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The mechanism of alpha-chymotrypsin acylation by N-acylimidazoles

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The mechanism of alpha-chymotrypsin acylation by N-acylimidazoles

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Content THE MECHANISM OF
ALPHA-CHYMOTRYPSIN ACYLATION
BY N-ACYLIMIDAZOLES
by
Robert Linn Kogan
A D i s s e r t a t i o n P resen ted to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In P a r t i a l F u lf i ll m e n t of the
Requirements f o r the Degree
DOCTOR OF PHILOSOPHY
(B io c h e m istry )
September 1983
UNIVERSITY O F S O U T H E R N CALIFORN IA
T H E G R A D U A T E S C H O O L
U N IV E R S IT Y PA R K
L O S A N G E L E S . C A LI P O R N IA 9 0 0 0 7
This dissertation, written by
under the direction of h%$.... Dissertation Com
mittee, and approved by all its members, has
been presented to and accepted by The Graduate
School, in partial fulfillment of requirements of
the degree of
p i o
' 0 3
\<1S
ftobert.. .Linn. .Kogan.
D O C T O R OF P H I L O S O P H Y
Dean
DISSERTATION COMMITTEE
Chairman
DEDICATION
To my d a r l i n g w ife S h e rri whose lov e and su p p o rt have
c a r r i e d me through the many long y e a rs of hard work, and to
my sons Aaron and Ethan who a re my most p rec io u s p o sses
s io n s . To Ph il and Martha, w ithout whom none of t h i s would
have been p o s s ib le .
ACKNOWLEDGEMENTS
The author wishes to g r a t e f u l l y acknowledge Dr. T.H.
F ife for the many y e a rs of guidance and i n s t r u c t i o n which
have culm inated in th e p re s e n t work. Also to Dr. T .J.
P rz y sta s fo r h i s c o n tin u a l w i l li n g n e s s to o f f e r h e lp f u l
c r i t i c i s m and a r e c e p t i v e e a r. F in a ll y , to Dr. R.
N atarajan and Eleanor Kwong fo r t h e i r honesty, s i n c e r i t y ,
and f r i e n d s h i p .
ABSTRACT
The r a t e s of a c y l a t i o n of alpha-chym otrypsin by a s e r i e s
of N -acy lim id azo les have been measured using a l i p h a t i c and
s u b s t i t u t e d benzoyl acyl groups. The v a lu e s of k2 /Km have
been determ ined in th e pH range 5 .0 - 9 .0 ( 4 .0 - 9 .0 fo r se
l e c t e d benzoyl d e r i v a t i v e s ) a t 30°C. A cy latio n of the ac
t i v e s i t e was measured by the p r o f l a v i n displacem ent te c h
nique. S p e c i f i c i t y for the a c t i v e s i t e was shown by the
r a t e of d e a c y la tio n a t pH 7 .0 - 8 .0 which was e s s e n t i a l l y
i d e n t i c a l in each case w ith t h a t of the acyl enzyme d e r iv a
t i v e prepared w ith th e co rrespo nd ing p - n itr o p h e n y l e s t e r .
The r a t e of a c y l a t i o n i n c r e a s e s w ith the le n g th of the acyl
group and i t s h y d ro p h o b icity (as measured by the Hansch pi
v a lu e s ) , hydrocinnam oylim idazole having k2 /Km=1 . 2 x 1 0 ^
M “ ^sec"^ and c y clo h e x y lp ro p io n y lim id a z o le having
1 .28x10^M” ^sec"^ a t pH 7 .5 . P lo ts of k y s k / [ S ] 0 have i n
f i n i t e slo p e s i n d i c a t i n g Km i s very la r g e r e l a t i v e to the
range of CS] 0 employed in t h i s study. The v a lu e s of k2 /Km
were n early independent of pH except f o r the N '-m e th y la te d
d e r i v a t i v e s where k2 /Km in c r e a s e s w ith pH u n t i l a maximum
i s reached (pKgpp=6 . 5 - 6 . 6 ). This d i f f e r e n c e r e f l e c t s th e
iv
f a c t t h a t r e a c t i o n of the N' -m e th y la te d d e r i v a t i v e i s v ia a
p o s i t i v e l y charged s p e c ie s a t a l l pH v a lu e s, whereas the
unm ethylated compounds r e a c t through both the n e u tr a l spe
c i e s and c o n ju g a te a c id s w ith s i m il a r r a t e c o n s ta n ts . The
r a t e c o n s ta n ts fo r the corresp on din g m ethy lated and unmeth
y la te d d e r i v a t i v e s e x t r a p o l a t e to the same value a t approx
im ately pH 4 .0 , showing t h a t the r e a c t i o n a t low pH i n
volves a c y l a t i o n of the enzyme by the N -acylim idazole con
ju g a te a c id . The l i m i t i n g value of ^2^^m ^or ^he
N-3»3“ d im e th y lb u ty ry l-N ’-m ethyl-im idazolium ion was 3.1
tim es slower in D2 O than in HgO ( 2 . 1 tim es slower fo r
N -(p-dim ethylam ino)benzoyl-N '-m ethy-im idazolium i o n ) . Thus
h i s t i d i n e - 5 7 in th e a c t i v e s i t e i s p a r t i c i p a t i n g in the
r e a c t i o n in th e base form, and th e r e a c t i o n in v o lv e s proton
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 . I t can be concluded t h a t
the nearly pH-independent r e a c t i o n s of the unm ethylated
d e r i v a t i v e s a t pH>7 must a ls o r e p r e s e n t n e u tr a l h i s t i
dine-57 c a ta ly z e d a c y l a t i o n of se rin e-1 9 5 by n e u tr a l N-a-
c y lim id a z o le s . Thus, h i s t i d i n e - 5 7 p a r t i c i p a t e s in the
r e a c t i o n s as a general base most l i k e l y by p a r t i a l l y ab
s t r a c t i n g a proton from the s e r in e hydroxyl in th e t r a n s i
t io n s t a t e . K i n e t i c a l l y e q u iv a le n t mechanisms in v o lv in g
the h i s t i d i n e - 5 7 co n ju g ate acid a c t in g as a g en eral acid
can be r u le d out. Hammett rho v a lu e s fo r a c y l a t i o n by sub
v
s t i t u t e d N -benzoylim idazoles a re 0.9 a t pH 7.5 and zero a t
pH 4 . 0 - 5 . 0 . These v a lu e s a re l e s s than those fo r a c y l a t i o n
r e a c t i o n s where the l e a v in g group pKa i s much lower than
im idazole anion. Thus much l e s s charge development occurs
in th e t r a n s i t i o n s t a t e r e l a t i v e to o th er s u b s t r a t e s .
P lo ts of l o g ( k 2 /Km) .vs pi ( th e Hansch h y d ro p h o b icity con
s t a n t s ) and Es (th e T a ft s t e r i c c o n s ta n ts ) show t h a t a c y la
t io n i s g r e a t ly in flu e n c e d by h y d rop ho bicity and much l e s s
so by s t e r i c f a c t o r s , a r e s u l t which i s j u s t th e r e v e r s e
f o r p -n itr o p h e n y l e s t e r s . Thus N -acy lim id azo les a c y l a t e
alp ha-ch ym o try p sin through q u i t e a d i f f e r e n t mechanism,
i . e . , t r a n s i t i o n s t a t e , than n itr o p h e n y l e s t e r s .
