A pproxim ate b u f f e r r a t i o 5 : 1 / J* = 4*0 M. ^A pproxim ate b u f f e r r a t i o 4 :1 . Q A pproxim ate b u f f e r r a t i o 1 :1 . 24 TABU! k SECOND-ORDER RATE CONSTANTS FOR IMIDAZOLE CAIALTSIS A ND PSEUDO-FIRST-ORDER RATE CONSTANTS FOR SPONTANEOUS HXDROLTSIS OF ESTERS OF N-ACETmKRINAMIDE* No. E s te r T em perature ( ° c .) *1* X 10® (Kf1 m irT1 ) k x 103 8 (mln_ 1 ) 1 A c e ta te 95 13.6 + l . k 2 .6 3 1 1 . ob 1 .6 b 2 B u ty ra te 9-33 + 1 .4 1.66 3 I s o b u ty r a te 7 .5 7 ± 1 .5 1.98 If I s o v a le r a te 7 .0 0 + O.85 0.805 5 T rim e th y la c e ta te 2 .1 0 0 .5 4 3 A c e ta te 85 7-75 ± 1 .6 B u ty ra te 4.85 ± 1 .3 I s o b u ty r a te 6 .6 3 + 0.01 I s o y a le r a te 8 .4 7 + 1.6 A c e ta te 75 3 .4 6 + 1 .2 B u ty ra te 1 .4 9 + 1 .2 I s o b u ty r a te 5 . 4o + 1 .3 %, - 1 .0 M. ^ R eferen ce 6 , p = 0 .9 M, pH * 7*1/ 100°. 25 Computer s o lu tio n (U8 ) o f e q u a tio n (9) was em ployed to g iv e th e b e s t v a lu e s o f p* and 6 . A lthough 6 co u ld be d eterm in ed q u ite p r e c is e ly , th e v a lu e o f p* c o n ta in e d la r g e u n c e r ta in ty presum ably becau se o f th e sm a ll ran g e o f o* v a lu e s (0 . 3 ) . I t can be shovn t h a t 6 1b f a i r l y in s e n s itiv e to any v a lu e o f p* chosen. T h e re fo re a l l s t e r i c e f f e c t s d a ta w ere p l o t t e d a c c o rd in g t o th e fo llo w in g e q u a tio n : lo g k ' k ’° b Efi (10) The v a lu e s o f Eg u sed w ere d eterm in e d from th e h y d r o ly s is o f e s t e r s o f th e ty p e BOOOEt. The v a lu e s o f E^ u sed a re g iv e n in T ab le 5- TABLE 5 TAFT ALIPHATIC STERIC CONSTANTS (kj) R E s C H3- 0 .0 0 C H 3C H 2— — 0 •07 C H3 (CHe — 0 .36 CHevCSHe}^— — O .^i-O (CH3 )c CH- - 0.47 (CHg^CSHCRs— “ 0*93 (CH3 ) 3C — — 1 .5 ^ (CH3 )3CCHa- - 1 .7 1 * - A p l o t o f lo g ( k ^ / k ^ 0 ) , where k^m ° i s th e s e c o n d -o rd e r r a t e c o n s ta n t f o r th e a c e ta te e s t e r , v s . E a t 95* f ° r th e e s t e r s o f N- ““ B a c e ty ls e rin a m id e i s shown in F ig u re 2 . The slo p e i s O.^t-9 + 0 .1 8 ( c o r- r e l a t i o n c o e f f i c i e n t , O .96) . A p l o t o f lo g (k / k ° ) , where k ° i s th e 8 6 B p s e u d o - f ir s t- o r d e r r a t e c o n s ta n t f o r th e sp o n tan eo u s h y d r o ly s is o f th e a c e ta te e s t e r , v s . E i s shown in F ig u re 3 . The Blope o f t h i s p lo t i s 0.!*8 + O .lk ( c o r r e la tio n c o e f f i c i e n t , 0 .9 7 )* a v a lu e alm o st i d e n t i c a l to t h a t f o r th e im id azo le p l o t . Fig. 2.— Plot of log (kT T n /kT T n °) for the Imidazole-catalyzed hydrolyslB o f e s te r s o f N -acety lserin am ld e a t 95° y s . E . The numbers s r e f e r to th e e s t e r s in T able 4 . 26 27 inO F ig . 3*— - • P lo t o f lo g (k / k ° ) f o r th e spo n tan eo u s ( v a te r and S B h y d ro x id e io n ) h y d r o ly s is o f e s t e r s o f N -a c e ty lse rin a m id e a t pH 6 .30 and 9 5 °» The numbers r e f e r to th e e s t e r s in T able 4 . 28 log (kg/kg0 ) m o - 6s 30 S ince r a t e s f o r th r e e o f th e e s t e r s v e re s tu d ie d a t th r e e d i f f e r e n t te m p e ra tu re s , i t was p o s s ib le to c a lc u la te a c t iv a tio n p a ra m e te rs f o r th e s e th r e e compounds from th e A rrh e n iu s e q u a tio n : W - A When In ho ts(i l s p l o t t e d v s . l/T ° K ., th e s lo p e o f th e r e s u l t i n g s tr a i g h t l i n e i s E /R , where E i s th e a c t i v a t i o n en erg y in c a l/m o le , an d R i s a a th e gas c o n s ta n t. A H*, th e e n th a lp y o f a c t i v a t i o n i s Eft - RT, T b e in g th e a b s o lu te te m p e ra tu re a t w hich th e a c t i v a t i o n p a ra m e te rs a re c a lc u l a t e d , u s u a lly 2 9 8 .2 o r 303. 2 0 K. A S*, th e e n tro p y o f a c t i v a t i o n , i s th e n d eterm in ed from th e r e la tio n s h ip a c c o rd in g to a b s o lu te r a t e th e o ry (^9 ) w hich fo llo w s : k = ^ exp (-A F*/RT) (12) where A F* = A H* - T A S*, (13) k i s th e r a t e c o n s ta n t c a lc u la te d in u n i t s o f se c a t th e te m p e ra tu re a t w hich th e a c t i v a t i o n p a ra m e te rs a r e c a lc u la te d , and k and h a re th e Boltzm ann and P la n c k c o n s ta n ts , r e s p e c tiv e ly . A c tiv a tio n p a ra m e te rs f o r th e im id azo le r a t e s a r e g iv e n in T able 6 . There i s q u ite a ran g e o f v a r ia tio n in th e v a lu e s . I f A H* i s p l o t t e d v s . A S*, a l i n e can be drawn betw een them w ith a slo p e o f 364 ( c o r r e la tio n c o e f f i c i e n t , 0 . 9998)* 31 TABLE 6 ACTIVATION PARAMETERS FOR THE IMIDAZOLE-CATALYZED HYDROLYSIS OF ESTERS OF N-ACETYLSERINAMIDE, CALCULATED AT 93* C. E s te r A H * , k c a l/m o le A &*, e u A c e ta te 1 6 .7 + 0 .6 - 5 0 .2 + 1 .7 B u ty ra te 2 2 ,7 + 1 .4 - 1 4 .7 + 3 .8 I s o b u ty r a te 5-38 + 0 .2 - 6 7 . I + 0 .5 I f th e p lo t o f A H* y b . A S* r e s u l t s In a s t r a i g h t l i n e , t h i s I n d ic a te s a r e l a t io n s h i p such a s A H* = A Hq* + P A S * (14) i s b e in g fo llo w e d , w here 0 h a s th e u n i t s o f a b s o lu te te m p e ra tu re . Com b in in g e q u a tio n s (13 ) and (1 4 ), A F* = A H q* + (P - T) A S*. (15) I f e q u a tio n (15) i s o p e ra te d on by B, an o p e r a to r r e p re s e n tin g changes i n s u b s tr a te s tr u c t u r e o r r e a c tio n medium, 6 A F * = (P - T) 6 A S * . (16) When T = 0 , 6 A F* « 0 , and th e r e a c tio n r a t e does n o t change w ith changing s tr u c tu r e o r c o n d itio n s . The ty p e o f r e la tio n s h ip d e s c rib e d , i n which a p l o t o f A H* yb. A S* g iv e s a s t r a i g h t l i n e , i s known aB an is o k i n e t i c r e l a t io n s h i p (50 ) . A s lo p e , 0 , o f j6k* K. co rre sp o n d s to a te m p e ra tu re o f 91° C .j t h a t i s , a l l th r e e compounds sh o u ld h y d ro ly z e a t th e same r a t e o f 91 *. 32 H ovever, s in c e th e o r d e r o f e s t e r s alo n g th e l i n e i s n o t a r a t i o n a l o n e, and. s in c e th e r e a re o n ly th r e e compounds i n th e s e r i e s , i t i s im p o ssib le to say v i t h c e r t a i n t y vh.eth.er such an i s o k in e tic r e la tio n s h ip does in f a c t e x i s t b e tv e e n th e s e th r e e e s t e r s . The Im idazole-*C atalyzed H y d ro ly sis o f E s te r s o f £ -N itro p h e n o l I t h a s a lre a d y been e s ta b lis h e d t h a t Im id azo le a c t s a s a n u cleo — p h i l i c c a t a l y s t in th e h y d r o ly s is o f e s t e r s o f p - n itr o p h e n o l, and t h a t t h e b a s ic s p e c ie s i s th e a c tiv e form (1 8 ). T h e re fo re , th e r a t e s v e re fo llo w e d a t o n ly one pH, a t f iv e im id a z o le c o n c e n tr a tio n s . The p seu d o - f i r s t - o r d e r r a t e c o n s ta n ts a re p re s e n te d in T ab le 7> a t th e th r e e te m p e ra tu re s a t v h lc h th e r e a c tio n v a s s tu d ie d . The v a lu e s o f k T m a t th e d i f f e r e n t te m p e ra tu re s a r e g iv en in T able 8 . A lso in c lu d e d a r e d a ta c o lle c te d a t 30* by F if e (1 8 ,5 1 ). The d a ta f o r th e tr im e th y la c e ta te e s t e r , a ls o c o lle c te d by T. H. F i f e , a re in c lu d e d f o r com parison. The i n t e r c e p t s a t zero im id azo le c o n c e n tra t i o n a re in m ost c a se s zero v l t h i n e x p e rim e n ta l e r r o r , in d ic a tin g t h a t th e r a t e c o n s ta n t f o r sp o n tan eo u s h y d r o ly s is i s v e ry sm a ll. In F ig u re k i s shovn a p l o t o f lo g ( t ^ A * , 0 ) IE - f o r th e d a ta a t J0° f o r th e h y d r o ly s is o f th e p - n itr o p h e n y l e s t e r s . The p l o t h a s a s lo p e o f 1 .3 5 ( c o r r e la tio n c o e f f i c i e n t , 0.97)* In T able 9 a re p re s e n te d a c t i v a t i o n p a ra m e te rs d eterm in ed from th e d a ta g a th e re d in t h i s stu d y a s v e i l a s from th e d a ta a t 30*. The a c t iv a tio n p a ra m e te rs f o r th e tr im e th y la c e ta te e s t e r a s v e i l a s th o s e d eterm in e d e a r l i e r f o r th e a c e ta te e s t e r u n d er som evhat d i f f e r e n t con d i t i o n s a re p re s e n te d a ls o . A H * rem ain s e s s e n t i a l l y c o n s ta n t v a ry in g H ff o o ' & (D C+ (V g P f t p i ? c+ p c+ p H m o £ p P <+ p 4=- O o o o o o o o o o o o o o o o o o o o o • • • • • • • • * • • I • « t • • • • • O O O O O O Q O O O O O O O O O OO O M H ro 4 ^\ji h ro -p-on h rov» .p-v/i vx -p-vji o V J l O H H W W • # OvO O - q Q £ r o g > I + I + I + I + I + o o o o o OOHHIO • * * » * * r\c ro 00 - r ^ C 0 - a P I + 1 + I + I + I + o o o o o • I « « » o o p g £ O H H W 'J • • • • * pv r o v o y i h ro oo w ■r I + I + I + I + I + o o o o o B ' S 8 > & f e v o o o o o • • • « • h H ro v -p- -p-\p C\ QD -J vo ^-vo JP h I + I + I + 1 + 1 + o o o o o • « • • • o o o o o O ro H V > 4 4 = " C D & £ f t C+* 0) g & p o p <+ & p ro o o o o o o o o o o o o o o o o o o o o o rovx ?-vn-4 VJ1 OQO OO h ro v> - p-v ji Q O O O O rovjj 4=-on -4 O O O H H V O O v - P * I + I + I + I + 1 + O O O O O • • • • • o o o o ro H Hvnoi V n o o o o o • • • • * H V J J V J 1 - 3 VO V O 0\ pr ^ V j j - q VJi ^ - C D I + I + I + I + I + o o o o o « « » • * o o o o o O f H->I HVx O o O H H • • • t « £-JVO H C X > & r3-g o d o C K O v O O I + I + I + I + I + o o o o o w Q> c+ p ► 1 a o s: & c t f o o ' 1% V _ K l Vj j 54 TABU! 7— C ontinued E s te r Tem perature C c .) (Im)t (M) kobsd (m lp"1 ) I s o v a le r a te 40 0.10 1.14 + 0.02 0.075 0.894 + 0.04 0.05 0.624 + 0.03 o .o 4 0.489 + 0.02 0.03 0.369 + 0.01 A c e ta te 50 0.05 4.47 + o.o 4 0 .0 4 3.44 + 0.15 0.03 2.62 + 0.08 0.02 1.82 + 0.03 0.01 0.978 + 0.03 B u ty ra te 0.05 2.89 + 0.11 o.o 4 2.28 + 0.07 0.03 1.76 + 0.07 0.02 1 .1 4 + 0.02 0.01 0.559 + 0.02 Is o b u ty r a te 0.05 3.69 + 0.16 0 .0 4 2 .8 4 + 0.14 0.03 2.09 + 0.06 0.02 1.45 + 0.06 0.01 0.714 + 0.01 I s o v a le r a te 0.10 1.56 + 0.06 0.075 1.23 + 0.06 0.05 0.816 + 0.03 0.04 0.599 + 0 0.03 0.479 + 0.05 1.64^6 d io x an e. 35 TABLE 8 SECOND-ORDER RATE CONSTANTS FOR THE IMIDAZDIE-CATALIZED HYDROLYSIS OF ESTERS OF p-NITRDPHENOLa T em perature k Im No. E s te r ( #c .) (M- 1 m in -1 ) I n te r c e p t 1 A c e ta te 20 30 40 50 28,2. 49. ? 73-3 102 C V J O OH • • • O OO + 1 +1+1 0.01 0.03 0.08 2 B u ty ra te 20 3° 40 50 2 2 .4 31.8C 57-5 68.7 + 0.02 + o . l 4 + o .o 4 -0 .0 1 - 0 ,0 7 -0.02 3 I s o b u ty r a te 20 30 1 * 0 50 28.1 4o . 3c 66.3 87.0 + o.o 4 + 0.08 + o . l l 0.01 0.01 -0.05 4 I s o v a le r a te 20 % 50 5 . 52. 6.02° 13.0 16.2 + 0.03 + 0.03 + 0.06 0.02 0.05 0.18 5 T rim e th y la c e ta te 20 30 4o 50 °* 3 3 q 0 . 55° ° . 9lJ 1. 3? aji * 1 .0 M, 1.64}£ d io x an e. ^ R eferen ce 51. 0 R eferen ce 18. P ig . 4 .