TABLE OF CONTENTS
DEDICATION...................... . . . . ................................................. i i
ACKNOWLEDGEMENTS ....................................................... i i i
ABSTRACT...................................................................... iv
Chapter page
I . INTRODUCTION .......................................................................................... 1
I I . EXPERIMENTAL.............................. . . 12
I I I . RESULTS.............................................................................................................24
IV. D ISCUSSION................................ ...........................................................61
SELECTED BIBLIOGRAPHY ... . .. ................................................... 89
LIST OF TABLES
Table page
1. C o n c en tratio n s of R eactants in th e A cylation of
Chymotrypsin by N-Acylimidazoles . . ............................ 20
2. The Log(ko/K ) a t pH 8.0 of Several N-Acylimidazoles
With tne co rrespo nd in g Pi and Eg Values. . . . . 44
vii
LIST OF FIGURES
F igure page
1. S t r u c t u r e s of N-Acylim idazoles and N'-Methyl N-
A c y lim id az o le s.............................. 10
2. P lo ts of k i s k / [ S ] Q f o r a c y l a t i o n of a lp h a -
chymotrypsin a t pH 6.48 by N - is o v a le r y lim id a z o le
and by N -b u ty ry lim id az o le a t pH 5.98 a t 30°C. . 27
3. P lo ts of k .ys k / [ S ] 0 f o r th e a c y l a t i o n of a lp h a -
chymotrypsin a t pH 7.0 by N-(p-methoxy)
b enzo ylim idazole and a t pH 8.0 by N-(p-
c h lo ro )b e n z o y lim id a z o le a t 30°C...................................... 29
4. P lo ts of k .ys [S]» f o r the a c y l a t i o n of alpha-
chymotrypsin a t pH 6.48 by N - is o v a le r y lim id a z o le
and a t pH 5.98 by N -b u ty ry lim id az o le a t 3 0 °C. . 31
5. P lo t of k i s CS30 fo r a c y l a t i o n of alph a-
chymotrypsin a t pH 7.0 by N-(p-methoxy)
b enzo ylim idazo le a t 30°C.................................................. . 33
6 . P lo t s of log(ko/K m) vs. pH fo r the a c y l a t i o n of
alph a-chy m otryp sin by N -acy lim idazoles a t 30°C. 37
7. P lo t s of log(kp/K m) x s pH for the a c y l a t i o n of
alpha-chym otrypsin by N -benzoylim idazoles a t
30 C................................................................................................... 39
8 . P l o ts of log(kp/K m) \rs pH fo r the a c y l a t i o n of
alph a-chym otrypsin by N^-cyclohexylacylimidazoles
a t 30°C.................................................................................................41
9. P lo t of l o g ( k 2 /Km) vs pi c o n s ta n ts l i s t e d in Table
2 . 45
10. P lo t of l o g ( k 2 /Km) _ys Es c o n s ta n ts l i s t e d in Table
viii
11. Log(k2 /Km) ( N -acylim id azole) vs l o g ( k 2 /Km)
( n itr o p h e n y l e s t e r s ) ........................................................................49
12. P lo ts of l o g ( k 2 /Km) a t 30°C f o r a c y l a t i o n of a lp h a -
chymotrypsin by s u b s t i t u t e d N -ben zoy l-im id azoles
vs th e Hammett c o n sta n t (sigma) a t pH 5.0 and a t
pH 7 . 5 ............................................................................................................52
13. P lo t of p -n itr o p h e n y l i s o v a l e r a t e h y d r o l y s i s by
alph a-chy m otry psin with (lower l i n e ) and w ith
out (upper l i n e ) N - i s o v a l e r y l im i d a z o le ...........................54
14. P lo ts of lo g kQb_ pH fo r h yd roly s i s of N-(p-
dim ethylam ino)benzoyl-N ’-m eth y l-im -id azo liu m ion
and N -(p -d im e th y la m in o )-b e n zo ly lim id az o le in H20
a t 30°C, 0.01 M b u f f e r , 0 . 1 1 ............................................ 7 59
ix
Chapter I
INTRODUCTION
The u ltim a te aim of enzyme mechanism re s e a rc h must be
to c l e a r l y d e fin e those param eters which c o n tro l enzymatic
c a t a l y s i s such t h a t th e a c tio n of an enzyme on a given sub
s t r a t e can be p r e d ic te d q u a n t i t a t i v e l y . Alpha-chym otrypsin
i s one of the most e x te n s iv e ly s tu d ie d enzymes in the
s c i e n t i f i c l i t e r a t u r e . I t has been so because i t s r e l a
t i v e l y simple s t r u c t u r e , ease of p u r i f i c a t i o n and i t s ap
p a r e n tly s t r a ig h t f o r w a r d r e a c t i o n scheme make i t one of the
most l i k e l y enzymes f o r which a com pletely q u a n t i t a t i v e
m e c h a n istic model could be fo rm u la te d . Yet, although a
g r e a t deal has been l e a r n e d about i t s mechanism of a c tio n
through the use of simple e s t e r s and amides as w ell as o th
er unique s u b s t r a t e s , a d e f i n i t i v e m e c h a n istic model for
chymotrypsin*s h y d r o l y ti c a c t io n has not been developed.
Alpha-Chymotrypsin.^ A lpha-chym otrypsin i s a p ro te o
l y t i c enzyme which i s m anufactured in th e acinous c e l l s of
the pancreas as a c a t a l y t i c a l l y i n e r t zymogen, chymotryp-
sinogen. Chymotrypsinogen i s then converted in th e small
Hess, G. P ., "The Enzymes", P.D. Boyer, Ed.; Academic
P re s s : New York, 1970, vol. 3, ch ap ter 7, p . 213.
1
i n t e s t i n e to th e a c t i v e enzyme by p r o t e o l y t i c cleavage.
Cleavage by t r y p s i n between a r g i n i n e 15 and i s o l e u c i n e 16
g e n e r a te s a f u l l y a c t i v e enzyme. F u rth e r cleavage by chy-
m otrypsin between le u c in e 13 and s e r i n e 14 l i b e r a t e s th e
s e r - 1 4 / a r g - 1 5 d ip e p tid e and r e s u l t s in th e form atio n of
d elta-c h y rao try p sin . Subsequent chym otryptic cleavage l i b
e r a t e s th e d ip e p tid e th r e o n i n e - 1 4 7 / a s p a r a g i n e - 1 48, y i e l d i n g
the w ell known form of the enzyme, alp ha-chy m otryp sin.
Alpha-chym otrypsin has a m olecu lar w eight of ap p ro x i
mately 25,000 d a lto n s and c o n ta in s 241 amino a c id r e s i d u e s .
The p r o t e o l y t i c a c t i v a t i o n of the zymogen a lre a d y d e scrib e d
c r e a t e s t h r e e d i s t i n c t p e p tid e c h ain s. The A chain con
t a i n s 13 amino a c id r e s i d u e s (1 -1 3 ), th e B chain c o n ta in s
131 r e s id u e s (1 6-146), and th e C chain c o n ta in s 97 r e s id u e s
(149-245). (The numbering of amino a c id r e s i d u e s in a lp h a -
chymotrypsin co rresponds to the chymotrypsinogen primary
seq uen ce.) A lpha-chym otrypsin i s an en d o p ep tid a se , c le a v
ing i n t e r n a l p e p tid e bonds, as opposed to e x o p e p tid a se s
such as c arb o x y p ep tid ase and am inopeptidase which r e q u i r e a
f r e e carboxyl or amino group, r e s p e c t i v e l y , a t the s i t e of
cleavag e. Chymotrypsin c le a v e s e s t e r and amide bonds in
which the r e a c t i v e carbonyl belongs to the L-amino a c id s
try p to p h a n , t y r o s i n e , and p h e n y la la n in e altho ug h i t w i l l
p
a ls o cleave a t la r g e hydrophobic r e s i d u e s such as leucine*-
and m eth io n in e .^
Chymotrypsin belongs to th e c l a s s of enzymes known as
s e r i n e p r o t e a s e s . These enzymes a re so c a l le d because they
po ssess a r e a c t i v e s e r i n e which becomes a c y la te d d urin g th e
^enzymatic r e a c t i o n . The major p a n c r e a t ic enzymes t r y p s i n ,
chymotrypsin, and e l a s t a s e as w ell as thrombin in th e blood
c l o t t i n g cascade po ssess a common tr im e r around th e a c t i v e
s e r i n e , a s p a r t a t e - s e r i n e - g l y c i n e , c o rresp on din g to r e s i d u e s
194-196 in chym otrypsin. A h i s t i d i n e r e s id u e , co rre sp o n d
ing to p o s i t i o n 5 7 in chymotrypsin, i s a ls o in vo lv ed in th e
c a t a l y t i c s i t e s of these s e r i n e p r o t e a s e s . F i n a ll y , an a s
p a r t a t e i s common to th e c a t a l y t i c s i t e of these enzymes,
co rrespo nd ing to p o s i t i o n 102 in chymotrypsin. The s e r in e ,
h i s t i d i n e , and a s p a r t a t e r e s i d u e s in th e s e enzymes a l l have
a s i m i l a r s p a t i a l o r i e n t a t i o n . The c o n se rv a tio n of such
s t r u c t u r a l homology among so many d i f f e r e n t enzymes would
seem to i n d i c a t e t h a t th ese elem ents a re fundamental to th e
c a t a l y t i c p ro c e ss . This c a t a l y t i c t r i a d of s e r i n e , h i s t i
d in e, and a s p a r t a t e has been given th e name 1 c h a rg e - r e la y
s y s te m '. This im p lie s a net charge t r a n s f e r from the a s-
p
Rovery, M.; P o ilro u x , M.; Yoshida, A.; D esnuelle, P.
Biochim. Biophvs. Acta 1957, £3, 608.
3 Brenner, N. ; M uller, H.R.; P f i s t e r , R.W. H elv. Chim.
Acta 1950, 33, 568.
3
p a r t a t e to the s e r in e to enhance th e n u c l e o p h i l i c i t y of the
s e r in e , and in f a c t an a s p a r t a t e pK h ig h er than h is -5 7 has
been p r o p o se d .2 * This scheme has been ch alle n g ed on both ex
perim ental and t h e o r e t i c a l grounds,^ and r e c e n t ^N-NMR
and neutron d i f f r a c t i o n s tu d ie s ^ would seem to su gg est a
r o le fo r the a s p a r t a t e which i s more in keeping with i t s
normal chemical p r o p e r t i e s .
Alpha-chym otrypsin c a ta ly z e d r e a c t i o n s of both s p e c i
f i c and n o n - s p e c i f i c e s t e r and amide s u b s t r a t e s follow the
scheme of Equation 1, where ES’ i s an acyl enzyme in te rm e
d ia te .
k* kp ko
E + S ^ = ± ± E S >ES» + P,-----—>E + P2 (1)
k -1
(a) H u n k a p ille r, M.W.; Smallcombe, S.H .; W hitaker, D.R.;
R ichards, J.H. B iochem istry 1973, 1£, 4732. (b) Blow,
D.M.; B i r k t o f t , J . J . ; H a rtle y , B.S. Nature ( London)
1969, £ £ 1 , 337.
^ (a) Matthews, D .A .; Alden, R.A.; B i r k t o f t , J . J . ; F re e r,
S .T .; K r a u t,J . J^. B io l. Chem. 1977, £ 5 £ , 8875. (b)
Nakagawa, S. ; Umeyama, H. ; Kudo, T. Chem. Pharm. B u l l .
Jpn. 1980, 2 3 , 1342. (c) Kraut, J. Ann. Rev. Biochem.
1977, M l, 331. (d) Desmeules, P. T h esis, P rin c eto n Univ
e r s i t y , 1 9 8 0 .
^ (a) K o ssiak o ff, A.A.; Spencer, S.A. B iochem istry 1981,
2 0 , 6462. (b) Bachovchin, W.W. ; R oberts, J.D. J_a. Am.
Chem. Soc. 1978, 1 M , 8041.
4
I t has been w e ll e s t a b l i s h e d t h a t an acy l enzyme in te r m e d i
a te i s formed during th e h y d r o l y s i s of both s p e c i f i c and
n o n - s p e c i f i c e s t e r and amide s u b s tr a te s * The acyl enzyme
i s undoubtedly an e s t e r of s e r i n e - 1 9 5 J H is t i d in e - 5 7 , a ls o
lo c a te d a t the a c t i v e s i t e , i s a c t i v e in th e base form fo r
usual e s t e r s u b s t r a t e s , but may a ls o f u n c tio n as a general
acid c a t a l y s t by a s s i s t i n g d e p a r tu re of the l e a v in g group
Q
in a c y l a t i o n r e a c t i o n s of amide s u b s t r a t e s .
N-A cylim idazoles. The h y d r o l y s i s of N -acy lim id azo les
having branched a lk y l c h ain s in th e acyl group i s marked by
s t e r i c e f f e c t s t h a t a re abnormal fo r b im o lecular r e a c
t i o n s . ^ The r a t e s of g e n eral base, general a cid , and hy
droxide ion c a ta ly z e d r e a c t i o n s a re not r e t a r d e d by branch
ing a t th e alpha carbon but a re a c t u a l l y a c c e l e r a t e d .
Branching a t th e beta carbon does produce a r a t e d ecrease
but to a l e s s e r e x te n t than found fo r n u c le o p h il i c r e a c -
7 (a) B ruice, T .C .; Benkovic, S. "Bioorganic R eaction Me
chanism s"; W.A. Benjamin: New Y ork,. 1966. (b) Bender,
M.L.; Kezdy, F .J . J. Am. Chem. Soc. 1964, M , 3704. (c)
Gutfreund, H. ; S t u r t e v a n t , J.M. Biochem. J . 1956, 6 3 ,
656. (d) Bender, M.L.; Z erner, B. Am. Chem. Soc.
1962, M , 2550. (e) Zerner, B. ; Bender, M.L. i*. Am.
Chem. Soc. 1964, 8 &, 3669. ( f ) Zerner, B . ; Bond,
R.P.M. ; Bender, M.L. J . Am. Chem. Soc. 1964, 8 6 ., 3690.
® Wang, J.H. Science 1968, 161 r 3 2 8 .Also r e f e r e n c e s 7(a)
and (b ).