— -P lo t o f lo g ( k ^ / k ^ 0 ) r s . E0 f o r th e im id a z o le - c a ta ly z e d h y d r o ly s is o f e s t e r s o f p - n itr o p h e n o l, 3 0 .0 ° . The numbers r e f e r to th o s e e s t e r s l i s t e d in T able 8 . % i°g (kjm/k|m) L L* p c j i o b i o m Q o- 38 TABU! 9 ACTIVATION PARAMETERS FOR THE IMIDAZO IE-CATALYZED HYDROLYSIS OF ESTERS OF p-NITROPHENOL, CALCULATED AT 50° E s te r A H*, Itcal/m o le A 8* , e u A c e ta te 7-4 ± 0 .5 7 .L + 0.5 7 . ^ - ^ • 5 . + 0 .9 - 27.2 B u ty ra te 6 .9 + 0 .5 - 37.2 + 1.5 Is o b u ty ra te 6 .7 + 0.2 - 37.2 + 0 .8 I s o v a le r a te 6 .9 + 0 .8 - 4o .4 + 2 .8 T rim e th y la c e ta te 8 .5 + 0 . 1° - 4o .6 + 0 . 3° R e f e r e n c e 14, 5?6 d io x a n e , < 0 . 0 2 . ^ R eferen ce 15 , 2 8 .5 ^ e th a n o l- v a te r ( r / v ) , 0 .2 M p h o sp h ate b u f f e r , c a lc u la te d a t 50*. cR eferen ce 5 1 , 39 o v e r o n ly 1 .6 k c a l, w h ile A 8* d e c re a s e s 6 .1 eu o v e r th e e n t i r e s e r i e s . The P e a c y la tio n o f A cyl-Q j-chym otrypslns The r a t e c o n s ta n ts f o r th e d e a o y la tio n o f acyl-Cfc-chymotrypBin d e r iv a tiv e s a t 25.0® a r e g iv e n in T ahle 10 a t s e v e ra l pH v a lu e s . V alues o f th e pH -Independent r a t e c o n s ta n ts , k 3, and th e a p p a re n t d is s o c ia tio n c o n s ta n ts o f th e e s s e n t i a l group a t th e a c tiv e s i t e w ere c a lc u la te d from p l o t s o f k3 * v s . k 3 1a g . These p l o t s were l i n e a r w ith o r d in a te i n t e r c e p t , k3, and s lo p e , The v a lu e s o f k 3 and pK^ a re g iv e n in T able 10. The pK^ v a lu e f o r th e a c e t y l d e r iv a tiv e i s t h a t de te rm in e d by Kezdy and Bender (3*0* A lso g iv e n in T ab le 10 f o r com parison w ith k3 v a lu e s a re th e v a lu e s o f k3 ' a t pH 8 .8 0 , w here th e r a t e sh o u ld be in th e pH -independ e n t r e g io n . These v a lu e s a r e much h ig h e r th a n k 3 . However, when ex tr a p o la te d to zero T r is c o n c e n tra tio n (u sin g th e d a ta in T able l l ) , th e d e riv e d v a lu e s a r e much more com parable to k 3 . Kezdy and B ender (3*0 r e p o r t a k 3 v a lu e f o r th e d e a c e ty la tio n o f acety l-ce-ch y m o try p sin o f 6 .8 X 10 s e c , w hich i s h ig h e r th a n t h a t 2 d eterm in e d in t h i s stu d y , p o s s ib ly b ecau se in th e p re v io u s stu d y T r is b u f f e r s may have been u sed . I t i s I n te r e s t i n g t h a t th e k 3 v a lu e ob t a i n e d by B ender and H am ilton ( 2 j) f o r th e tr im e th y la c e ty l d e r iv a tiv e , — X 1 .3 X 10 sec , i s q u ite c lo s e to t h a t d eterm in e d in t h i s stu d y even th ough T r is b u f f e r s w ere u sed f o r th e h ig h pH’s i n th e fo n a e r stu d y . T r is b u f f e r h as a pronounced e f f e c t on th e m agnitude o f th e d e a c y la tlo n r a t e c o n s ta n t, a s can be seen from T able 11. F a l l e r and 2 l -.3 — 1 4 .5 X 10 sec , by e x tr a p o la tio n to ze ro T ris c o n c e n tra tio n u s in g th e d a ta in T able 11. TABIZ 10 RATE CONSTANTS FOR THE BEACYLATION OF ACIL-O-CHYMDTRYPSINS AT VARIOUS pH VALUES* 40 k 'a X 10* k 3 X 10* No. A cyl Oroup pH (se c ” ) (se c " * ) PKa 1 A c e ty l 7.66 3 4 .7 + 2 .0 7.28 8 . 80. 8 5 .4 + 1 .8 8.80 4 5 .0 2 P ro p io n y l 5.88 4 .5 4 + 0 .1 79.2 7 .1 0 7.28 4 8 .4 + 0 .3 7.66 6 2 .1 + 3 .2 8 . 80. 110 + 2 .4 8.80 71.0 B u ty ry l H exanoyl 5.88 6.20 7.28 7.66 8 . 80. 8.8ox 5.88 6.20 7.28 7.66 8.8o. 8 .8 o 2.61 ^•77 3 2 .3 kp.8 9 4 .0 56.0 + + + + + 0.1 0.1 0 .4 1.3 0.9 59.8 7.22 1 2 .1 + 0 .4 2 3 .8 + 0.5 161 + 5 .5 252 + 13 525 + 10 450 364 7.35 I s o b u ty r y l 5.88 6.20 7-28 7.66 8 . 80. 8.80 1.29 + 0 .1 3.08 + 0 .1 1 6 .1 + 0 .7 18.1 + 0 .2 2 8 .1 + 0 .6 20.5 2 4 .8 7.11 I bov a le r y 1 5.88 7.28 7.66 8 . 80. 8 . 801 0.620 + o .o 4 6.62 + o.o 4 9.27 + 0.3 23.8 + 0.9 15.0 12.5 7.18 TABUS 10— C ontinued No. A cyl Group PH E 'a X (sec" 10^ *> k3 X 104 (sec**1 ) pKa 7 T rim e th y la c e ty l 5.88 6.20 7.28 7.66 8 . 80. 8.80 0.180 + 0.336 + 1 .1 9 + 1 . 4 l + 1 .8 7 + l.4o 0 0.01 0.02 0.02 0.06 1.60 6 .7 5 8 3*3 -D lm e th y lb u ty ry l 5.88 7.28 7.66 8 .80. 8.80 0.2 4 6 + 1.25 + 1.34 + 1.50 + 1 .3 5 0.01 0.002 0.01 0.01 1 .5 0 6.60 a2 5 . 0 % = 0.075 M, 1 .6 # a c e t o n i t r i l e . ^ E x tra p o la te d to zero T rig b u f f e r c o n c e n tra tio n . TABLE 11 VARIATION OF RATE OF DEACXLATION OF ACXL-OS-CHXMOTRYPSINS WITH TRIS BUFFER CONCENTRATION8- R ate C o n stan t a t In d ic a te d T r is C o n c e n tra tio n A cy l Group 0.075 M 0 .1 0 M 0 .2 0 M 0 .3 0 M A c e ty l 8 5 .4 + 1 .8 122 133 + 7 .0 213 P ro p io n y l 110 + 2 .0 184 + 5 .0 230 + 3 .0 B u ty ry l 9 4 .0 + 0 .9 180 + 4 .0 229 + 7 .0 H exanoyl 525 996 1130 + 31 1228 + 33 I s o b u ty r y l 2 8 .1 + 0 .6 52.0 + 0 .1 6 6 .7 + 1 .9 I s o v a le r y l 2 3 .9 + 0 .8 38.4 + 0 .7 4 3 .6 + 1 .4 T rlm e th y l- a c e ty l 1.87 2.32 2 .7 0 + 0 .5 2 .9 3 3 >3-Dlme th y 1- b u ty r y l 1.49 + 0.01 2 .2 1 + 0 .1 2.65 + 0 .1 a 2 5 .0 % 1 .6 ^ a c e t o n i t r i l e , k3 ' X 104 , sec” 1 . *3 S tu rte v a n t ( 38) have p re v io u s ly shown t h a t th e d e a c e ty la tio n o f a c e t y l - a -c h y m o try p sin i s g r e a tly in c re a s e d by in c re a s in g c o n c e n tra tio n s o f T r i s . They c a lc u la te d th a t T r is was 600 tim e s more r e a p tlv e th a n w a te r i n d e a c y la tio n , o r , i f o n ly th e u n p ro to n a te d s p e c ie s i s p a r t i c i p a t i n g , th e n i t i s 6000 tim e s more r e a c tiv e th a n v a t e r . The e f f e c t o f T ris on d e a c y la tio n i s s u b je c t t o a m arked s t e r i c e f f e c t . There i s c o n s id e ra b ly s m a lle r r a t e a c c e le r a tio n when th e a c y l group i s h ig h ly b ran ch ed , as in th e case o f th e tr im e th y la c e ty l o r 3>3- d im e th y lb u ty ry l d e r iv a tiv e s . T h is i s e v id e n t from an ex am in atio n o f T able 10, a s w e ll a s from a c o n s id e ra tio n o f th e re p o rte d v a lu e s o f B ender, e t a l . above, in com parison to th o se d eterm in e d in t h i s stu d y . A n o th er f a c t o r w hich a p p e a rs to be a f f e c te d by extrem e s t e r i c b u lk i s th e a p p a re n t io n iz a tio n c o n s ta n t o f th e e s s e n t i a l group a t th e a c tiv e s i t e , w hich i s re p o rte d in T ab le 10. Though th e k3 '--pH p r o f i l e s a re e s s e n t i a l l y sig m o id al f o r a l l th e acyl-Cfc-chym otrypsins s tu d ie d , th e pK c a lc u la te d from th e d a ta i s lo w er f o r th e two d e r iv a tiv e s in w hich a th e a c y l group i s v e ry h ig h ly b ran ch ed . In F ig u re 5 I s shown a p lo t o f lo g (k3 '/ k 3 10), where k3 '° i s th e r a t e c o n s ta n t f o r th e d e a c e ty la tio n o f a cety l-C t-ch y m o try p sin . D ata a t pH 7*66 w ere u sed a s th e pH -independent r a t e c o n s ta n t f o r th e a c e ty l d e r iv a tiv e d eterm in ed from d a ta a t s e v e r a l pH's was n o t a v a ila b le u nder th e c o n d itio n s o f t h i s stu d y . A s im ila r p l o t u s in g lo g k3 was a ls o l i n e a r w ith th e same s lo p e . The s lo p e was 1.05 + 0 .2 1 ( c o r r e la tio n c o e f f i c i e n t , 0 .9 9 ) when th e p o in t f o r th e a c e t y l d e r iv a tiv e was n o t in c lu d e d on th e p l o t . With th e in c lu s io n o f th e a c e t y l compound, th e slo p e was 0.98* F ig . 5 . —P lo t o f lo g Qt3 '/ k 3 '°) v s . EB f o r th e d e a c y la tio n o f acyl-C t-ch y m o tiy p sin s a t 2 5.O0, pH 7*66, 1 .6 £ a c e t o n i t r i l e . The numbers r e f e r to th e compounds in T able 10. kk 45 C V J ooQ 46 B a te s w ere m easured a t f o u r te m p e ra tu re s : 1 5 ", 25 ° , 35 ° , and 4 0 °. The d a ta a r e g iv e n in T a b le 12 . The e x p e rim e n ta l pH was 7 . 6 6 . In T a b le s 13 and 14 a re p re s e n te d th e d a ta f o r th e te m p e ra tu re depend ence o f th e io n iz a tio n o f th e e s s e n t i a l group a t th e a c tiv e s i t e . In T ab le 15 a r e g iv e n t h e a c t i v a t i o n p a ra m e te rs f o r th e d e a c y la tio n o f th e a cy 1-C t-chym otrypsinb w ith and w ith o u t th e c o r r e c tio n f o r th e h e a t o f i o n iz a tio n o f th e e s s e n t i a l group a t th e a c tiv e s i t e . The c o r r e c tio n was made u s in g a A o f 6 .4 h e a l, s in c e t h i s i s th e mavimim c o r r e c tio n f a c t o r found e x p e rim e n ta lly . Then th e t o t a l c o r r e c tio n on A H*, A , was th e fo llo w in g : (A = A (17) w here a^ was th e n e g a tiv e a n tilo g a r ith m o f th e pH v a lu e , 1-66, and K& was d ete rm in e d from th e pK v a lu e s l i s t e d In T ab le 1 0 .^ I t can be seen a t h a t th e c o r r e c tio n s a r e v e ry sm a ll— a maxi mum o f - 2 .2 k c a l i n A H * and - 6 .7 eu i n A S * . A s t r a i g h t l i n e r e l a t i o n s h i p betw een A H* and A S* i s o b ta in e d f o r th e s e r i e s w ith th e e x c e p tio n o f th e n -h e x a n o y l d e r i v a t iv e , w hich d e v ia te s n e g a tiv e ly , and th e tr im e th y la c e ty l and 3, 3 -d im e th y lb u ty ry l d e r i v a t iv e s , w hich d e v ia te p o s i t i v e l y , a s can b e seen from F ig u re s 6 and 7 . The s lo p e s o f th e s t r a i g h t l i n e p o r tio n s in F ig u re s 6 and 7; o r ^Cunningham an d Brown (52) p r e v io u s ly r e p o r te d a A % o f U k c a l / mole w hich had b een d e te rm in e d from d a ta a t two te m p e ra tu re s , 11 ° a p a r t , f o r th e h y d r o ly s is o f t h e s p e c i f i c s u b s t r a te , N - a c e ty l- ty r o s in e e th y l e s t e r . However, s in c e A H i was d e term in e d o v e r a ran g e o f 25° in t h i s s tu d y , and f o r th e s u b s t r a te s to w hich i t was to be a p p lie d , t h i s v a lu e was th o u g h t t o be more a p p r o p r ia te . No e x p la n a tio n can be o f f e r e d f o r th e d e g ree o f v a r i a t i o n in t h e d e term in e d v a lu e s . 