9 (a) F if e , T.H. Am,. Chem. Soc. 1965, fil, 4597. (b)
Fee, J.A. ; F i f e , T.H. O re. Chem. 1966, 31, 2343.
(c) Fee, J .A .; F i f e , T.H. Phvs. Chem. 1966, 70 f
3268.
5
t i o n s of e s t e r s . S im ila r r e l a t i v e r a t e r a t i o s a re found in
th e h y d r o l y s i s of N-acylim idazolium io ns, which r u l e s out
th e s t e r i c i n h i b i t i o n of resonance as th e cause of the ab
normal s t e r i c o rd e r. In c o n t r a s t , am in o ly sis of N-acylim-
i d a z o le s by d ieth y lam in e giv es r i s e to a normal s t e r i c o rd
er r e l a t i v e to the e s t e r h y d r o l y s i s . I t was su gg ested t h a t
the unusual s t e r i c e f f e c t s in h y d r o l y s i s could be due to
r e l i e f of s t r a i n in a t r a n s i t i o n s t a t e i n which th e carbon-
n itr o g e n bond i s breaking./*® Exchange of i n t o th e a c y l-
im idazole carbonyl i s not observed in h y d r o l y s i s r e a c t i o n s
11 1 P
of e i t h e r the p r o t o n a t e d 11 or n e u tr a l s p e c i e s ' of N-acyl-
im idazole d e r i v a t i v e s of a l i p h a t i c c a rb o x y lic a c id s .
T h ere fo re , e i t h e r n u c le o p h il i c a t t a c k i s s t r i c t l y r a t e lim
i t i n g , or bond making and bond b reak ing a re c o n ce rte d as
suggested by the abnormal s t e r i c e f f e c t s . I f C-N bond
breaking i s p a r t of the r a t e - d e t e r m i n in g s te p , then a s t a
b le t e t r a h e d r a l I n te r m e d ia te cannot e x i s t . The r e a c t i o n s
of t r i f l u o r o e t h o x i d e ion w ith N -a c e ty lim id a z o le and N-ace-
ty lim id a zo liu m ion were a ls o concluded to proceed by a con
c e r t e d pathway or v ia an a d d it i o n in te r m e d ia te whose l i f e
time i s too s h o r t f o r i t to reach e q u ilib riu m w ith r e s p e c t
see r e f e r e n c e 9 ( a ) .
^ Bunton, C.A. J. Chem. Soc. 1963» 6045.
Fee, J.A. T h esis, U n iv e rs ity of Southern C a li f o r n ia ,
1 9 6 7 , and u n pu blish ed d a ta .
6
to proton t r a n s f e r . 1®
S t e r i c e f f e c t s a re normal in a c y l a t i o n of alpha-chymo
t r y p s i n by p - n itr o p h e n y l e s t e r s 1 *1 and in d e a c y la tio n of the
acyl enzyme i n t e r m e d i a t e . 1® E le c t r o n i c e f f e c t s have been
determ ined, u sin g th e Hammett s u b s t i t u e n t con- s t a n t s , 1® in
d e a c y la tio n of s u b s t i t u t e d benzoyl c h y m o t r y p s i n s 1^ and a-
c y l a t i o n by s u b s t i t u t e d p h e n o lic e s t e r s 1® and a n i l i d e s . 1 ^
A c y latio n of the enzyme by e s t e r s u b s t r a t e s i s c h a r a c t e r
ized by rho v a lu e s which are l a r g e and p o s i t i v e (+ 1 . 8 with
s u b s t i t u t e d phenyl a c e t a t e s 2 0 ), whereas a c y l a t i o n by
s u b s t i t u t e d a n i l i d e s g iv es a h ig h ly n e g a tiv e rho value
( - 2 . 0 ) . 21 This d i f f e r e n c e could r e f l e c t an in c r e a s e d impor
tance of g e n eral acid c a t a l y s i s in r e a c t i o n s - o f amides. A
1® O akenfull, D .G .; Jen cks, W.P. J^. Am. Chem. Soc. 1971,
a a , 178.
1* * M ils te in , J . B . ; F if e , T.H. B iochem istry 1969, J3, 623.
1® F i f e , T.H.; M ils t e i n , J.B. B iochem istry 1967, &, 2901.
1® Hammett, L.P. "P hysical Organic Chem istry";M cG raw -H ill:
New York, 1940,Chapt. 7.
1^ Caplow, M. ; Jenck s, W.P. B iochem istry 1962, 1, 8 8 3 .
1® (a) Bender, M .L.; Nakamura, K. J . Am. Chem. Soc. 1962,
84, 2577. (b) Hubbard, C.D.; K irsch, J .F . B iochem istry
1972, 11, 2483.
19 Inagami, T. ; York, S .S .; P a tc h o rn ik , A. J_ * . Am. Chem.
Soc. 1965, 8 1 , 126.
2 0 see r e f e r e n c e 1 8 ( a ) .
p 1
1 see r e f e r e n c e 19.
7
n itr o g e n i s o to p e e f f e c t in a lp h a-chy m otry psin c a ta ly z e d hy
d r o l y s i s of N -acety l-L -try p to p h an am id e r e q u i r e s t h a t the
C-N bond of th e amide i s broken in th e r a t e - d e t e r m i n i n g
s t e p . ^ Breakdown of t e t r a h e d r a l in te r m e d ia te formed in
a c y l a t i o n r e a c t i o n s of amide s u b s t r a t e s might t h e r e f o r e i n
volve the h i s t i d i n e - 5 7 c o n ju g a te a c id s in c e p r o to n a tio n of
the l e a v in g group i s a req uirem en t in h y d r o l y s i s r e a c t i o n s
of amides to avoid e x p u ls io n of an amine anion.
An i n v e s t i g a t i o n of the a c y l a t i o n of alpha-chym otryp
s in by N -acy lim id azo les would allow d e te rm in a tio n of wheth
er h i s t i d i n e 5 7 w i l l p a r t i c i p a t e in th e r e a c t i o n s o f amides
which do not form k i n e t i c a l l y s i g n i f i c a n t t e t r a h e d r a l i n
te r m e d ia te s . Such p a r t i c i p a t i o n should only occur in th e
s te p in which n u c le o p h il i c a t t a c k o c cu rs, u n lik e a c y l a t i o n
r e a c t i o n s of c o n v en tio n a l amide s u b s t r a t e s . Thus, the de
t e r m in a tio n of the p a t t e r n of s t e r i c e f f e c t s in a c y l a t i o n
r e a c t i o n s of th ese compounds w ith alph a-ch ym otry psin i s of
c o n s id e r a b le m e c h a n istic i n t e r e s t . F u rth e r, the d e te rm in a
t i o n of Hammett s u b s t i t u e n t e f f e c t s in a c y l a t i o n r e a c t i o n s
of these amides w i l l give m e c h a n istic in fo rm a tio n t h a t i s
d i r e c t l y a p p li c a b l e to th e n u c le o p h il i c a t t a c k s te p , uncom
p l i c a t e d by o th e r f u n c t i o n a l group i n t e r a c t i o n s . There
f o r e , the r a t e c o n s ta n ts f o r a number of a l i p h a t i c N -acyl-
^ O'Leary, M.H.; K luetz, M.D. J L * . Am. Chem. Soc. 1972,
M , 3585.