1 - (K + ' a 47 TABLE 12 RATE CONSTANTS FOR THE DEACYLATION OF ACYL-O-CHYM0TR3CPSINS AT VARIOUS TEMPERATURESa A cyl Qroup T em perature (* c .) k3 ' X 104 (s e c ’’* 1 ') A c e ty l 15.0 13*7 + 1.0 25.0 34*7 + 2.0 55*0 8 6 .7 + 0.5 59.5 113 + 4 .6 P ro p io n y l 15.0 22.5 + 0.6 1 25.0 62.1 + 3 .2 55.0 137 + 1.1 4o .o 204 + 6 .9 B u ty ry l 15.0 1 6.5 + 0.8 25.0 4 5 .8 + 1 .3 35*0 107 + 1 .9 4 0 .0 1^7 + 5 .2 H exanoyl 15.0 96.5 + 1.5 25.O 252 + 13 34.5 611 + 7 .4 39.2 861 + 11 I s o b u ty r y l 15.0 5-57 + 0.2 25.O 18.2 + 0.2 35*0 4o .6 + 0 .4 4o .o 50.2 + ■ 1.2 I s o y a le r y l 15.0 2.58 + 0.1 25.0 9.27 + 0.3 35*0 2 4 . k + 0.2 4o .o 34.9 + 1.0 T rlm e th y la c e ty l 15.0 0.584 + 0.01 25.0 1 .4 1 + 0.02 35*0 5*56 + 0.1 4o .o 5.80 + 0 .1 5 , 5 -B im e th y lb u ty ry l 15.0 0.457 + 0.02 25.0 1 .5 4 + 0.01 3 5.0 2.87 + 0.2 4o .o 4.71 + 0.1 apH 7*66, p = 0.075 M, 1 .6% a c e t o n l t r i l e . p H A N D T E M PE R A T U R E D E PE N D E N C E O F T H E H A T E S O F D EA C Y LA T IO N O F SO M E ACYL-O-CHIM O TRYPSINS8 , 3 H r| I X O « > at P ) "—t M W P i i H o < O KNO * • ft • O O O H +I+I+I+I f t f t f t f t O H OS K'v H C V J o «o ft • • O O KV4- +I+I+I+I -4 t-r-i f t f t O S C V J O H O f o o o o +I+I+I+I 8cb O J c- k Ssq o o it s f t f t f t f t O O tOir\ O OJ c* - o j f t • f t f t O O O 1 - 1 +I+I+I+I Os <M 0 0 CO OJ f t f t f t f t irs O O Os H irsiA H r l r l U S o o o o .... o o o o +I+I+I+I -4" t— t r \ On-4 - 0 0 H OJ rC US ft ft ft • O O OJ OJ K S O H O S O f t f t f t f t O O O rH +1+1+I+I r-( 00 0J ir \K V 4 - K S K S S O 0 J C '-V Q ksvo o t - v o 0 J KAVO o c — v q o j O C-vO oj rf'i'O S o O J t'-vo K S V O s l/SVO N N LfSVO t — f— ITS VO t— LfNVO C — t~~ u s 'O fr— ITS VO t*— ITS r l O -4- LTS r l O - 4 - -P O J t) <: p & O ( 0 H iH ft ■ 3 I u ,= L aJ 0.075 M , 1.656 a c e t o n i t r i l e . TABLE Ik TEMPERATURE DEPENDENCE OF THE IONIZATION CONSTANTS OF THE ESSENTIAL G RO U P AT THE ACTIVE SITE IN THE DEACYLATION OF SO M E ACYL-O-CHYMDTRYPSINS A cyl Group T em perature (°C.) PKa A Ht (kc& l/m ole) A c e ty l 15 7*36 6 . 1 + 25 7.28 i + o 6.98 I s o b u ty r y l 15 7 .1 9 5-8 25 7.08 l+ o 6.96 I e o y a le r y l 15 7 . 21+ 3-5 25 7.1 9 1 + 0 7 .0 5 R e f e r e n c e $k. 50 TABLE 15 ACTIVATION PARAMETERS FOR THE DEACYLATION OF ACYL-Ct-CHYM O TRYPSINS, UNCORRECTED AND CORRECTED FOR THE HEAT OF IONIZATION OF THE ESSENTIAL C ffiD U P AT THE ACTIVE SITE, CALCULATED AT 25* A H * A S * A H * A S * ( k c a l) (eu ) ( k c a l) (eu) No. A cyl Croup Unconnected. C o rre c te d 1 A c e ty l 1 5 .0 + 0 .3 — 19.1 + 1.0 13.0 - 25.5 2 P ro p io n y l 15.2 + 0 .3 - 1 7 .7 + 1 .0 13.8 - 21.8 5 B u ty ry l 1 5 .3 + 0 .3 - 17.9 + 1.0 13.5 “<23.5 4 H exanoyl 1 5 .7 + 0 .3 - 13.1 + 1.0 13.5 - 19.8 5 I s o b u ty r y l 16.4 + 0.5 - 16 .1 + 1.6 1 4 .9 - 20.5 6 I s o v a le r y l 18.0 + 0 .7 - 12.0 + 2 .3 16.3 -1 7 .1 7 T rim e th y l- a c e ty l I 8 .7 + 0 .4 - 13.4 + 1 .4 18.0 -1 5 .4 8 3, >-Diine th y 1 - b u ty r y l 16.0 + 0 .4 - 22.5 + 1 .3 15 .5 -2 4 .2 F ig . 6 .— P lo t o f A H * y b . A S*, u n c o rre c te d , f o r th e d e a c y la tio n o f acyl-^JM ihym otrypsins. The numbers r e f e r to th e compounds in T able 15• 51 m I < 18 17 16 15 4 8 - 1 6 AS* (e.u.) -12 -22 -20 -14 VJ 1 r o F ig . 7 . —P lo t o f A H* v s . A S*, c o r re c te d f o r th e h e a t o f i o n i z a tio n o f th e e s s e n t i a l group a t th e a c tiv e s i t e , f o r th e d e a c y la tio n o f acy l-Q f-ch y m o try p sin s. The num bers r e f e r to th e compounds l i s t e d in T able 15. 53 A H * ( k cal.) -26 -24 -22 ^20 = 1 8 AS* (e.u.) -16 -14 55 {J, th e i s o k in e tic te m p e ra tu re s , a r e 448° K. and 405° K ., r e s p e c tiv e ly . Thus, though th e c o r r e c tio n do es cause some d if f e r e n c e s in th e a b s o lu te v a lu e s o f A H* and A S* r e p o r te d , i t cau ses no d if f e r e n c e in th e q u a li t a t i v e o b s e rv a tio n s and o n ly a sm a ll d iff e r e n c e in th e slo p e o f th e is o k i n e t i c p l o t . The te m p e ra tu re dependence o f th e r a t e c o n s ta n ts was a ls o de te rm in e d in T r is b u f f e r a t pH 8 .8 0 . These d a ta a re p re s e n te d in T able 1 6. T able 17 shows th e v a lu e s o f th e a c t iv a tio n p a ra m e te rs d e te r m ined in T r is . The d a ta f o r th e 5 , 3 -d im e th y lb u ty ry l d e r iv a tiv e d id n o t g iv e a l i n e a r A rrh e n iu s p l o t , and so t h i s compound was o m itte d . A p l o t o f A H* v s , A S* f o r th e s e v a lu e s g iv e s th e same ty p e o f f i t as F ig u re s 6 and 7 , w ith th e slo p e 568° K. T h is i s o k in e tic r e l a t io n s h i p may be f o r t u i t o u s s in c e th e p a r t i c i p a t i o n o f T r is d e f i n i t e l y h as an e f f e c t on th e a c t i v a t i o n p a ra m e te rs f o r t h i s r e a c tio n . Thus i t i s be lie v e d t h a t a c t iv a tio n p a ra m e te rs d eterm in e d in p h o sp h ate b u f f e r and c o r re c te d f o r th e h e a t o f io n iz a tio n o f th e group a t th e a c tiv e s i t e , o r d eterm in e d in T r is b u f f e r and e x tr a p o la te d to zero T r is c o n c e n tra t i o n , p ro b a b ly g iv e a t r u e r r e p r e s e n ta tio n o f th e s e p a ra m e te rs th a n th o se m erely d eterm in ed in T r is b u f f e r w ith o u t c o r r e c tio n . A c y la tio n o f th e enzyme co u ld n o t be ac h ie v e d when p - n i t r o - p h e n y l t r i e t h y l a c e t a t e was used a s a s u b s tr a te . No r e a c tio n co u ld be o b serv ed a f t e r th e r e a c tio n m ix tu re had sto o d f o r fo u r h o u rs a t 25 °. Thus th e t r i e t h y l a c e t y l group im poses s t e r i c re q u ire m e n ts t h a t a re to o se v e re f o r th e r e a c tio n to o c c u r. 56 T A B L E 16 TEMPERATURE DEFENIKNCE OF THE RATE OF DEACXLATION OF ACYL-OS-CfflCM OTrcCPSINB IN TRIS BUFFER8 , A cyl Group T em perature ( #C .) k 3 ' X 10* (se c ” 1 ) A c e ty l 15.0 3I+.0 + 0 . 1 + 2 5 .0 85.5 + 1 .8 55.0 ik k + 2 .6 PropJLonyl 15.0 60,6 + 1 . 1 + 25.0 110 + 2 . 1 + 35.0 219 + 3.0 B u ty ry l 15.0 1 + 6 .2 + 1 .0 25.0 9^.0 + 0 .9 55.0 195 + 10 59.5 267 H exanoyl 15.0 335 0 • J- +1 25.5 525 55.0 1259 ± 93 39.5 1791 I s o b u ty r y l 15.0 H + .l + 0 .6 2 5 .0 2 8 .1 + 0 .6 35.0 6 1 .1 + 0.5 I s o v a le r y l 15.0 7.21 + 0 .1 25.75 23.8 + 0.8 35.0 3 5 .3 + 0 .9 T rlm e th y la c e ty l 15.0 0 .6 2 6 + 0.0 2 2 5 .0 1 .8 7 + 0.06 35.0 1+.28 + 0.07 apH 8 .8 0 , n = O.075 1 .6 ^ a c e t o n i t r i l e . 57 TABLE 17 ACTIVATION PARAMETERS DETERMINED IN TRIS BUFFER FOR THE DEACYLATION OF ACYL-O-CHYMDTKYPSINS, CALCULATED AT 25 °a A cyl Group l . i : 1 t 1 ea=ea3= x = a a * s = a = = . 1:1 1 r-u s A H* (k c a l/m o le ) A 3* (eu) A c e ty l 9 .7 “ 2<>.5b -3 6 .5 P ro p io n y l 10.8 - 31.2 B u ty ry l 12.2 — 26 .8 H exanoyl 11-3 -2 6 .4 I s o b u ty r y l 12.8 - 28.2 I s o v a le r y l 1^.1 - 23.0 T rlm e th y la c e ty l 16.7 -1 9 .5 apH 8 . 80, ^ = 0.075 1 .6 $ a c e t o n i t r i l e . ^R eferen ce 53, 0 .1 M T r is . The A c y la tio n o f O-ChyinotrypBin by E s te r s o f p-N itroph.en.ol As d is c u s s e d i n th e I n tr o d u c tio n , an E adle p l o t o f e q u a tio n ( 2 ), k* v s . 'is* /(S )Q, w here k* * k - k a , sh o u ld g iv e a s tr a i g h t l i n e w ith slo p e e q u a l to — and a b s c is s a in te r c e p t e q u a l to k e . F ig u re 8 shows th e r e s u l t s o f a p l o t o f th e d a ta made in t h i s m anner. The slo p e i s i n f i n i t e . T h is im p lie s t h a t i s much l a r g e r th a n th e h ig h e s t sub s t r a t e c o n c e n tra tio n s tu d ie d so t h a t th e c o n c e n tra tio n o f ES i s ex p e rim e n ta lly u n d e te c ta b le . T h e re fo re th e d a ta w ere t r e a t e d a c c o rd in g to se c o n d -o rd e r k i n e t i c s . I n T a b le s 18-25 a re g iv e n th e pseudo— f i r s t - o r d e r r a t e con s t a n t s , k* , f o r th e a c y la tio n o f O ^chym otrypsin by th e in d ic a te d e s t e r s a t th e pH v a lu e s s tu d ie d , k* i s th e o b serv ed p s e u d o - f ir s t- o r d e r r a t e c o n s ta n t l e s s k3 , th e d e a c y la tio n r a t e c o n s ta n t. k * /(S )Q sh o u ld be ap p ro x im a te ly c o n s ta n t. In th e case o f a se c o n d -o rd e r r e a c tio n , a p l o t o f k* v s . (S )Q sh o u ld have an o r d in a te i n te r c e p t o f z e ro , and th e s lo p e o f t h i s p l o t sh o u ld be e q u a l to (k ^ /K ^ )1, th e pH -dependent B econd-order a c y la tio n r a t e c o n s ta n t (35)* A t y p i c a l p l o t i s shown in F ig u re 9* d a ta a re p re s e n te d i n T able 2 6 . The (ke/K m) ' v a lu e s a re th e s lo p e s o f th e r e g re s s io n l i n e s . A lso p re s e n te d in T ab le 26 a re th e av erag e v a lu e s o f k * /(S ) Q, w hich should be e q u a l to (ka/K m) (5*0) th e in t e r c e p t v a lu e s ; and th e s ta n d a rd e r r o r s o f th e e s tim a te , S^.. The i n te r c e p t v a lu e s sh o u ld be zero w ith in th e l i m i t s o f e x p e rim e n ta l e r r o r a s de te rm in e d by th e s ta n d a rd e r r o r o f th e e s tim a te . D e v ia tio n s from ex p e c te d b e h a v io r may be due to th e fo llo w in g : (a ) a t h ig h s u b s tr a te 59 i c 1 ) 3 5 -------------- 1 ■ i - • 2 c 3 0 f t u Si C M o c D 2 5 - 0 - X * * 1 - 2 0 - 0 ‘ 1 i 1 5 1 - ..-L , 0 1 1 ________________ I - - L 1- 1 ■ 1 2 12 14 k*/(S^ x 10'2 (M'1sec.‘1) P ig . 8 . —Eadie p lo ts f o r th e a c y la tio n o f Ct-chymotrypain by e ste rB o f p -n ± tro p h en o l, y. « 0.075 M, 2 5 .0 ° , 4 .6 8 ^ a c e to n i t r il e ; O, tr im e th y la c e ta te e s t e r , pH 7*99; 0 j a c e ta te e s t e r , pH 7*58. 6o TABLE 18 PSEUDO-FIRST-ORDER RATE CONSTANTS FOR THE ACXLAUON OF QM2HIM0 TRIPSIN BX ESTERS OF p-NITROPHENOL, pH 5*92, PHOSPHATE BUFFER8 , E s te r (E) X 1O0 ' o (M) (S )o X 10* (M) k* x 10® (se c "* ) W ( S )0 x (M~* sec" A c e ta te 8.28 0 .9 3 8 4 .4 1 4 .7 0 8.10 1 .1 3 5 .3 4 4 .7 4 If 1 .4 1 4 .1 7 2 .9 7 8 .^ 4 1.88 7.88 4 .2 0 8.28 2.32 7 .8 7 3-39 •1 2.81 11.6 4 .1 4 P ro p io n a te 1 3 .4 I .38 9.00 6 .5 3 l 4 .0 1 .4 3 6.8 9 4.83 13.4 1 .9 0 10.5 5 .5 1 II 2.21 12.6 5 .7 2 i4 .o 2.85 1 7 .3 6.18 B u ty ra te 9.10 0 .9 3 1 7.32 7.86 9.11 l .4 o 9-01 6 .4 5 if 1 .5 4 9.80 6.36 9.10 1.88 12.7 6 .7 9 8.91 2.34 17.2 7.3 4 9 .1 1 3.08 20.1 6.52 H exanoate 4 .2 4 0.512 16.8 32.7 It 0.7 9 4 23.8 30.0 II 0.9 3 8 32.1 3 4 .3 1! 1.02 3 0 .1 29.4 tf 1.27 4 4 .4 3 4 .9 I s o tm ty ra te 9.10 1.0 1 6 .2 4 6.18 8 .9 1 1.07 6 .4 0 5-99 9 .1 1 1.34 3.82 2.86 II 1.78 6 A 3 3.61 9.10 2.21 7.05 3*19 II 2 .6 7 12.0 4 .4 8 I s o v a le r a te 9.10 1.00 2.63 2.62 9 . H 1.45 2 .3 9 1.65 Tf 1.74 3-29 1.89 If 1.95 3-15 1.62 8 .9 1 2 .1 8 3.72 1.71 TAJBIE 18— C ontinued 6 1 (E) X 10® ' 0 ( s ) o X 10® k* X 102 W (s)Q X 10-* E s te r (M) (M) (se c 1 ) (M "1 s e c " 1 ) T rim e th y l 9 .1 0 1.01 0 .3 2 9 0.325 a c e ta te 9 . H 1 .5 0 0 .4 0 1 0.308 ff 1 .4 0 0 .4 4 6 0.318 tl 1.68 0.4 8 9 0.291 3 , >*Dime t h y l - 9.10 1.00 0.136 O .I36 h u ty r a te 9 . H 1.21 0.130 0.108 It 1.32 0.137 0 .1 0 4 9.10 1.50 0.166 o . l l i I t 1.63 0.171 0.105 a2 5 .0 ° , ^ = 0.0 7 5 M, 4 .6 8 # a c e t o n i t r i l e . TABIE 19 PSEUDO-FIRST-ORDER RATE CONSTANTS FOR THE ACYLATION OF Ct-CHYMQ TRYPSIN BY ESTERS OF p-NITROPHENOL, pH 6.19 PHOSPHATE BUFFER8' E s te r (E) x io a '0 (M) ( s ) o x 10* (M) k* X 10® (se c ” 1 ) W ( 8 ) 0 X 1 (M _1 sec*"*1 A c e ta te 13.'K 1 . 1+ 1 8.81 6.27 1 1 1.88 8 .8 9 I+.7I+ ft 2.13 11.0 5 .1 7 ft 2.52 13 . 1 + 5 .3 0 tf 2 .8 1 13.2 1+.69 P ro p io n a te 12 . 1 + 1 .1 8 11-7 8 . 31+ 13.1+ 2.36 16.3 6 .9 2 1 1 2.85 20.3 7 .1 2 ft 3 .5 3 25.5 7.21 B u ty ra te 1 3 .4 1 . 1 + 0 17.2 12.3 If 1 . 