8
im id a zo les ( I - V I I ,F i g u r e 1) and s u b s t i t u t e d N-benzoylim ida-
z o le s (VIII-XIV, Figure 1) have been determ ined. In a d d i
t io n th e N '-m ethyl d e r i v a t i v e s of VII and XIII (XX and XIX
r e s p e c t i v e l y , F ig ure 1) have been s tu d ie d . F i n a l l y , a more
e x te n s iv e s e r i e s of hydrophobic N -'cyxlohexylacylim idazoles
(XV-XVIII, F igure 1) have been in clu d ed to b e t t e r determ ine
the r e l a t i v e im portance of h y d ro p h o b icity and s t e r i c e f
f e c t s in th e a c y l a t i o n of alp ha-chy m otryp sin by th ese com
pounds using th e Hansch pi c o n s t a n t s 2 ^ ancj T a f t ' s Eg param
e t e r s . 2 4
23 F u j i t a , T. ; J u n k ic h i, I. ; Hansch, C. i*. Am. Chem. Soc.
1964, M , 5175.
2 4 T a ft, R.W., " S t e r i c E f f e c t s in Organic Chemistry", M.S.
Newman, Ed.; W iley: New York, 1956, p . 644.
Figure 1 S t r u c t u r e s of N -A cylim idazoles and N'-M ethyl N-
A cylim idazoles.
10
R — C - N T ' ^ N
S 1 = 1
I ,R = C H ,-
II, r = c h ,(c h ,) 2 -
iii, r = ( c h 3 ) 3c -
iv, r = ( c h 3] 2c h c h , -
V , r = c h 3 (c h 2) 4-
VI, r = ( c h 3) 3 c c h 3-
V|l,
r C
o
V III, X = H
ix, x = p - o c h 3
X , X = m - N 0 2
X I,X = p - C l
xii,x = p - c h 3
xiii,x = p - n (c h 3) 2
XIV,X = P -C N
p H 2 )n - C 'N '
o L
X V , n:
XVI,n =
XVII,n:
X V III, n:
0
1
2
3
N R—C— CH3
o l = = J
* IX ,R = (C H 3 ) 2N -h ( Q ^ -
xx, r = ( c h 3 ) 3c c h 2 -
11
Chapter I I
EXPERIMENTAL
M a te r ia ls . A lpha-chym otrypsin, th r e e tim es c r y s t a l
l i z e d , was o b ta in e d from W orthington Biochemical Corp. or
Sigma Chemical Co. (typ e I I ) . 3 , 6 -d ia m in o a c rid in e ( p r o f l a
vin) h y d ro c h lo rid e (A ld rich Chemical Co.) and p y r id in e
(J .T . Baker Chemical Co.) were used w ith o u t f u r t h e r p u r i
f i c a t i o n . Im idazole (Sigma grade I) and p - n itr o p h e n o l
(A ld rich ) were sublimed b e fo re use. D2 O was o b ta in e d from
Bio-Rad L a b o r a to r i e s . N - tr a n s -.cinnamovlimidazole was p re
pared a cc o rd in g t o th e method of Schonbaum e t a l . 2^
Two methods were used to p rep are th e N -a cy lim id a zo les
and p - n itr o p h e n y l e s t e r s . The a cid c h lo r i d e method i n
volved slowly adding dropwise one of th e r e a c t a n t s , u s u a lly
the a cid c h lo r i d e , to a r e f l u x i n g benzene s o l u t i o n of the
o th er r e a c t a n t ( s ) and r e f l u x i n g f o r s e v e r a l hours t h e r e
a f t e r .T h e a c y lim id a z o le p r e p a r a t i o n s used a 1 : 2 molar r a t i o
of a c id c h lo r id e to im id azo le w h ile the p - n itr o p h e n y l e s
t e r p r e p a r a t i o n s used a 1 : 1 : 1 molar r a t i o of p y rid in e and
th e o th er r e a c t a n t s . A fter the r e a c t i o n was complete the
2^ Schonbaum, G.R.; Z erner, B. ; Bender, M.L. .« L _ B i o l .
Chem. 1961, 2 3 6 , 2930.
12
hot m ix tu re was f i l t e r e d to remove im idazo le h y d ro c h lo rid e
(or p y rid in e h y d ro c h lo rid e ) and th e benzene was removed
from the f i l t r a t e by r o ta r y e v a p o ra tio n . The r e s i d u a l pro
duct was then e i t h e r vacuum d i s t i l l e d or r e c r y s t a l l i z e d
from an a p p r o p r i a te s o lv e n t. The second method used N,N-
d ic y c lo h e x y lc a rb o d iim id e (DCC). DCC was mixed w ith the ap
p r o p r i a t e a cid and im id azo le in a 1 : 1 : 1 molar r a t i o in a
s o lv e n t which was u s u a lly methylene c h lo r id e , although
chloroform , t e t r a h y r o f u r a n and e th y l a c e t a t e were o c c a sio n
a l l y used. The r e a c t i o n was complete in s e v e ra l hours as
was evidenced by the appearance of N, N -d icy clo h ex y lu rea
p r e c i p i t a t e . The m ix tu re was f i l t e r e d to remove th e urea
and th e so lv e n t was removed by r o ta r y e v a p o ra tio n . The r e
s id u a l p roduct was th en e i t h e r vacuum d i s t i l l e d or r e c r y s
t a l l i z e d from an a p p r o p r i a te s o lv e n t.
The fo llo w in g compounds w i l l be l a b e l l e d a c id c h lo r i d e
(AC) or DCC to d e s ig n a te which techn iq ue was used in th e
s y n t h e s i s of the compound. N -a c e ty lim id a z o le (I) (AC) mp
103-105°C ( l i t . mp 104-105°C); N -n -b u ty ry lim id a z o le ( I I )
(AC) bp 88-92°C a t 2-3mm Hg ( l i t . bp 90-95°C under reduced
p r e s s u r e ) ; N - t r i m e th y la c e ty lim id a z o le ( I I I ) (AC) mp 54-56°C
r e c r y s t a l l i z e d from hexane ( l i t . mp 55-56°C); N -n-hexanoyl-
im idazole (V) (AC) mp 33-35°C r e c r y s t a l l i z e d from hexane
13
( l i t . mp 3 5 °C );2® N - is o v a le ry lim id a z o le (IV) (AC) bp
88-91°C a t 1-2mm Hg ( l i t . 2 7 bp 71-73°C a t 0.5mm Hg);
N - 3 ,3 -d im e th y lb u ty ry lim id a z o le (VI) (AC) mp 69-71°C r e c r y s
t a l l i z e d from hexane ( l i t . 2 ® mp 6 9 -6 9 .5°C); N-hydroeinnam-
o y lim id az o le (VII) (DCC) mp 54-56°C r e c r y s t a l l i z e d from
e t h e r / p e t e th e r , a n a l y s i s c a l c u l a t e d f o r C.j2 H.|2 N2 0 :
C=7 1.98, H=6.04, N=13.99 I found C=72.28, H=6.30, N=14.15.
The fo llo w in g s u b s t i t u t e d N -benzoylim idazoles were
made: p-H (V III) (DCC) bp 106-108°C a t 0.2mm Hg ( l i t . 2 9 bp
104-105°C a t 0.25mm Hg); P-OCH3 (IX) (DCC) mp 68-70°C r e
c r y s t a l l i z e d from cyclohexane ( l i t . mp 69-71°C); m-N02 (X)
(AC) mp 83-84°C r e c r y s t a l l i z e d from cyclohexane ( l i t . mp
8 5 . 5 - 8 6 ,5 ° C ) ; p-Cl (XI) (AC) mp 85-86°C r e c r y s t a l l i z e d from
pet e th e r ( l i t . mp 8 5 - 8 6 .5°C); P-CH3 (XII) (DCC) mp 69-71°C
r e c r y s t a l l i z e d from pet e th e r ( l i t . mp 69-71°C ) ; 3®
p-N(CH3 ) 2 (X III) (DCC) mp 109-111°C r e c r y s t a l l i z e d from cy
clohexane ( l i t . 31 mp 108-109°C). p-CN (XIV) (AC) mp
111— 113°C r e c r y s t a l l i z e d from cyclohexane, a n a l y s i s c a lc u -
2 6 Staab, H.A. Chem. Ber. 1956, M , 1927, 2088.
2 7 see r e f e r e n c e 9 ( b ) .
p Q
see r e f e r e n c e 9 ( a ) .
2 9 see r e f e r e n c e 2 7 .
3 0 see r e f e r e n c e 17.
31 Staab, H .A .; Luking, M.; Durr, F.H. Chem. Ber. 1962,
25., 1275.
14
l a t e d f o r C ^ H ^ O : C=67.00, H=3.58, N=21.31; found
C=66 .20, H=3.93, N=20.86.
N-3, 3 -d im e th y lb u ty ry l-N '-m e th y l-im id a z o liu m c h lo r id e
(XX) and N -C p-dim ethylam inoJbenzoyl-N '-m ethyl-im idazolium
c h lo r id e ( X I X ) were prep ared as f o l l o w s . 3 , 3-d im eth ylb u-
t y r y l c h lo r id e and (p-dim ethylam ino)benzoyl c h lo r id e were
d is s o lv e d i n dry e th e r and t e t r a h y d r o f u r a n r e s p e c t i v e l y and
an equim olar amount of N -m ethylim idazole was d is s o lv e d in
th e same so lv e n t and added dropwise to th e r a p id ly s t i r r i n g
s o l u t i o n of acid c h lo r i d e . The product p r e c i p i t a t e d i n
s ta n ta n e o u s ly . F u rth e r p u r i f i c a t i o n in vo lv ed removing a
sample under s o lv e n t and washing and d e ca n tin g w ith the
same so lv e n t s e v e r a l tim es b e fo re drying under vacuum.
Dried samples had to be used im m ediately because of the
h yg roscop ic n a tu r e of th ese compounds. N -(p -d im eth y l-
am inoJ-b en zoy l-N '-m eth yl-im id azo liu m c h lo r i d e mp 138-140°C,
a n a l y s i s c a l c u l a t e d f o r C ^H ^C IN ^O : C=58.76, H=6.07,
N=1 5 . 8 1 ; found C=5.8.57 H=6.25, N=15.53. N-3, 3 -d im e th y lb u -
t y r y l - N 1-m ethy1-im idazolium c h lo r id e mp 111 — 113°C. Analy
s i s of t h i s u n s ta b le compound was preclud ed by the le n g th
of time i t took to send th e sample to G a lb r a ith L abo rato
r i e s in K no xv ille, Tenn.
32 Wolfenden, R. ; Jenck s, W.P. JL a. Am. Chem. Soc. 1961,
M , 4390.
15
The fo llo w in g N -c y c lo h e x y la c y lim id a z o le s were
p rep ared : N -c y clo h ex y lc arb o n y lim id az o le (DCC) mp 87-88°C
r e c r y s t a l l i z e d from pet e th e r , a n a l y s i s c a l c u l a t e d fo r
C10H14N20: c=67.39, H=7.92, N=15.72; found C=67.52, H=7.85,
N=15.73. N -c y c lo h e x y la c e ty lim id a z o le (DCC) mp 71-72°C re -
c r y s t a l l i z e d from pet e th e r , a n a l y s i s c a l c u l a t e d for
C11H16N20: C=68.72, H=8.39, N=14.57; found C=68.96, H=8 . 3 8 ,
N=14.95. N -c y clo h ex y lp ro p io n y lim id a z o le (DCC) bp 135-137°C
a t 0.02mm Hg,mp 47-49°C, a n a l y s i s c a l c u l a t e d f o r C ^ ^ i g ^ O :
C=69. 8 7 , H=8 .79, N=13.58; found C=69.89 , H=8.89, N=13.34.
N -c y c lo h e x y lb u ty ry lim id a z o le (DCC) mp 7 5.5-77.5°C r e c r y s
t a l l i z e d from cyclohexane, a n a l y s i s c a l c u l a t e d fo r
c 13H20N20: C=70.87, H=9.15, N=1 2 . 7 2 ; found C=70.49,
H=9 • 04, N=12.61.
The fo llo w in g p - n itr o p h e n y l e s t e r s were p rep a red : p-
nitro'phenyl p-CN benzoate (AC) mp 193-194°C r e c r y s t a l l i z e d
from acetone ( l i t . 33 mp 195-196°C); p-n i tro p h en y l p-NCCH^^
benzoate (DCC) 191-193°C r e c r y s t a l l i z e d from acetone
( l i t . 3^ mp 195-196,5 ° C ) ; p - n itr o p h e n y l c y clo h ex y lca rb o x y 1-
a te (DCC) mp 50-52°C r e c r y s t a l l i z e d from eth an o l ( l i t . 35 mp
49-51°C); p - n itr o p h e n y l c y c lo h e x y la c e ta te (DCC) mp
33 see r e f e r e n c e 1 8 (b ) .
3^ K irsch, J . F . ; C lew ell, W . ; Simon, A. J_t Ore. Chem.
1968 11, 127.
3 5 S i l v e r , M.S. J . Am. Chem. Soc. 1966, M , 4247.
16
2 5 .5 -26 .5 °C r e c r y s t a l l i z e d from e t h a n o l . 3 3 p -N itro p h en y l
c y c lo h e x y lp ro p io n a te (DCC) mp 80-82°C r e c r y s t a l l i z e d from
e th a n o l. p-N itro p h en y l c y c lo h e x y lb u ty r a te (DCC) mp 29-31°C
r e c r y s t a l l i z e d from e th a n o l. p -N itro p h en y l hydrocinnamate
(DCC) mp 97-99°C r e c r y s t a l l i z e d from chloroform /hexane
( l i t . 3 7 mp 97-98°C).
B u ffe rs used were sodium a c e t a t e (MCB), sodium phos
phate d i b a s ic ( M a lli n c k r o d t ), T r is (Sigma), and
2 -a m in o -2 -m e th y l-1 , 3 -p ro p an e d io l (ammediol) (Sigma). Buf
f e r s were r e a g e n t grade and p rep a red in d i s t i l l e d w a te r.