51* 18.8 12.2 ft 1.88 19.8 10.6 It 2.32 26 . 1 + 11.1+ It 3.08 30.8 9 .9 9 H exanoate 1 +.21+ 0.512 23 .5 1*5-9 It 0 . 791+ 2 0 . 1 + 2 5 .7 It 0.938 3Q-9 1*1.5 ff 1.02 52 .5 51.2 If 1 .1 6 1 + 6 .2 3 9 .7 I so "butyrate 9 .1 1 1.07 -5 .7 1 5 . 3I* ff 1 . 3I + 7 .2 6 5.1*3 It 1.78 7.23 1+.06 9.10 2 .2 1 10.0 i*-55 tr 2 .6 7 1 1 .1 1+.16 I s o v a le r a te 9.10 1.00 3.28 3.26 9.11 1.28 3.23 2 . 1+ 9 ti 1 . 1 + 5 1+.22 2 .9 1 ft 1.9 5 5 .2 8 2 .7 1 T rim e th y l 9.10 1 .0 1 0.1+35 0 .1+29 a c e ta te 9.11 1.30 0 .6 4 7 0 .1+97 it 1 . 1 + 0 O.588 0.1+20 it 1.68 O.878 0.522 9.10 2.17 1 .2 0 0 .5 5 1 TABLE 19— Continued 6 3 E s te r (E )q X 10® (M) (S) X 10* ' '0 (M) k* X 108 ( s e c " 1 ) k»/(B)o X 10"* (M 1 Bee"1 ) 3 , 3“ D im etiiyl- 9.10 1.00 0.142 0 .1 4 2 b u ty ra te 9 . H 1.58 0.184 0.116 9 .1 0 1.63 0.175 0.107 ft 2 .0 4 0.226 0 .1 1 1 a2 5 . 0 % (j. * * 0.075 M, 4 .6 8 $ a c e t o n i t r i l e . 64 TABLE 20 PSEUDO—FIRST-ORDER RATE CONSTANTS FOR THE ACYLATION OF O-CHYMDTHYPSIN BY ESTERS OF p-NITROPHENOL, pH 7 .3 4 PHOSPHATE BUFFER8 , E B ter (E) x 10® °(M) (s} o X 104 (M) k* X 102 (sec**1 ) k * / ( s ) o ^ 10“* (M “ X Bee**1 ) A c e ta te 1 3 .4 1 .4 l 25.2 1 7 .9 II 1.88 2 3 . I 12.3 12.8 2.15 2 4 .7 11.6 tr 2.52 37-4 1 4 .9 15.4 2 .8 1 3 6 .4 12.9 P ro p io n a te 8.34 1 .0 7 2 1 .5 2 0 .1 if 1.28 3 4 .7 27.0 ff 1 .4 3 5 2 .9 37*1 rt 1.88 3 6 .7 19.6 it 2.13 72.1 33-8 it 2.81 6 4 .1 2 2 .8 B u ty ra te 8 .^ 4 0 .9 2 4 2 9 .3 3 1 .7 if 0.938 3 0 .4 3 2 .4 if 1.15 37*5 3 2 .6 ft 1 .1 7 35*1 30.0 if 1 .5 4 52.0 33-8 >i 1.56 39-2 2 5 .1 it I .92 37*7 1 9 .6 11 1.9 5 57*3 2 9 .3 it 2 .3 1 50.1 21.7 >i 2.34 62.0 2 6 .4 H exanoate 5 .5 5 O.625 5 4 .4 87.1 ti 0.781 6 5 .4 83*7 it 0.938 65*7 70.0 11 I .25 8 8 .4 7 0 .7 I s o b u ty r a te 1 3 .4 1.34 11.5 8.62 IT 1.52 1 7 .1 11*5 1 1 I .78 15*1 8 .4 6 12.8 2 .2 1 2 5 .4 11.5 If 2.67 35*7 13.4 I s o v a le r a te 9 . H 1.30 7.26 5.60 It 1-95 10.9 5.62 8 .9 9 2 .4 0 12.8 5 .3 6 TABIE 20— Continued 65 E s te r (E )q X 10® (M) (s) x 10* (M) k* X 10* (s e c " 1 ) W ( s ) G X 10“* (M -1 sec” 1 ) T rim e th y l 9.H 1 .1 2 1 .4 7 1 .3 1 a c e ta te f t 1 .3 0 1 .8 8 1.45 ir i . 4o 1 .8 7 1 .3 3 n 1 .6 8 2 .3 5 1 .4 2 3 , 3 -D im eth y l- 8 .9 9 1 .0 0 0.3 8 4 0.383 b u ty ra te 9.11 1 .2 1 0 .4 6 4 0.382 it 1 .3 2 0.592 0.449 tt 1 .5 8 0 .5 9 1 0.373 8 .9 9 2 .0 4 0.882 0.432 a2 5 . 0 ° , j j i . = 0.075 M, 4 .6 8 # a c e t o n i t r i l e . 66 TABUS 21 PSEUDO-FIRST-OREKR RATE CONSTANTS FOR THE ACYLATION OF Ot-CHYMDTRYPSIN BY ESTERS OF p-NITROPHEHOL, pH 7-58 PHOSPHATE BUFFER8 , E s te r (E)q X 10® (M) ( s ) o X 1o4 (M) k* X 108 (s e c - 1 ) k * / ( s ) o X (M 1 sec A c e ta te 8 .3 4 0 .9 3 8 1 3 .1 14.0 IV l . 4 l 19.8 1 4 .1 If 1 .8 8 25.2 1 3 .9 ft 2.34 32.5 1 3 .9 It 2 .8 1 3 5 .8 1 2 .8 P ro p io n a te 8.34 0.938 33*0 35 .2 II 1 .4 l 3 8 .2 27.2 tt 1 .8 8 5 5 .6 2 9 .7 ff 2.34 65.6 28.0 rr 2 .8 1 8 0 .1 28.5 B u ty ra te 8.34 0 .9 3 8 2 4 .1 2 5 .7 it 1 .1 7 31.6 2 6 .9 it 1.56 3 9 .0 25.0 ti 2 .3 4 52.8 22.5 H exanoate 5-55 0.625 6 5 .7 105 it 0.781 6 7 .4 86.2 it 0 .9 3 8 102 108 it 1.25 137 110 I s o b u ty r a te 8.34 0.890 1 1 .2 12.6 it 0 .9 7 9 11.6 1 1 .8 it I .07 15.9 1 4 .9 tt 1.25 17.8 14.3 11 1 . 3^ 19.1 1 4 .3 I s o v a le r a te 8.34 0.871 4 .2 2 4.8 5 ti 1 .1 2 5 .6 2 5.00 tt 1.1*5 1 0 .3 7 .0 9 it 1 .8 1 1 2.5 6.92 rr 2 .1 8 1 3 .3 6 .1 1 T rim eth y l 9.24 0.981 2 .2 2 2 .2 6 a c e ta te tt 1.1 2 3 .6 4 3.25 11 1 .1 2 2.70 2 .4 1 it 1 .4 0 3 .7 9 2.70 it 1 .6 8 3 .1 3 1 .8 6 it 2.17 4 .4 o 2.03 TABI£ 21— Continued 67 E s te r (E ) x 10« (M) ( s ) o X 10* (M) k* X 102 (s e c - 1 ) * * / ( s )0 x 10-2 (M -1 se c ” 1 ) 5 ,5 — Dime th y 1 - 9 .2 4 0 .9 2 5 O .76I 0.825 b u ty r a te I f 1.06 0 .6 8 0 0 .6 4 4 I I 1 .5 2 1.2 2 0.928 It 1 .5 8 1 .5 0 0 .8 2 4 It 2 .0 4 1 .2 1 0 .5 9 1 a2 5 .0 ° , p , = 0.075 M, 4 .6 8 £ a c e t o n i t r i l e . 68 TABLE 22 PSEUDO-FIRST-ORDER BATE CONSTANTS FOR THE ACYLATION OF O-CHYMDTRYPSIN B Y ESTERS OF p-NITRDPHENOL, pH 7 .9 9 PHOSPHATE BUFFER8 , E s te r (E) X 10® °(M ) (S )o X 10* (M) k* X 108 (s e c - x ) k * /(s)Q X 10“2 (M- i sec” 1 ) A c e ta te 7.95 O.958 2 5 .8 2 5 .4 11 1 .4 1 2 8 .0 19-9 11 1.88 2 7.5 1 4 .6 II 2 .^ 4 5 2 .5 13.8 If 2 .8 1 55-4 12.6 P ro p io n a te 7.95 1 .4 1 2 5 .6 18 .2 tl 1 .8 8 57-0 1 9 .7 ft 2.54 38.8 16.5 ft 2 .8 1 45.5 1 6 .2 B u ty ra te 7-95 0.859 25.2 29.4 11 1 .1 7 3 3 .4 2 8 .5 n 1.56 3 9 .8 2 5 .5 it 1.95 4o.8 2 0 .9 H exanoate 7.95 0.859 4 9 .3 57*3 ir 0.958 65.6 70.0 ti 1.09 89.0 8 1 .4 ft 1 .4 1 7 8 .4 55-8 I s o b u ty r a te 7.95 0.890 1 1 .1 12 .5 it 0 .9 7 9 1 5 .1 1 5 .4 ti I .07 1 9 . I 1 7 .9 ti 1.25 2 1 .6 1 7 .4 ti 1 .5 4 2 0 .2 1 5 .1 I s o v a le r a te 7.95 0 .8 2 1 5.2 5 6 .3 9 it 1 .1 2 4 .7 0 4 .1 9 it 1.45 7 .0 8 4 .8 8 it 1 .8 1 8 .6 4 4 .7 7 11 2 .1 8 1 0 .2 4 .6 8 a25 . 0 % p = 0.075 M, 4 .6896 a c e t o n i t r i l e . 69 TABLE 2 3 PSEUDO-FIRST-ORDER RATE CONSTANTS FOR THE ACYLATION OF Ct~CHYMOTRYPSIN BY ESTERS OF p-NITRDPHENOL, pH 7.99 TRIS BUFFER8 , (E )o X 10® (S )q X 104 k* X 102 W (s)Q X Uf“ E s te r (M) (m) (se c - 1 ) (M"1 se c - 1 ) A c e ta te 7-95 0.958 2 0 .9 22.3 If l . 4 l 22.3 1 5 .9 ft l .88 27 .5 1 4 .7 fl 2.34 3 6 .4 15.6 fl 2 .8 1 40.5 l 4 .4 P ro p io n a te 7.95 0.938 28.6 30.6 If l . 4 l 4 4 .7 31.8 If 1.88 66.2 35 .3 If 2 .3 4 89.0 38.0 II 2 .8 1 9 2 .3 32.8 B u ty ra te 7-95 0 .8 5 9 31.2 3 6 .3 If 1 .1 7 32.1 2 7 .4 ft 1.56 4 3 .3 27.7 It 1 .9 5 50.2 2 5 .7 II 2 .3 4 6 4 .2 27.4 H exanoate 7.95 0.859 7 4 .3 86.5 ft 0 .9 3 8 94.2 100 If 1 ,0 9 101 92.3 If I .25 123 98.4 If 1 . 4 l 127 90.4 Is o h u ty r a te 7-95 0.890 1 5 .3 17.4 ft 0 .9 7 9 1 5 .9 16.3 ft 1.07 1 8 .0 1 6 .8 ft 1 .3 4 23.6 1 7 .7 I s o v a le r a te 7-95 O.871 6 .5 9 7 .5 7 ft 1 .1 2 10.6 9.45 fl 1.45 9 .2 4 6 .3 7 ft 1 .8 1 1 4 .1 7-79 II 2 .1 8 1 1 .6 5 .3 5 T rim e th y l 9 .2 4 O .98I 1.43 1 .4 6 a c e ta te If 1.1 2 1.72 1 .5 3 If 1 .4 0 2 .0 0 1 .4 2 If 1 .6 8 2.50 1 .4 8 70 TABLE 23— Continued (S )o X 10® (8 )o X 10* k* x io 2 W ( s )0 X 10- E s te r C m) (M) ( s e c " 1 ) (M-J“ s e c " 1 ) 3 , 3 -D im eth y l- 9.24 0.923 O .606 0.656 b u ty ra te I T 1.06 0.654 0.610 tt 1.32 o.8o4 0 .6 1 0 r r 1 .5 8 0.924 0.584 a25 . 0 °i f n = 0.075 Mj 4.6856 a e e to n l t r l l e . 71 TABLE 24 PSEUDO-FIRST-ORDER RATE CONSTANTS FOR THE ACYLATION OF Ct-CHYMO TRYPSIN BY ESTERS OF p-NITROPHBNOL, pH 8.12 TRIS BUFFER* E a te r (E)q X 10® (M) (S) X 104 °(M) k* X 10® (sec™1 ) W (s)0 X ] (M~X sec” 3 A c e ta te 7.95 0.958 15.7 16.8 r r l . 4l 27.4 19.5 t r 1.88 27.O 14.4 ti 2.54 33.7 14.4 H 2 .8 1 44.5 15.8 P ro p io n a te 7.95 0.938 29.7 31.7 1 1 1.41 45.1 30.6 I I 1.88 54.8 29.2 I f 2.54 68.0 29.0 t r 2.81 70.2 25.0 B u ty ra te 7-95 0.859 25.4 27.2 1 1 1.17 27.O 25.0 ff 1.56 54.4 22.0 If 1.95 53-8 27.6 I I 2 .5 4 68.0 29.0 H exanoate 7-95 0.859 89.2 104 tf 0.958 102 109 1 1 1.09 142 130 I f 1.25 162 150 I s o b u ty r a te 7-95 0.979 17.4 17.8 r r 1.07 15.1 14.1 ir 1.25 18.0 15.4 « 1.34 20.2 15.2 I s o v a le r a te 7.95 0.871 6 .8 1 7.82 II 1.12 7.88 7.02 II 1.45 7.11 4.90 It 1.81 8 .7 1 4.80 II 2.18 9.26 4.26 T rim eth y l 9.24 O.98I 1.76 1.80 a c e ta te It 1.12 2.27 2.02 If 1.40 5.04 2.17 1 1 1.68 3.12 1.85 1 1 2.17 5.42 1.58 72 TABLE 24—-Continued E s te r (E) X 10* ' 'o (M) (s) X 10* (M) k* X 102 (se c * ■ ) W ( s ) Q x 10“* (M"1 see""*) 3 , 3-Dime th y 1 - 9.2 4 0 .9 2 3 0 .5 1 7 O.56O b u ty ra te II 1.06 o.6o4 0 .5 7 ^ II 1 .5 2 0.858 0.650 It 1 .5 8 0.802 0 .5 0 7 If 2 .0 4 0.963 0.472 a25 . 0 °j n = 0.075 M, 4.68% a c e t o n i t r i l e . 75 TABLE 25 PSEUDO-FIRST-ORDER RATE CONSTANTS FOR THE ACYIATION OF O-CHXIOTRYPSIN BY ESTERS OF p-NITHOPHENOL, pH 8 .90 TRIS BUFFER8 , (E) X 10° (S )o X 10* k* x 10® k*/(s)0 x 10"* E s te r (M) (M) (Bee"*1 ) (M*1 sec” 1 ) A c e ta te 7.95 0 .9 5 8 18.7 20.0 1 1 1 .4 1 23.2 16.5 ! ! 1.88 2 6 .1 1 5 .9 I I 2 .5 4 59.2 1 6 .7 I I 2 .8 1 4 1 .8 1 4 .9 P ro p io n a te 7-95 0.9 5 8 4 l .o 4 3 .7 1 1 1 .4 1 4 4 .7 31.8 It 1.88 50.0 2 6 .7 B u ty ra te 7-95 0 .8 5 9 25.8 30.1 n 1 .1 7 2 9 .1 2 4 .8 tt 1.56 5 6 .9 23.6 1 1 1 .9 5 4 8 .8 25.0 r r 2 .^ 4 4 9 .4 21.1 Hex&noate 7-95 0 .8 5 9 9 6 .7 112 ii 1 .0 9 101 9 2 .3 1 1 I .25 123 98.2 I s o t u t y r a te 7.95 O.890 8.95 10.0 1 1 0 .9 7 9 12.6 12.9 1 1 1.07 7 .8 4 7 .5 4 if 1.25 12.0 9 .6 7 1 1 1 .5 4 12.5 9 .5 8 I s o v a le r a te 7.95 1.45 5 .7 8 5 .1 9 1 1 1 .8 1 6 .5 9 3 .6 4 1 1 2 .1 8 7 .5 5 5 .5 7 a2 5 . 0 ° / n = 0.075 M, 4 .6 8 # a c e t o n i t r l l e . F ig . 9*— S eco n d -o rd er p l o t f o r th e a c y la tio n o f CX-chymotrypsin by p - n itro p h e n y l b u ty r a te , 2 5 .0 ° , y. = 0*075 M, l+.68$ a c e t o n i t r i l e , pH 5-Sff. 7 ^ 0 1 2 3 (S). x 104 (M) 76 TABIE 26 SECOND-ORDER RATE CONSTANTS FOR THE ACYLATION OF Ct-CHXMQ TRYPSIN BY ESTERS OF p-NITROPHENOLa E s te r pH ( W \ > (M"1 1 se c -1 X W (s)0 10- ) I n te r c e p t S y A c e ta te 5-92 3-66 + 2.0 4.02 0.01 0.01 6.19 3.77 + 1 .7 3.23 0.03 0.01 7-34 10.4 + 0.8 13.9 0.07 o .o 4 7.58 12.4 + 2.0 13.7 0.02 0 .0 1 7 -99* 5.90 + 2.6 17.3 0.18 0.01 7.99 11.4 + 4.0 16.6 0.08 0.02 8.12 15.6 + 6.2 16.2 0.04 0.03 8.90 13.3 ± 5.3 16.4 0.05 0.02 P ro p io n a te 5.92 6.52 + 1-9 5.75 - 0.01 0.01 6.19 5.79 + 0 .2 7 7 . 4o o .o 4 0.01 7 0 ^ 25.0 + 24.2 26.7 0.06 0.11 7.58 25.9 + 5.7 29.7 0.06 0.02 7-99 56.6 + 10.9 33.7 -0 .0 5 0.05 8.12 22.6 + 6.9 2 9 .I 0.11 0.03 B u ty ra te 5.92 6.49 ± 1*7 6 .8 9 0.01 0 .0 1 6 .1 9 8.32 ± 2 *3 H .3 0.06 0.01 7.34 17.8 + 11.3 28.2 0.15 0.06 7.58 19.8 + 3 .4 25.0 0.07 0.01 7 .99* 14.2 + 6.1 26.1 0.15 0.02 7.99 22.6 + 6 .0 28.9 0.09 0.03 8.12 31.2 + 8 .9 25.8 - 0.08 o .o 4 8.90 17.8 + 5.4 24.9 0.10 0.02 Hexanoate 5-92 35*6 + 5.1 32.2 -0 .0 3 0.02 6.19 46.0 + 17.0 40.8 ~o.o4 0 .0 7 7-34 51.9 + 8 .7 77.9 0.22 0.0 3 7.58 123 + 19.7 102 - 0.18 0 .0 7 7 . 99, 47.0 + 32.4 66.1 0.20 0.11 7-99 93.4 + 12.3 93.6 0.29 0.05 8.12 195 + 11.9 118 -O.78 0.05 8.90 6 3 .1 ± 33.6 101 0.39 0.05 Is o b u ty ra te 5.92 3.10 + 3-3 4.38 0.02 0.02 6.19 3.32 + 1.2 4.71 0.02 0 .0 1 7-34 17.2 ± 5.5 10.7 - 0.12 0.02 7 -58^ 18.6 + 2 .1 13.6 -0 .0 5 0.01 7 -99° 2 0 .7 + 4.1 15.7 -0 .0 5 0.02 7.99 19.2 ± 1 *1 + 17.0 - 0.02 0.01 8.12 9.58 + 3*6 15.1 0 .0 7 0.01 8.90 6.25 + 4.0 9.86 0.04 0.02 TABLE 26— Continued 77 E p te r PH < W * * ) ' (M"1 1 sec ” 1 X W (s)0 io-“) I n te r c e p t ® s r I s o v a le r a te 5.92 0.981 + 0 .6 1 1.90 0 .0 1 0.003 6 .1 9 2 .3 1 + 0 .7 7 2.84 0 .0 1 0.003 7 - 3^ 5 .1 2 + 1 .1 5.52 0 .0 1 0.002 T*58b 7*53 + 2 .3 6.00 -0 .0 2 0.01 7 . 99: 4.09 + 1 .3 4.98 0 .0 1 0.01 7.99 4.00 + 3 .9 7.31 0.04 0.02 8 .1 2 1.74 + 1 .0 5.76 0.05 o .o o 4 8.90 2.14 + 0 .1 2 3 . 