Stock s o l u t i o n s of p r o f l a v i n h y d ro c h lo rid e were p rep ared in
d i s t i l l e d w a te r. P r o f l a v in s o l u t i o n s were p r o te c te d from
l i g h t a t 5°C and used f o r p e rio d s of up to th r e e weeks be
fo re d is c a r d in g . Stock s o l u t i o n s of alp ha-chy m otryp sin
were p rep ared in pH 5.0 a c e t a t e b u f f e r 0.1M, and w ith an
i o n ic s t r e n g t h of 0.5M ( 0 .5 1 ) . The n orm ality of a c t i v e
s i t e s in th e sto c k enzyme s o l u t i o n s was determ ined by t i
t r a t i o n of the s o l u t i o n w ith N - tr a n s -cin n am o v lim id azo le a t
310 nm (method A, Schonbaum e t a l ) . 3 3
33 Murakami, Y. ; Aoyama, Y. ; Kida, M. ; Nakano, A. B u l l .
Chem. Soc. Jpn. 1977, 5H, 3365.
37 I n g le s , D.W. ;Knowles, J.R . Biochem. J. 1968, 108., 561 .
33 see r e f e r e n c e 25.
17
K in e tic Methods. Rate c o n s ta n ts f o r a c y l a t i o n of a l -
pha-chym otrypsin by N -a ey lim id a zo les a t 30°C were d e t e r
mined in th e presence of p r o f l a v i n . The dye forms a 1:1
complex w ith the a c t i v e s i t e of a lp h a -c h y m o try p s in ,39 which
r e s u l t s in a s p e c t r a l s h i f t . The maximum absorbance d i f
fe re n c e between complexed and uncomplexed dye occurs a t 465
nm, thereby a llo w in g r e a c t i o n s t h a t by them selves may ex
h i b i t l i t t l e s p e c t r a l change to be c o n v e n ie n tly follow ed
s p e c t r o p h o t o m e t r i c a l l y . A cylation of the enzyme r e s u l t s
in d isplacem en t of p r o f l a v i n , which c on seq uen tly giv es r i s e
to a l a r g e absorbance change a t 465 nm. All k i n e t i c runs
were in 0 . 1 M b u f f e r s ( 0 . 5 D except w ith N -a c e ty lim id a z o le
and N - 3 ^ - d i m e t h y l b u t y r y l - N ’-m ethyl-im id azoliu m c h lo r id e
w ith which r a t e d e te r m in a tio n s were c a r r i e d out in 0.05M
b u f fe r (0 .5 1 ) and in 0.3M b u f f e r ( 0 . 8 1 ) , r e s p e c t i v e l y . The
d i f f e r e n t b u f f e r s y i e ld e d c o n s i s t e n t r e s u l t s when employed
a t the same pH. The c o n c e n tr a tio n s of r e a c t a n t s f o r each
compound s t u d i e d a re l i s t e d in Table 1. A c y latio n r a t e s
were follow ed to com pletion between 465 and 490 nm a t 30°C,
employing e i t h e r a Beckman Model 25 sp e ctro p h o to m e ter or a
39 Bernhard, S. A.; Lee, B .F .; T a s jia n , Z.H, J_t Mol. B i o l .
1966, 1 8 , 405.
210 For examples see: (a) Brandt, K.G. ; Himoe, A.; Hess,
G. P. B io l. Chem. 1967, £ 1 £ , 3973. (b) G u i l la i n ,
F. ; T husius, D. J.. Am. Chem. Soc. 1970, 22., 5534. (c)
H utchins, J . E . C .; F i f e , T.H. J_i Am. Chem. Soc. 1972,
21, 8848.
18
Durrum Model D-110 sto pped-flow sp e ctro p h o to m e ter. Equa
t i o n s a re given in r e f e r e n c e 4 0 ( a ) . To f u r t h e r v e r i f y th e
r e l i a b i l i t y of the p r o f l a v i n displacem en t tec h n iq u e a de
t e r m in a tio n of the r a t e c o n s ta n t f o r N - tr a n s -cin nam ov lim id-
a z o le was made. The r a t e in pH8.0 T r is b u ff e r (0.1M, 0 .51 )
a t 3 0 °C with a s u b s t r a t e range of 3.5x10”^M to 3.5x10_2*M
and enzyme and p r o f l a v i n c o n c e n tr a ti o n s of 1.6x10'"^M and
7.7x10“ ^M, r e s p e c t i v e l y , were c o n s i s t e n t w ith p rev io u s mea
s u re m e n ts .2 ^ I t has p r e v io u s ly been shown in r e a c t i o n s of
e s t e r s u b s t r a t e s t h a t the r a t e c o n s t a n t s o b ta in e d by pro
f l a v i n displacem en t a re i d e n t i c a l w ith those o b tain ed by
lip
independent methods. D if f e r e n c e s in th e r a t e c o n s ta n ts
o b ta in e d by d i r e c t o b s e r v a tio n of the a c y lim id a z o le a t 245
nm (25°C) and by the p r o f l a v i n method a re not l a r g e ( u s u a l
ly approxim ately a f a c t o r of 2) and th e pH dependence i s
i d e n t i c a l . 2 ^ However, measurements a t 245 nm are s u b j e c t to
g r e a t e r e r r o r due to th e absorbance of the enzyme a t the
same w avelength.
^ Bender, M.L. ; Schonbaum, G.R.; Z erner, B. J.*. Am. Chem.
Soc. 1962, M , 2562.
Himoe, A.; B randt, K.G.; DeSa, R . J . ; Hess, G.P. J.
B io l. Chem. 1969, £ M , 3483.
^3 Kogan, R.L. ; Fee, J.A. ; F i f e , T.H. J_t Am. Chem. Soc.
1 9 8 2 , 1Q 4, 3569.
19
TABLE 1
C o n c e n tra tio n s of R e ac ta n ts in th e A c y latio n of
Chymotrypsin by N-Acylim idazoles
Compound [N -A c y lim id a z o le ], [Enzyme], [ P r o f l a v i n ] ,
x ( 1 0 )M x ( 1 0 )M x ( 10 ) M
I 100-1600 16.5 77
I I 260-6100 16.5 77
I I I 500-5400 16.5 77
IV 260-6200 16.5 77
V 1 0 0 - 1 0 0 0 16 .5 77
VI 130-5100 16.5 77
VII 7.5-120 4.0
39
VIII 150-3500 16.5 77
IX 1 9 0 - 3 0 0 0 16.5 77
X 2 5 0 - 2 5 0 0 16.5 77
XI 75-2500 16.5 77
XII 2 0 0 - 3 0 0 0 16.5 77
XIII 1 0 0 - 1 0 0 0 16.5 77
XIV 150-9400 16.5 77
XV 5 0 - 8 0 0 6 . 0 77
XVI 5 0 - 8 0 0 6 . 0
77
XVII 10-400 3.0
77
XVIII 25-200 3.0
77
XIX 1 0 0 - 1 0 0 0 16.5 77
XX 3 0 0 - 5 8 0 0 16.5 77
20
In a t y p i c a l run employing th e Beckman spectrophotom e
t e r 3.0 mL of b u f f e r was added to a c u v e tte and a l i q u o t s of
enzyme and p r o f l a v i n sto c k s o l u t i o n s were added. A fter a l
lowing ap pro xim ately ten m inutes f o r the tem p era tu re to
e q u i l i b r a t e , an a l i q u o t of the s u b s t r a t e sto c k s o l u t i o n was
added w ith a Hamilton sy rin g e . In a t y p i c a l run employing
the stop ped -flow sp ectro p h o to m e ter two s e p a r a te s o l u t i o n s
were p rep a red . One s o l u t i o n was a b u f f e r which c o n ta in e d
twice the f i n a l c o n c e n tr a ti o n s of enzyme and p r o f l a v i n .