4o 0.03 0.0002 T rim eth y l 5.92 0.244 + 0.03 0.310 0 .0 0 1 0.0001 a c e ta te 6 .1 9 0.667 + 0 .1 0 0.484 - 0.003 o.ooo4 7 -3^ 1.50 + 0 .1 8 1.38 -0 .002 0.001 7.58 1.32 + 1 .0 2.42 0 .0 1 0.01 7-99 1.45 + o . i 4 1.48 0.0003 0.001 8 .1 2 1.31 + 0.60 1.88 0 .0 1 0.003 3 , 3-D im ethy1 - 5.92 0.0667 + 0.0 2 0.112 0.0 0 1 0.0001 b u ty ra te 6 .1 9 0.0787 + 0 .0 2 0.119 0.0 0 1 0.0001 7 -3^ 0.460 + 0 .0 1 o .4o4 -0 .0 0 1 o.ooo4 7.58 0.498 + o .4o 0.762 0.003 0.002 7-99 0.495 + 0 .0 2 0.615 0 .0 0 1 0.0001 8 .1 2 0.375 + 0 .1 6 0.553 0.0 0 2 0.001 a2 5 .0 ° , ix = 0.0 7 5 M, 4 .6 8 # a c e t o n l t r l l e . b P h o sp h ate b u f f e r . C T r ls b u f f e r . 78 c o n c e n tra tio n s lo v s o l u b i l i t y may r e s u l t in some o f th e s u b s tr a te p re c i p i t a t i n g o u t o f s o lu tio n , c a u sin g th e r a t e c o n s ta n t a t t h i s s u b s tr a te c o n c e n tra tio n to be to o lo v j (b) a t lo v s u b s tr a te c o n c e n tra tio n , where th e m agnitude o f th e b u r s t i s l e s s , from e q u a tio n ( 5 ), th e r e i s more e r r o r i n th e method o f e x tr a p o la tio n s in c e h e re th e ste a d y s t a t e r a t e i s la r g e r e l a t i v e to th e b u r s t , and (OD)^ i s n o t v e ry much d i f f e r e n t from (OD)^. In t h i s ca se any e r r o r in th e d e te rm in a tio n o f (OD)^ i s more pronounced a t low th a n a t h ig h s u b s tr a te c o n c e n tra tio n s (35)* T h e o re tic a l c u rv e s have been f i t t e d t o th e p H -ra te d a ta , u s in g th e fo llo w in g e q u a tio n : 1 5 & 1 .V K m Cl + H/Kj. + Ka/H} ( 1 8) w here Kj. and Ha a re th e io n iz a tio n c o n s ta n ts o f th e e s s e n t i a l groups n e c e s sa ry f o r en zy m atic c a t a l y s is and (ka/Kjn) i s th e pH -independent a c y la tio n r a t e c o n s ta n t. The m ethod o f A lb e rty (55) was u sed to c a lc u l a t e Kx and Ka. The pH v a lu e s f o r th e pH optimum o f th e r a t e v s . pH f u n c tio n as w e ll s b th e p o in ts a t which t h i s fu n c tio n was a t h a l f th e maximum h e ig h t on e i t h e r s id e o f th e optimum w ere e s tim a te d . These v a lu e s co rresp o n d t o - lo g H q, - lo g % , and - lo g H®, r e s p e c tiv e ly . Then + Ha - KX + Ifflo, (19) and Ha = H^/Kx. (20) A f te r Kj. and Ke have been d e te rm in e d , th e y a re s u b s titu te d back in to e q u a tio n (1 8 ), alo n g w ith th e e s tim a te d v a lu e o f (k e /K ^ ). The c a lc u la - t i o n I s I t e r a t e d u n t i l th e YalueB o f Kj., Kg, and (k^/KjO w hich g iv e th e b e s t f i t to th e e x p e rim e n ta l p o in ts a re fo u n d . The c a lc u la te d v a lu e s o f pKX/ pK^, pHoptimuau 3 X 1 (1 (kaA m ) a r e shown in T able 2 7 , f o r each o f th e compounds s tu d ie d . A t y p i c a l p H -ra te p r o f i l e i s shown i n F ig u re 10. The p l o t o f lo g ( W ^ r e l " h e re ( W W r e l = (W K m )/(W K m )° and (ke/Km)° i s th e se c o n d -o rd e r a c y la tio n r a t e c o n s ta n t f o r th e a c e ta te e s t e r , v s . EB , was made u s in g th e pH -independent v a lu e s o f k a / l ^ g iv en in T able 2 7 . The p lo t i s shown in F ig u re 11. The p o in t f o r th e ace t a t e e s t e r h a s been o m itte d f o r com parison w ith th e d eacy l& tio n d a ta . A ll d a ta on th e p l o t e x c e p t f o r th e h ex an o ate e B te r a re c o r r e la te d w ith a Blope o f 0.95 + 0 .2 2 ( c o r r e l a t i o n c o e f f i c i e n t , 0 . 98)- I f th e p o in t f o r th e a c e ta te e s t e r i s in c lu d e d , th e s lo p e i s 0 .8 2 + 0 .J 8 ( c o r r e la t i o n c o e f f i c i e n t , 0.9 * 0 • 8o TABLE 27 THEORETICAL pK VALUES, pH OPTIMA, AND pH-INDEPENDENT RATE CONSTANTS DETERMINED FOR THE ACYLATIQN OF C X -C H Y M Q TRYPSIN BY ESTERS OF p-NITRDPHENOLa No. E s te r -rn 1 1 I 1 J uj - T r: PKi PK^ P^optlmum. ( W kJ (M _1 sec- 1 ) 1 A c e ta te 6 .6 8 10.12 8 .4 0 1550 2 P ro p io n a te 6 .7 3 9 .2 7 8 .0 0 3200 3 B u ty ra te 6 .5 8 9 .4 2 8 .0 0 2825 4 H exanoate 6 .9 9 9 .0 1 8 .0 0 i4ooo 5 I s o b u ty r a te 6 .7 9 8 .8 2 7 .8 0 1850 6 I s o v a le r a te 6 .6 6 8 .5 4 7 .6 0 800 7 T rim eth y l a c e ta te 6 .8 4 8 .5 6 7 .7 0 225 8 3 , 3-Dlme th y 1 - b u ty ra te 7 .0 7 8 .4 3 7-75 80 a 2 5 . 0 ° , n = 0.075 M, 4 .6 8 $ a c e t o n l t r i l e . 8 1 I____________ I __________ ■ ' ■ 5 6 7 8 9 pH F ig . 1 0 .— p H -ra te p r o f i l e f o r th e a c y la tlo n o f a -c h y n jo try p sin by p - n itr o p h e n y l a c e t a t e , 2 5 .0 * , u * 0.075 M, 4 .6 6 ^ a c e t o n l t r i l e ; O f e x p e rim s n ta l p o i n t s ; s o l i d l i n e i s cu rv e c a lc u la te d u s in g th e c o n s ta n ts l i s t e d in T ab le 2J. F ig . 1 1 .—P lo t o f l o t (k a/K n Jp ei v s . Es f o r th e a c y la tio n o f O -chym otrypsin by e s t e r s o f p - n itr o p h e n o l, 2 5 . 0 °, n = 0.075 M, 4 .6 8 $ a c e t o n i t r i l e . The num bers r e f e r to th e e s t e r s l i s t e d in T able 27. 82 0 .5 < b L. % E -0 .5 o > o - 1.0 -2 .0 -1.6 -1.2 -0 8 -0.4 Es 0 0 DISCUSSION OF RESULTS S t e r i c E f f e c ts From a com parison o f th e T a ft s t e r i c e f f e c t s p l o t s f o r th e a c y - l a t i o n and d e a c y la tio n o f o -c h y m o try p sin , and f o r th e tv o n o n -en zy m atic system s s tu d ie d , i t can he seen t h a t th e enzyme p l o t s have s lo p e s o f ab o u t 1 .0 , -while th e m odel system s have s lo p e s o f 0 .5 and 1 .4 . Thus th e enzyme r e a c tio n i s in te rm e d ia te betw een n u c le o p h ilic c a t a l y s i s by im id a zo le and c l a s s i c a l g e n e ra l b ase c a t a l y s i s by im id azo le in i t s s u s c e p t i b i l i t y to s t e r i c e f f e c t s . I t m ight b e .e x p e c te d t h a t s t e r i c e f f e c t s w ould be l e s s im por t a n t in a g e n e ra l b a s e -c a ta ly z e d r e a c tio n s in c e an i n c ip ie n t h y d ro x id e io n i s c o n s id e ra b ly s m a lle r th a n im id a z o le . The s lo p e much l e s s th a n u n ity found f o r th e T a ft p l o t f o r N -a c e ty lse rin a m id e e s t e r s i s , how ever, s u r p r is in g s in c e th e r e a c tio n m ight re a s o n a b ly be ex p e c te d to have n e a r ly th e same s u s c e p t i b i l i t y t o s t e r i c e f f e c t s a s th e h y d ro x id e io n o r hydronium io n -c a ta ly z e d h y d r o ly s is o f e th y l e s t e r s . The Es con s ta n t s w ere d e riv e d from e th y l e s t e r h y d r o ly s is d a ta (4 7 ), w hich Im p l i e s a 6 o f 1 .0 f o r t h i s r e a c tio n . The ra te B o f enzym atic a c y la tio n and d e a c y la tio n w ere m easured a t 2 5 °, and th e im id a z o le -c a ta ly z e d h y d r o ly s is o f th e p - n ltr o p h e n y l e s t e r s was s tu d ie d a t 3 0 °, bo th e s e two s e t s o f d a ta a re d i r e c t l y com parable. The T a ft p l o t f o r th e N -a c e ty lse rin a m ld e e s t e r s , how ever, 84 was made from d a ta c o lle c te d a t 95 “ • I f s ig n i f i c a n t d if f e r e n c e s ex iB t i n th e a c t i v a t i o n e n e rg ie s f o r th e v a rio u s compounds in th e s e r e a c tio n s , th e n th e s lo p e s o f th e T a ft p l o t s w ould be m arkedly dependent on tem p er- a t u r e . T3ie a c t iv a tio n e n e rg ie s f o r th e h y d r o ly s is o f th e p -n itro p h e n y l e s t e r s a re n e a r ly c o n s ta n t (T able 9 )/ so th e slo p e sh o u ld be n e a r ly 1.14- o v e r a wide te m p e ra tu re ra n g e . U sing th e a c tiv a tio n e n e rg ie s ^ d e te r m ined f o r th e d e a c y la tio n o f acyl-C t-chym otrypsins^ th e k3 v a lu e s have been e x tr a p o la te d from 2 5 ° to 95 "• The k 3 v a lu e s a t 95° a re g iv en in T able 28 . TABLE 28 RATE CONSTANTS EXTRAPOLATED TO 95° FOR THE DEACYLATION OP ACXL-C8-CHYM0 TRYPSINS No. A cyl Group k3 X 102 (sec**1 ) 1 A c e ty l 3 5 .2 2 P ro p io n y l 6 5 .9 3 B u ty ry l 5 ^ .8 5 I s o b u ty r y l 35-6 6 I s o v a le r y l 2 8 .1 7 T rim e th y la c e ty l 6 .2 0 The v a lu e f o r prop io n y l-0 6 -ch y m o try p sin h as b een in c lu d e d be cau se a c e ty l-O -c h y m o try p sin f a l l s o f f th e T a ft p l o t , a s n o te d in th e o th e r enzym atic r e a c tio n s . The r e s u l t i n g p l o t o f lo g ( k a '/ k ^ 0 ) v s . Es a t 95° i s shown i n F ig u re 12. The slo p e i s 0 .6 8 + 0 .2 1 ( c o r r e la tio n c o e f f i c i e n t , 0 .97)* Thus, com paring T a ft p l o t s a t s im ila r te m p e ra tu re s ^These v a lu e s can be d e riv e d from th e A H* v a lu e s i n T able 15. 2 These v a lu e s a re u n d o u b ted ly more a c c u ra te th a n th o s e d e te r m ined f o r th e h y d ro ly s is o f e s t e r s o f N -a c e ty lse rin a m id e b ecau se o f th e e x tre m e ly slow r a t e s o f h y d r o ly s is f o r th e l a t t e r s e r i e s o f compounds. F ig . 1 2 .—P lo t o f lo g Qt3 '/ k 3 '° ) v s . Es f o r th e d e a c y la tio n o f acy l-C t-ch y m o try p sin s, e x tr a p o la te d to 95 °. The numbers r e f e r t o th o s e compounds l i s t e d in T ab le 28. 86 Z'Hlt'A) Bo| 0.2 o - 0.2 - 0.6 07 - 0A - 0 . 8 Ec - 1.2 - 1.6 88 shows t h a t th e d if f e r e n c e in s lo p e i s due m ainly to th e d iff e r e n c e in x e x p e rim e n ta l te m p e ra tu re s . The s lo p e s a t 9 5 while com parable f o r th e e s t e r s o f N -a c e ty lse rin a m id e and f o r th e d e a c y la tio n o f acyl-C t- ch y m o try p sin s, a r e much s m a lle r th a n th e slo p e f o r th e Im id azo le n u c le o p h ile - c a ta ly z e d h y d ro ly s is o f p - n itr o p h e n y l e s t e r s . The enzym atic r e a c tio n , th e r e f o r e , m ust more n e a r ly resem b le a g e n e ra l b a s e -c a ta ly z e d r e a c tio n in re g a rd to s e n s i t i v i t y to s t e r i c e f f e c t s . I t w ould seem p o s s ib le , a p r i o r i , from a c o n s id e ra tio n o f th e co m p lex ity o f th e enzym atic r e a c tio n , t h a t th e r a t e c o n s ta n ts w ould be s u b je c t t o la r g e s t e r i c in flu e n c e s due to th e p r o te in en v iro n m en t. T hat t h i s i s n o t th e c a s e , and t h a t th e r a t e d e c re a se s a re p r im a r ily r e f l e c t i n g s t e r i c h in d ra n c e to ap p ro ach o f a n u c le o p h ile , i s shown by th e l i n e a r i t y o f th e T a ft p l o t (F ig u re s 5 /H ) * The s t e r i c c o n s ta n ts , EB, w ere o b ta in e d from e s t e r h y d r o ly s is d a ta (V f), and can be con s id e re d a m easure o f p o te n tia l- e n e r g y and li n e t i c - e n e r g y e f f e c t s r e s u lt i n g from in c r e a s in g h in d ra n c e to n u c le o p h ilic a tta c k a t th e e s t e r c a rb o n y l. A d d itio n a l in flu e n c e s such bb c o n fo rm a tio n a l changes in duced by i n t e r a c t io n o f th e a c y l group w ith th e p r o te in w ould, t h e r e f o r e , be e x p ec te d to cau se d e v ia tio n from th e o b serv ed l i n e u n le s s such i n t e r a c t i o n produced e f f e c t s on th e r a t e t h a t w ere a ls o a l i n e a r fu n c t i o n o f th e Es c o n s ta n ts , w hich w ould seem u n lik e ly . x ^A te m p e ra tu re o f 95° i s , o f c o u rse , f a r above any e x p e r i m e n ta lly f e a s i b l e w ith th e enzyme due to p h y s ic a l changes in th e p ro t e i n w hich ta k e p la c e a t h ig h te m p e ra tu re s . The assu m p tio n i s made i n t h i s e x tr a p o la tio n t h a t th e s t e r i c e f f e c t s a re due to th e n an -en zy m atic, n o t th e p r o te in , c h a r a c te r o f th e r e a c tio n , and th e r e f o r e th e A rrh e n iu s p l o t w ould be l i n e a r o v er t h i s te m p e ra tu re ra n g e . 89 S u p p o rtin g t h i s Y ie v p o in t i s th e f a c t t h a t th e p o in t f o r th e n - h ex an o y l d e r i v a t iv e , w hich h a s th e f a s t e s t r a t e o f a c y la tio n and deac y la tio n i n th e s e r i e s , shows a m arked d e v ia tio n from th e l i n e . The f a s t r a t e o f a c y la tio n and d e a c y la tio n o f t h i s d e r iv a tiv e i s v e ry l i k e l y due to i n t e r a c t io n o f th e lo n g c h a in a c y l group w ith a h y d ro p h o b ic re g io n n e a r th e a c tiv e s i t e , p e rh a p s in d u c in g a c o n fo rm a tio n a l change so t h a t u n fa v o ra b le e x p e n d itu re s o f f r e e en erg y can be av o id ed i n a t t a i n i n g th e t r a n s i t i o n s t a t e . I n t r a n s e s t e r i f i c a t i o n r e a c tio n s th e r a t e h a s b een fo u n d t o in c r e a s e w ith in c r e a s in g c h a in le n g th o f th e p rim ary a lc o h o l (56, 5 7) in d ic a tin g t h a t th e b in d in g o f th e n u c le o p h ile It- o c c u rs a t a h y d rophobic re g io n a d ja c e n t to th e a c tiv e s i t e . I t sh o u ld be n o te d t h a t th e r e i s a s l i g h t d if f e r e n c e i n th e r e l a t i v e r a t e r a t i o s f o r a c y la tio n and f o r d e a c y la tio n . These v a lu e s a r e shown i n T ab le 2 9 . I t can be seen t h a t th e r a t i o s o f th e r e l a t i v e r a t e s f o r a c y la tio n t o th o s e f o r d e a c y la tio n a re c lo s e to u n ity f o r th e s t r a i g h t c h a in compounds. However, f o r th e b ran ch ed compounds th e r e seems to be a g r e a te r d if f e r e n c e betw een a c y la tio n and d e a c y la t i o n cau sed by b in d in g .'* The r a t i o K /K ° , where K ° I s th e K v a lu e J mf m ' m m f o r th e a c e ta te e s t e r , I s th e in v e rs e o f th e r a t i o o f th e r e l a t i v e r a t e f o r a c y la tio n to t h a t f o r d e a c y la tio n , I . e . , k3r e l/(k e /K m )re l* Inw ard and Je n c k s (5 6 ) n o te t h a t th e r e i s no s a tu r a tio n e f f e c t due to in c r e a s in g c o n c e n tra tio n s o f th e n u c le o p h ile , s u g g e s tin g t h a t th e r e i s no b in d in g s i t e a s su ch , b u t a re g io n w hich I n t e r a c t s fa v o r a b ly w ith th e n u c le o p h ile , p ro b a b ly by hydrophobic b o n d s. 5 I t sh o u ld be remembered t h a t th e d e a c y la tio n r a t e c o n s ta n t i s k 3 , w h ile th e a c y la tio n r a t e c o n s ta n t In c lu d e s b o th k a , w hich i s th e r a t e c o n s ta n t f o r th e r e a c tio n w hich i s th e m ic ro sc o p ic r e v e rs e o f th e k 3 r e a c tio n (5 5)t an^- Kj^, th e " b in d in g " c o n s ta n t. 90 TABLE 29 C O M P A R IS O N O P T H E RELATIVE R A T E RATIOS F O R T H E A C YLATIO N O P ESTERS O F p-NITRQPHENOL B Y O -CH YM Q TR X PSIN W IT H T H O S E F O R T H E D EA C Y L A TIO N O F T H E RESULTING ACYL-O-CHIM0TRYPSINSa E s te r ( W ^ r e l ” k3r e l C (ke/Km) r e l / l t 3 r e l A c e ta te 1 .0 1 .0 1 .0 P ro p io n a te 2 .1 1 .6 1 .3 B u ty ra te 1 .8 1 .2 1 .5 H exanoate 9 .0 7 .4 1 .2 I s o b u ty r a te 1 .2 0 .5 2 2 .3 I s o v a le r a te 0 .5 2 0.25 2 .0 T rim e th y la c e ta te 0.15 0 .0 3 3 .0 5 , 3-Dim ethy1 - b u ty r a te 0.05 0 .0 3 1 .7 a2 5 .0 ° , n = 0.075 M. t>(ka /Km) / ( k l 2/KJ 1 1 ) " # T able 27, 4 .6 8 $ a c e t o n i t r i l e . clc.$/hiS0, T able 10, 1 .6 $ a c e t o n i t r i l e . 91 These r a t i o s f o r a l l th e compounds p l o t t e d a g a in s t Es g iv e a maximum s lo p e o f ab o u t 0.3* Thus b ra n c h in g h a s a sm a ll e f f e c t on b in d in g . H o fstee (2 9 ) s tu d ie d th e h y d r o ly s is o f n - f a t t y a c id e s t e r s o f hydroxy ben z o ic a c id s by chym otrypsin a t pH 8 . He fo u n d t h a t v h ile in c re a s e d e x p o n e n tia lly from C 2 --C7, was e s s e n t i a l l y c o n s ta n t up to a c h a in le n g th o f C7 . Thus i t seems t h a t th e v e ry f a s t r a te o f h y d ro ly s i s o f p - n itr o p h e n y l h ex an o ate i s due to an e f f e c t on th e c a t a l y t i c p ro c e ss i t s e l f , n o t on b in d in g . T h is co u ld be due to a hydrophobic i n t e r a c t io n o f th e a l i p h a t i c c h a in w ith th e p r o te in v h ic h in some vay makes th lB e s t e r more s u s c e p tib le to n u c le o p h lllc a tt a c k , as m entioned above. T hat t h i s co u ld be an e f f e c t o f o r ie n ta ti o n i s seen by th e more fa v o ra b le e n tro p y o f a c t i v a t i o n o f th e h ex an o ate e s t e r a s compared to th e b u ty r a te e s t e r , v h ile th e r e i s no d if f e r e n c e in t h e i r a c tiv a tio n e n e rg ie s (T ab le 1 5 ). A c tiv a tio n P a ra m ete rs S e v e ra l s tu d ie s have been made o f s t e r i c in flu e n c e s on th e a c t iv a tio n p a ra m e te rs i n h y d ro x id e io n and hydronium io n -c a ta ly z e d e s t e r h y d r o ly s is . Wien compounds o f th e ty p e C H 3COOR w ere s tu d ie d in 62$ a c e to n e , w ith s t e r i c h in d ra n c e v a r ie d in th e a lc o h o l p o r tio n o f th e e s t e r (58- 6° ) , a com pensation in th e a c t i v a t i o n p a ra m e te rs v a s n o te d , w ith P, th e i s o k i n e t i c te m p e ra tu re , b e in g around 3° ° ° K. (2 7 0 -33°* K.). I t h a s been su g g e ste d (5 0 ) t h a t t h i s com pensation may be due to s t e r i c in te r f e r e n c e w ith s o lv a tio n Bince th e t r a n s i t i o n s t a t e complex i s an a n io n , and s in c e th e v a lu e o f P 1b in a ran g e f r e q u e n tly a s s o c ia te d w ith s o lv a tio n e f f e c t s . I n c o n t r a s t , th e s a p o n if ic a tio n o f s a tu r a te d 92 a lk y l a c e ta te s In w a te r shows a c o n s ta n t en erg y o f a c tiv a tio n ( 6 l ) . S t e r i c e f f e c t s in th e a lk y l and a c y l p o r tio n s o f an e s t e r in v o lv e d i f f e r e n t s t e r i c i n t e r a c t io n m echanisms (5 0 ). Thus, v a r i a t io n o f B te ric h u lk in th e a c y l p a r t o f th e e s t e r g iv e s somewhat d i f f e r e n t r e s u l t s . In th e a c id - c a ta ly z e d e s t e r i f i c a t i o n o f v a rio u s c a rb o x y lic a c id s in m ethanol (6 2 ), A H* i s e s s e n t i a l l y c o n s ta n t, v h ile A S* de c re a s e s o v er a ran g e o f 4 .4 eu . H ighly b ran ch ed c a rb o x y lic a c id s such a s tr im e th y la c e tic and 3 , 5 -d im e th y lb u ty ric a re c h a r a c te r iz e d by A H* v a lu e s t h a t a re more u n fa v o ra b le th a n f o r s t r a i g h t ch ain a c id s . In a stu d y o f th e a lk a lin e h y d ro ly siB o f a s e r i e s o f e s te r s o f th e ty p e RCOaEt in 85$ e th a n o l (6 3 ) , th e d if f e r e n c e s in r a t e a re due alm o st en t i r e l y to changes in Ea , e x c ep t f o r compounds b ran ch ed a t th e Cl-carbon, w hich a ls o show changes in A S * . The v a r i a t io n in Ea i s o n ly ab o u t 3*2 k c a l o v er th e e n t i r e s e r i e s . I n a s tu d y o f th e a lk a lin e h y d ro ly s is o f th e same e s t e r s in J0$> a c e to n e , A 8* i s a g a in f a i r l y c o n s ta n t and Ea v a r ie s o v e r about 4 .5 k c a l f o r th e compounds s tu d ie d , w h ile i n th e case o f a c id - c a ta ly z e d h y d r o ly s is , b o th a c t i v a t i o n p a ra m e te rs v a ry s l i g h t l y w ith no ob v io u s tr e n d (6 4 ). Thus, i n s tu d ie s made o f th e e f f e c t on th e a c t i v a t i o n p a ra m e te rs o f v a ry in g th e s t e r i c b u lk in th e a c y l p o r tio n o f an e s t e r , e i t h e r A H* o r A S* o r b o th p a ra m e te rs become more u n fav o r a b le . I t 1b d i f f i c u l t t o be c e r t a i n w h eth er th e r e i s any r e a l tre n d i n th e case o f th o se r e a c tio n s in v h lc h one o f th e a c tiv a tio n param e t e r s becomes more u n fa v o ra b le b ecause o f th e sm a ll ran g e o v er which th e p a ra m e te rs v a ry (6 5) . The a c t i v a t i o n p a ra m e te rs f o r th e im id a z o le -c a ta ly z e d h y d ro ly s is o f a s e r i e s o f p -n itro p h e n y l e s t e r s (T able 9 ) show th e same ty p e o f 95 b e h a v io r. A H* i s n e a r ly c o n s ta n t, in c r e a s in g by a t o t a l o f o n ly 1 .6 k c a l o v er th e e n t i r e s e r i e s , and o n ly 0 .9 k c a l a s s t e r i c b u lk i s in c re a se d from a a e ty l to tr im e th y la c e ty l, w h ile A 8* becomes more nega t i v e o v er a ran g e o f 6 .1 eu a s s t e r i c h in d ra n c e in c r e a s e s . The im id a z o le -c a ta ly z e d h y d r o ly s is o f e s t e r s o f N -a c e ty lB e rin a - m lde shows a v id e ran g e o f v a r i a t io n in th e a c t iv a tio n p a ra m e te rs (T able 6 ) , A H* and A S* b o th d e c re a s in g a s s t e r i c b u lk I s in c re a s e d from a c e ty l o r b u ty r y l to is o b u ty r y l. A l i n e can be drawn betw een th e th r e e p o in ts on th e i s o k in e tic p l o t h av in g a s lo p e o f K. I t ap p e a rs t h a t th e r e may be an i s o k i n e t i c r e l a t io n s h i p betw een th e members o f t h i s s e r i e s . However, th e o rd e r i s n o t a r a t i o n a l on e; t h a t i s , i t i s n o t in th e o rd e r o f in c r e a s in g s t e r i c b u lk . There i s a v e ry sm a ll d if f e r e n c e in th e r a t e s o f h y d r o ly s is o f th e th r e e compounds a t 9 1 °/ 0 . The av erag e r a t e i s 9*5 x 10~3 M 1 min"1 w ith a v a r i a t i o n o f 20^. However, w h eth er o r n o t an is o k in e tic r e l a t io n s h i p e x i s t s , th e r e i s a la r g e v a r i a t io n in th e a c t i v a t i o n p a ra m e te rs in c o n tr a s t to th e im i d a z o le n u c le o p h ile - c a ta ly z e d r e a c tio n . I t was found t h a t a ama.11 com pensation i s o b serv ed f o r th e hy d r o ly s is o f N -acy lim id azo liu m io n s (6 6 ) , compounds whose h y d r o ly s is r e a c tio n s a re s u b je c t t o g e n e ra l c a t a l y s i s (6 7 ) . The com pensation was o v e r a range o f o n ly 1 .7 k c a l and ^.5 e u , how ever, w ith a slo p e o f 375° K. Pronounced s t e r i c b u lk a ls o cau ses m arked d e v ia tio n from th e is o k i n e t i c li n e in th e s e r e a c tio n s . There i s a d if f e r e n c e betw een th e b e h a v io r o f th e N -a c y llm ld a z o le s and t h a t o f s p e c if ic b a se o r n u c le o - p h i l i c c a t a l y s i s . A H* becomes more fa v o ra b le and A 8* , more u n fav o r a b le a s s t e r i c b u lk i s in c re a s e d in th e h y d r o ly s is o f N -acy llm id azo liu m 94 io n s , r a t h e r th a n b o th becom ing more u n fa v o ra b le . The a u th o rs a t t r i b u te d t h i s b e h a v io r to a c o n c e rte d mechanism. The d e a c y la tio n o f ac y l-O -ch y m o try p sln s shows a m arked compen s a tio n betw een th e a c t i v a t i o n p a ra m e te rs (3*3 k c a l, 8 .4 e u ) , w ith b o th A H* and A S* becom ing more p o s itiv e a s th e s t e r i c b u lk in th e a c y l group i s in c re a s e d (T able 15)* The slo p e o f th e com pensation p l o t f o r a c y l g roups t h a t a re n o t h ig h ly b ran ch ed i s 405° K. (F ig u re 7)* The n a tu re o f th e e f f e c t t h a t cau ses th e com pensation betw een A H* and A S* in th e d e a c y la tio n r e a c tio n i s n o t c l e a r . S o lv a tio n e f f e c t s co u ld be v e ry Im p o rta n t, a lth o u g h com pensation due to s o lv a tio n e f f e c t s i s u s u a lly marked by an i s o k i n e t i c te m p e ra tu re c lo s e r to room te m p e ra tu re (5 0 ). S ince th e l i n e a r i t y o f th e T a ft p l o t seems to r u le o u t hy d ro p h o b ic i n t e r a c t io n w ith th e p r o te in , i t i s u n lik e ly t h a t th e o b se rv e d com pensation i s due to any cau se o th e r th a n th e mechanism o f th e r e a c tio n . I t was n o t p o s s ib le to d eterm in e a c t i v a t i o n p a ra m e te rs f o r th e a c y la tio n r e a c tio n b ecau se o f th e l i m i t a t i o n s in B u b stra te s o l u b i l i t y and f e a s i b l e e x p e rim e n ta l te m p e ra tu re s w hich e x is te d . From th e d a ta g iv e n i t a p p e a rs t h a t th e n u c le o p h llic - and c l a s s i c a l g e n e ra l b a s e -c a ta ly z e d r e a c tio n s s tu d ie d d i f f e r in th e e f f e c t o f s t e r i c b u lk on th e a c t i v a t i o n p a ra m e te rs . The n u c le o p h ile - c a ta ly z e d r e a c tio n e x h ib its no co m pensation, and o n ly a sm a ll v a r i a t io n in a c tiv a tio n p a ra m e te rs , w h ile th e c l a s s i c a l g e n e ra l b a s e -c a ta ly z e d r e a c tio n may e x h ib it com pensation, and shows a l a r g e r v a r i a t i o n in a c t iv a tio n p a ra m e te rs . The Knzyme R e a ctio n O rganic S o lv en t E f f e c ts The optimum r a t e f o r th e a c y la tio n by p -n itr o p h e n y l a c e ta te o f Ofr-chymo t r y p s i n , 1550 se c” 1, com pares fa v o ra b ly w ith t h a t d eterm in e d by Kezdy and Bender (3 4 ): i 960 M 1 sec ”1 in 4$ a c a t o n i t r i l e , and 353° NT1 sec”1 In 1 .6 $ a c e t o n i t r i l e . The p re s e n t stu d y was p erfo rm ed in 4 .6 8 $ a c e t o n i t r i l e . There i s a l i n e a r d e c re a se in th e r a t e c o n s ta n ts w ith in c r e a s in g m o la rity o f a c e t o n i t r i l e , a s can be seen from th e d a ta o f Clement and B ender (6 8 ) in t h e i r s tu d ie s o f m ixed s o lv e n ts . There i s some d if f e r e n c e betw een th e e s tim a te d pK v a lu e s de term in e d in t h i s stu d y and p re v io u s ly d eterm in ed v a lu e s (T able 2 7 ) . R ep o rted v a lu e s by B ender, e t a l . (6 9 ) g iv e th e fo llo w in g pH dependence f o r p - n itr o p h e n y l a c e ta te in 1 . 6$ a c e t o n i t r i l e : f o r k g , pKj, = 6 . 5 9 # pKg = 8 . 6l , P-^opt 1 T m im * 7*®> f o r kg/K ^, pKj, * = 6 .8 5 , pKg = 9*04* pHoptinnaa = 7-9* Clement and B ender (6 8 ) p o s tu la te t h a t o rg a n ic s o l v e n t a f f e c t s o n ly K^. Then, th e t h e o r e t i c a l pK v a lu e s g iv en above f o r kg w ould be th e same a s th o se f o r k g / l ^ in p u re w a te r. I f t h i s i s th e c a s e , th e e f f e c t o f a c e t o n i t r i l e seems t o be t o in c r e a s e pKg (from 8 . 6 l to 10.12 w ith a change o f a c e t o n i t r i l e from 0 t o 4 .6 8 $ ) and to f l a t t e n th e pH -(kg/K ^) curve (a s can be seen by th e lo w e rin g o f th e o p t imum r a t e a s a c e t o n i t r i l e c o n c e n tra tio n i s In c re a s e d ) by changing 1 ^ . pKg r e f l e c t s th e pH dependence o f b in d in g ( 70) K aplan and ^B ender, e t a l . (70) show t h a t a lth o u g h pKg r e f l e c t s th e pH dependence o f Kq and t h a t ke shows no pH dependence f o r s p e c if ic sub s t r a t e s , p -n itr o p h e n y l a c e ta te shows no pH dependence o f Km, b u t o n ly o f kg. However, K aplan and L a id le r ( 71) f in d t h a t k s i s pH Independent on th e b a s ic s id e o f th e optimum, w h ile K^ i s pH dependent on th e b a s ic s id e , im p ly in g t h a t pKg i s a d i r e c t consequence o f th e pH dependence o f K m f o r p - n itr o p h e n y l a c e t a t e . 96 L a id le r (71) found t h a t In tr o d u c tio n o f o rg a n ic s o lv e n t p e r tu r b s pK^ to h ig h e r v a lu e s , up to g r e a te r th a n 11 in 26.7?t d io x an e. The e f f e c t o f o rg a n ic s o lv e n t a p p e a rs to d e c re a se a s b ran ch in g in c r e a s e s . The v a lu e o f k ^ / l ^ o f 225 M"1 s e c " 1 f o r th e trim e th y la c e t a t e e s t e r d eterm in e d in t h i s stu d y i s com parable to t h a t p re v io u s ly d eterm in e d (2 5 ) in 1 .6 £ a c e t o n i t r i l e , kg/Kja = 232 M 1 s e c " 1 . Comparison to Model S tu d ie s I t was o b serv ed above t h a t th e enzym atic r e a c tio n v a s much more lik e th e im id a zo le g e n e ra l b a s e -c a ta ly z e d r e a c tio n in s e n s i t i v i t y to s t e r i c e f f e c t s when compared a t th e same te m p e ra tu re th a n to th e im id a z o le n u c le o p h ile - c a ta ly z e d r e a c tio n . The b e h a v io r o f th e a c t i v a t i o n p a ra m e te rs due to changing s t e r i c b u lk f o r th e enzym atic r e a c tio n a ls o seems t o be more l i k e th e g e n e ra l b ase m echanism. I t may b e , how ever, t h a t th e p a tte r n s o f s t e r i c e f f e c t s and a c t iv a tio n p a ra m e te rs a re p a r t l y a consequence o f w h eth er c a t a l y s is in v o lv e s a t e t r a h e d r a l in te rm e d ia te o r a c o n c e rte d r e a c tio n , a s w e ll a s w h eth er th e r o le o f Im id azo le i s t h a t o f a g e n e ra l b ase o r o f a n u c le o p h ile . As m entioned above, h y d ro ly s is o f N -acy lim id azo liu m io n s prob a b ly goes by a c o n c e rte d m echanism , bond b re a k in g b e in g sim u ltan eo u s w ith a tta c k o f th e i n c ip ie n t h y d ro x id e io n . T h is mechanism was p ro posed f o r two re a s o n s : (a ) S t e r i c h in d ra n c e cau sed r a t e a c c e le r a tio n . T h is co u ld be e x p la in e d e i t h e r by a c o n c e rte d r e a c tio n o r by a mechan ism in v o lv in g th e r a t e — d e te rm in in g breakdown o f a t e t r a h e d r a l i n t e r m e d ia te . (b ) L i t t l e 0i a exchange v a s found (7 2 ), w hich w ould r u le o u t a k l n e t i c a l l y s ig n i f i c a n t t e t r a h e d r a l in te rm e d ia te . The p a tte r n o f th e a c t i v a t i o n p a ra m e te rs , in c re a s in g s t e r i c h u lk c au sin g A H* to become more fa v o ra b le and A S* to become more u n fa v o ra b le , i s c o n s is te n t v i t h t h i s . In c o n t r a s t , th e a lk a lin e h y d r o ly s is o f e th y l b en zo ate (7 3 ) t and p ro b a b ly t h a t o f a l l such sim ple e s t e r s (7*0/ p ro ceed s th ro u g h a t e t r a h e d r a l in te rm e d ia te a s d eterm in e d from 0 i 8 exchange v i t h th e s o l v e n t (75>76). T his ty p e o f r e a c tio n shovB a d i f f e r e n t p a t t e r n o f 9 " R-C-OR' OH a c t i v a t i o n p a ra m e te rs , A H* and A S* b o th becom ing more u n fa v o ra b le v i t h in c r e a s in g s t e r i c b u lk . A c tiv a tio n p a ra m e te rs f o r th e Im id azo le n u c le o p h ile -c a ta ly z e d h y d ro ly s is o f p - n itr o p h e n y l e s te r s shov s im ila r b e h a v io r. The a c tiv a tio n p a ra m e te rs f o r th e im id a z o le -c a ta ly z e d h y d ro ly s i s o f e s t e r s o f N -a c e ty lse rin a m id e do n o t shov a r a t i o n a l o rd e r; th e r e i s a d e c re a se in b o th A H* and A S* f o r th e is o b u ty r a te e s t e r a s com p a re d to th e a c e ta te e B te r, w h ile th e b u ty ra te e s t e r shows In c re a s e d A H* and A S* a s compared to th e a c e ta te e s t e r . I t may be t h a t b ran ch in g ca u ses th e compound to undergo a c o n c e rte d r e a c tio n w h ile th e s t r a i g h t c h a in e s t e r s more fa v o ra b ly h y d ro ly z e v ia t e t r a h e d r a l i n t e r m e d ia te s. The l i n e a r T a ft p l o t a t 95° seems to r u le o u t a change in m echanism . A lso , a s L e f f l e r (6 5 ) p o in ts o u t, an i s o k i n e t i c r e l a t i o n s h ip can be ex p ec te d to a p p ly o n ly to a s e rie B o f r e a c tio n s in w hich a change o f s tr u c tu r e does n o t cau se a change i n th e mechanism o f th e r e a c tio n o r in th e n a tu re o f th e t r a n s i t i o n s t a t e . B ecause o f th e la c k o f c e r t a i n t y co n c ern in g th e e x is te n c e o f an i s o k i n e t i c r e la tio n s h ip f o r th e serin am id e e s t e r s , i t i s d i f f i c u l t to d e c id e w h eth er a change in mechanism co u ld be o c c u rrin g h e r e . The T a ft p l o t f o r sp o n tan eo u s h y d ro ly s is g iv e s th e same slo p e a s t h a t f o r th e im id a z o le -c a ta ly z e d r e a c tio n a t 95°# and p re lim in a ry te m p e ra tu re s tu d ie s in d ic a te a p a t t e r n o f s t e r i c e f f e c t s such t h a t b ra n c h in g in c r e a s e s b o th A H * and A S*. Bender and Heck (77) found t h a t in th e a lk a lin e h y d r o ly s is o f fl-a c e ty l-Q -c in n a m o y l-se rin a m ld e -cin n am o y l-carb o n y l-Oi a f ca rb o n y l oxygen exchange w ith th e s o lv e n t ta k e s p la c e , su g g e stin g t h a t t h i s r e a c tio n p ro c e e d s v ia a t e t r a h e d r a l in te rm e d ia te . Thus, i n th e h y d r o ly s is o f N -a c e ty lse rin a m ld e e B te rs , i t i s l i k e l y t h a t th e mechanism in v o lv e s a tta c k o f an i n c ip ie n t h y d ro x id e io n a t th e c a rb o n y l t o form a t e t r a h e d r a l In te rm e d ia te , w hich th e n b re a k s down t o p r o d u c ts . The more fa v o ra b le e n tr o p ie s o f a c t i v a t i o n w hich may be cau sed by in c r e a s in g s t e r i c b u lk a re p o s s ib ly due to th e g e n e ra l b ase c h a r a c te r o f t h i s r e a c tio n , sin c e t h i s b e h a v io r i s n o t seen in th e n u c le o p h ilic r e a c tio n . 99 The enzym atic r e a c tio n h a s A H* becoming more u n fa v o ra b le and A S* becoming more fa v o ra b le a s s t e r i c b u lk in c r e a s e s . T h is b e h a v io r c o u ld be c o n s is te n t w ith a r e a c tio n in v o lv in g a t e t r a h e d r a l in te rm e d i a t e , an alo g o u s to t h a t p ro p o sed f o r th e N -a c e ty lse rin a m id e e s t e r s above. However, in th e d e a c y la tio n o f clnnam oyl-C t-chym otrypsin- c ln n a - m o y l-ca rb o n y l-O*®, 0*® exchange was n o t o b serv ed (77)* It* I s p o s s ib le t h a t no exchange co u ld be n o te d even though th e acyl-enzym e h y d ro ly zed th ro u g h a t e t r a h e d r a l in te rm e d ia te , i f th e two oxygens w hich would be in v o lv e d in th e exchange r e a c tio n n ev er become e q u iv a le n t, a s th e a u th o rs p o in t o u t. In any c a s e , th e enzym atic r e a c tio n p ro b a b ly in v o lv e s im id azo le a s a g e n e ra l b ase a c tin g to remove a p ro to n from w a te r, and a t e t r a h e d r a l in te rm e d ia te may be in v o lv e d in t h i s r e a c tio n . R-C-O-CH A nother d iff e r e n c e betw een th e enzym atic r e a c tio n and th e serin am id e model system b e s id e s th e exchange d a ta i s t h a t th e p o in t f o r th e a c e ty l d e r iv a tiv e in th e enzym atic r e a c tio n alw ays shows a n e g a tiv e d e v ia tio n on th e T a ft p l o t . P erh ap s t h i s r e f l e c t s th e e x is te n c e o f some minimum c h a in le n g th below w hich s e v e ra l o r ie n ta tio n s o f th e s u b s tr a te can le a d to u n p ro d u c tiv e b in d in g . 100 I n g le s and Knowles (7 8 ) p o s tu la te th r e e s i t e s o f a tta c h m e n t o f th e s u b s tr a te to th e enzyme: (a ) by a c o v a le n t bond t o th e s e rin e hy d ro x y l group o f th e enzyme; (b) by a h y d ro p h o b ic i n t e r a c t io n betw een th e s id e c h a in and i t s c o rre sp o n d in g lo c u s a t th e a c tiv e s i t e ; (c ) by th e hydrogen b o n d (s) o f th e p e p tid e l i n k o f th e a c y l amino gro u p . Any s u b s tr a te a tta c h in g to th e enzyme a t a l l th r e e s i t e s w ould show th e maximum p o s s ib le r a t e o f r e a c tio n . p -N itro p h e n y l e s t e r s have a t m ost th e p o s s i b i l i t i e s o f in t e r a c t io n s (a ) and ( b ), though th e T a ft p l o t s would seem t o in d ic a te t h a t o n ly th e h ex an o y l d e r iv a tiv e i s in v o lv e d in i n t e r a c t io n ( b ) . Then th e s u b s tr a te m oiety h as more d e g re e s o f freedom . T h is means a low er d e a c y la tio n r a t e c o n s ta n t, and would ex p la in an e n tr o p ic o r ig in o f th e d if f e r e n c e s in d e a c y la tio n r a t e s be tw een th e p -n itr o p h e n y l e s t e r s w hich f i t on th e T a ft p l o t and th e hex an o ate e s t e r . The f a c t t h a t th e enzyme i s a polym er made up o f many sm all m oleculeb p o in ts up th e inadequacy o f m odels. One consequence o f th e f a c t t h a t th e enzyme i s a p r o te in 1b th e " re d s h i f t " w hich alw ays oc c u rs in th e n a tiv e a c y l— enzyme r e l a t i v e to th e d e n a tu re d acyl-enzym e f o r s u b s tr a te s o f th e a r o y l ty p e (79)* Charney and B ernhard (80) have su g g e ste d t h a t t h i s co u ld be due to th e tw is tin g by th e enzyme o f th e s u b s tr a te from th e s - t r a n s to th e s - c i s form ab o u t th e a c r y lo y l s in g le bond. T h is i s r e l a t e d to a su g g e stio n o f B ru ice ( 8 l) t h a t th e enzyme may a c t to change an e s t e r from th e t r a n s to th e c i s form . T h is ty p e o f b e h a v io r i s , o f c o u rse , im p o ssib le in model sy stem s. However, th e l i n e a r i t y o f th e T a ft p l o t s and th e i s o k i n e t i c r e l a t io n s h i p sugge s t t h a t o n ly one I n te r a c tio n mechanism i s ta k in g p la c e (V7) , th u s making 101 i t l i k e l y t h a t t h i s ty p e o f b e h a v io r, w h ile p o s s ib le f o r s p e c if ic sub s t r a t e s , i s n o t a p p lic a b le to t h i s system . Summary SI an ex am in atio n o f th e im id a z o le -c a ta ly z e d h y d ro ly s is o f e s t e r s o f N -a c e ty lse rin a m id e , i n w hich im id az o le fu n c tio n s a s a c l a s s i c a l g e n e ra l b a s e , T a ft s t e r i c e f f e c t s p l o t s f o r b o th th e im id a z o le - c a ta ly z e d and th e spo n tan eo u s (w a te r p lu s h y d ro x id e io n ) r e a c tio n have a slo p e o f 0.5* The a c t i v a t i o n p a ra m e te rs f o r th e im id a z o le -c a ta ly z e d r e a c tio n shov a w ide ran g e o f v a lu e s . The h y d ro ly siB o f e s t e r s o f p -n itr o p h e n o l w ith im id azo le c a ta ly s i s , in w hich im id azo le fu n c tio n s a s a n u c le o p h ile , g iv e s a T a ft p lo t h a v in g a slo p e o f 1 .4 . D e te rm in a tio n o f a c tiv a tio n p a ra m e te rs f o r t h i s r e a c tio n shows t h a t A H* i s e s s e n t i a l l y c o n s ta n t w h ile A 8* b e comes more u n fa v o ra b le by ab o u t 6 e n tro p y u n i t s a s s t e r i c b u lk i s in c re a s e d . The d e a c y la tio n o f acyl-05-chym otrypsins a ls o g iv e s a l i n e a r T a ft p l o t , v i t h a slo p e o f 1 . 0 . ' The p o in t f o r hexanoyl-Q S-chym otrypsin d e v ia te s p o s it i v e l y . T h is r a t e a c c e le r a tio n may be due to a h y d ro - p h o b ic in t e r a c t io n o f th e a l i p h a t i c s id e c h a in w ith th e p r o te in . The a c t i v a t i o n p a ra m e te rs e x h ib it com pensation, 0 b e in g 405" K. However, h ig h ly b ran ch ed compounds, a s w e ll a s th e h ex an o y l d e r iv a tiv e , do n o t f i t on t h i s p l o t . When th e r a t e c o n s ta n ts a re e x tr a p o la te d to 95° Tor com parison w ith th e serln am ld e d a ta , th e slo p e o f th e T a ft p l o t i s 0 .7 , a v a lu e much c lo s e r to t h a t f o r th e g e n e ra l b a se r e a c tio n th a n to t h a t f o r th e n u c le o p h ilic r e a c tio n , 1 .4 , w hich sh o u ld be e s s e n t i a l l y in v a r i a n t o v er t h i s te m p e ra tu re ra n g e . 102 T rie b u f f e r I b seen to p a r t i c i p a t e in th e d e a c y la tio n r e a c tio n , and to a f f e c t th e a c t i v a t i o n p a ra m e te rs . S in ce th e T r is p a r t i c i p a t i o n i s s u b je c t to s t e r i c h in d ra n c e , i t was th o u g h t t h a t th e a c t i v a t i o n p a ra m e te rs a re b e t t e r d eterm in ed i n a b u f f e r t h a t does n o t p a r t i c i p a t e i n th e r e a c tio n . The a c y la tio n by e s t e r s o f p - n itro p h e n o l o f OM shymotrypsin a ls o g iv e s a l i n e a r T a ft p l o t v i t h a slo p e o f 1 .0 . S in ce th e a c y la tio n r a t e c o n s ta n t d e te im ln e d , X s /Kq , v a s a complex c o n s ta n t in c lu d in g Kj^, i t v a s concluded t h a t s t e r i c b u lk had l i t t l e e f f e c t on th e b in d in g r e a c tio n , a s i t v o u ld be e x p e cted t h a t a T a ft p l o t u s in g o n ly th e kg v a lu e s v o u ld have a slo p e i d e n t i c a l to t h a t f o r th e d e a c y la tio n r e a c tio n . S in ce th e slo p e f o r th e T a ft p l o t u s in g k g / i s n e a r ly id e n t i c a l to t h a t f o r th e d e a c y la tio n r e a c tio n , s t e r i c b u lk can have l i t t l e e f f e c t on O rganic s o lv e n t h a s an e f f e c t on 1 ^ , and in c r e a s in g o rg a n ic s o lv e n t r a i s e s pKg, and lo v e r s th e pH -in d ep en d en t (optimum) r a t e . The enzym atic r e a c tio n a p p e a rs to be m ost d i r e c t l y com parable t o th e im id azo le g e n e ra l b a s e -c a ta ly z e d r e a c tio n . Thus, i t v o u ld in v o lv e th e im id azo le r in g o f th e h i s t i d i n e a t th e a c tiv e s i t e o f th e enzyme a c tin g to v ith d r a v a p ro to n from v a t e r (o r from s e r i n e ) , fo m t- in g an in c ip ie n t h y d ro x id e io n (a lk o x id e io n ) v h ic h v o u ld a t t a c k th e acyl-enzym e ( e s t e r ) in d e a c y la tio n ( a c y la tio n ) . REFERENCES CITED 103 REFERENCES CITED 1 . B ender, M. L ., and Z e m e r, B ., J . Am. Chem. S oc. , 8£ , 2391 (1961) . 2 . B ender, M. L ., and Z e m e r, B ., I b i d . , 8 4 , 2550 (1-962). 3 . Epand, R. M ., and W llBon, I . B ., J . B io l. Chem. , 2^8, 1718 (1963)* 4. M erco u ro ff, J . , and H ess, G. P ., Biochem. B iophys. R es. Conmun. , 11, 283 (1963). 5 . S tru m ey er, D. H ., W hite, W. N ., and K oshland, D. E ., J r . , P ro c . W atl. Acad. S c i. U .S . , £ 0 , 931 ( I 963) . 6 . A nderson. B. M ., C ordes, E . H ., and J e n c k s, W. P . , J . B io l. Chem., 2^6 , 455 (1 9 6 1 ). 7 . S choellm ann, G ., and Shaw, E ., F ed . P r o c . , 2 1 , 232 (1962) . 8 . Schoellm ann. G ., and Shaw, E ., Blochem. B iophys. R es. Commun. , J , >5 (1962) . 9 . S choellm ann, G ., and Shaw, E ., B io c h e m lstry , 2 , 252 (1 9 6 3 ). 10. Ong, E. B ., Shaw, E ., and Schoellm ann, G ., J . Am. Chem. S oc. , 86, 1271 (1 9 6 4 ). 11. B ru ic e , T. C ., and B enkovic, S. J . , "B io o rg a n ic M echanism s," W. A. B enjam in, I n c ., New Y ork, 196 6 , V o l. I , Chap. 2 . 12. O u tfreu n d , H ., and S tu r te v a n t, J . M ., P ro c . N a tl. Acad. S c i. U .S . , 42, 719 (1956) . 1 3 . S p en cer, T ., and S tu r te v a n t, J . M., J . Am. Chem. S oc. , 8 1 , 1874 (1 9 5 9 ). ~ 14. B ender, M. L ., and T u rn q u e st, B. W ., i b i d . , J 9 , 1652, 1656 (1957)- 15. B ru ic e , T. C ., and Schm ir, G. L ., i b i d . , J g , 1663 (1957)* 16. J e n c k s, W. P ., and C a rriu o lo , J . , i b i d . , 8£ , 1743 ( l 9 6 l ) . 1 7 . B ru ic e , T. C ., F i f e , T. H ., B runo, J . J . , and B enkovic, P . , i b i d . , 84, 3012 (1962) . 104 105 18. F i f e , T. H ., i b i d . , 8j , 4597 (1965)* 19. Langenbeck, W ., and M abrwald, R ., Chem. B e r. ,

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Creator Milstien, Julie Block, 1942- (author)

Core Title Steric effects in imidazole and ɑ-chymotrypsin catalyzed ester hydrolysis

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1968-01

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), [Mogel], Harris S. (committee member), Mehl, John W. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-610835

Unique identifier UC11360246

Identifier 6810242.pdf (filename),usctheses-c18-610835 (legacy record id)

Legacy Identifier 6810242.pdf

Dmrecord 610835

Document Type Dissertation

Rights Milstien, Julie Block

Type texts

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

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

Repository Name University of Southern California Digital Library

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