This s o l u t i o n was in tro d u c e d i n t o one of two i d e n t i c a l
d r iv e s y r in g e s . The o th e r sy rin g e c o n ta in e d w ater to which
was added an a l i q u o t of s u b s t r a t e sto c k s o l u t i o n t o give a
c o n c e n tr a ti o n equal to tw ice the d e s ir e d f i n a l c o n c e n tra
t io n . The le n g th of tim e t h a t a s u b s t r a t e was in th e h o ld
ing s o l u t i o n be fo re an a c y l a t i o n r a t e was o b ta in e d was used
to c o r r e c t th e p s e u d o - f i r s t - o r d e r r a t e c o n s ta n ts o b ta in e d
f o r a c y l a t i o n by d iv id in g by e” ^°^, where kQ i s th e f i r s t -
order r a t e c o n s ta n t f o r the h y d r o l y s i s of the s u b s t r a t e in
w a ter, measured a t 245 nm a t 30°C, and t i s th e time the
s u b s t r a t e was in th e h o ld in g s o l u t i o n b e fo re the a c y l a t i o n
r e a c t i o n was commenced. This c o r r e c t i o n was n e g l i g i b l e in
c ases where spontaneous h y d r o l y s i s i s slow. The d r iv e sy
r i n g e s , mixing chamber, and c u v e tte were suspended in a wa
t e r trough whose te m p era tu re was m a in ta in e d a t 30±0.1°C.
21
O p t i c a l- d e n s i t y changes a f t e r mixing were rec o rd ed on a
H ew lett-P ack ard s to r a g e o s c ill o s c o p e (Model 1207B). With
each b u f fe r th r e e to fo u r r e a c t i o n s were t a b u l a t e d . The
r e a c t i o n s were fo llo w ed to com pletion. P s e u d o - f i r s t - o r d e r
r a t e c o n s ta n ts were c a l c u l a t e d u sin g e i t h e r a l i n e a r r e
g re s s io n program on a Texas In stru m e n ts TI-59 programmable
c a l c u l a t o r or a n o n - l in e a r l e a s t sq u ares a n a l y s i s on an IBM
370/168-3 computer. Good f i r s t - o r d e r k i n e t i c s were ob
ta i n e d in a l l c a se s. Reaction pH v a lu e s were o b tain ed w ith
a Radiometer Model 22 pH m eter or a Beckman Model 3500 d i g
i t a l pH m eter. The pD was determ ined u sin g th e g la s s e l e c
tro d e c o r r e c t i o n e q u a tio n of F if e and B r u i c e . 4 4
A cy latio n of th e A ctive S i t e w ith N -A cylim idazoles.
To ensure t h a t th e N -a cy lim id a zo les were a c y l a t i n g th e a c
t i v e s i t e of alp h a-ch y m o try p sin th e fo llo w in g experim ent
was c a r r i e d o u t. Using an a p p r o p r i a te b u f f e r the p - n i t r o -
phenyl e s t e r co rresp o n d in g t o th e N -acy lim id azo le was added
in ex cess a f t e r a c y l a t i o n of the enzyme w ith N -acylim ida
z o le . The appearance of p - n i t r o p h e n o l / p - n i t r o p h e n o l a t e an
ion was follo w ed a t 330 or 400 nm, whichever gave the
g r e a t e s t d e f l e c t i o n . The f i r s t - o r d e r r a t e c o n sta n t f o r dea-
c y l a t i o n was c a l c u l a t e d from the l i n e a r p o r tio n of the
t r a c i n g , the i n i t i a l c o n c e n tr a tio n of enzyme, and th e ex
4 4 F i f e , T.H.; B ru ice, T.C. jLl Phvs. Chem. 1961, £5.,
1079.
22
t i n c t i o n c o e f f i c i e n t of the p - n i t r o p h e n o l / p - n i t r o p h e n o l a t e
a n i o n . ^ The r a t e c o n s ta n ts measured were comparable to
those measured f o r d e a c y la tio n of th e co rresp o n d in g acy l
enzymes p repared from p - n itr o p h e n y l e s t e r s in d e p e n d e n tly .
Doub, L . ; V andenbelt, J.M. J . Am. Chem. Soc. 1947, 6 Q t
2714.
23
Chapter I'll
RESULTS
The scheme in e q u a tio n 1 y i e l d s e q u a tio n 2 ^ fo r r e a c
t i o n s of a lp h a -chy m otryp sin, where Km= (k_^+k2 ) /k - |, and k i s
a f i r s t - o r d e r r a t e c o n s ta n t governing th e p r e - s t e a d y - s t a t e
r e a c t i o n .
(k 2 + k3 ) [ S ] c + k3 Km k2 [S ] 0
" i s i o Z " £
+ k^ (2)
I f (k 2 +k3 ) [ S ] 0 >k3 Km, then e q u a tio n 2 can be s i m p l i f i e d to
e q u a tio n 3 , and th e data can be analyzed a cc o rd in g to con
v e n tio n a l M ichaelis-M enten k i n e t i c s .
( kp + k o ) [ S ] 0
k = -------1-------- (3)
[ S ] 0 + ^
E quations 2 and 3 may be re a r r a n g e d to e q u a tio n s 4 and 5
r e s p e c i v e l y .
see r e f e r e n c e 7 ( c ) .
24
k = (ko + k,)
[S3,
(4)
Ck - k 3 )Km
(k - kn) = k2 ---------------- --- -
J [S3.
(5)
A p l o t of k i s k /[S 3 0 or (k-k^) vs ( k - k 3 ) / [ S 3 0 should then
be a s t r a i g h t l i n e w ith a slope equal to -Km and an o r d i
n ate i n t e r c e p t equal to k2 . it7 At s u b s t r a t e c o n c e n tr a ti o n s
which are small in comparison to Km i t i s not p o s s ib l e to
M Q
determ ine the v a rio u s c o n s t a n t s by employing e q u a tio n 3 .
In such cases th e r e a c t i o n w i l l be e x p e rim e n ta lly second-
o rd e r.
Typical p l o t s a re shown in F ig u re s 2 and 3 of k vs
k /[S 3 0 , where k i s th e p s e u d o - f i r s t - o r d e r r a t e c o n s ta n t.
In each case th e p l o t s were v e r t i c a l as shown i n d i c a t i n g
t h a t the h ig h e s t s u b s t r a t e c o n c e n tr a tio n i s s t i l l much l e s s
than Km so t h a t an e n zy m e-su b strate complex i s experimen
t a l l y u n d e te c t a b l e . Under such c o n d itio n s th e r e a c t i o n can
be co n sid ere d se c o n d -o rd e r and th e [S30 term w i l l f a l l out
of the denominator in both e q u a tio n s 2 and 3. A p l o t of k
vs [S ] 0 or (k-k^) j£s [ S 3 0 should have an i n t e r c e p t of z ero
4 7 Eadie, G.S. J. B io l. Chem. 1942, 1 M , 85.
F a l l e r , L . ; S t u r t e v a n t , J.M. J. B io l. Chem. 1966, 2 4 1 T
4825. Also r e f e r e n c e 14.
25
and a slope equal to the pH-dependent seco nd -ord er r a t e
c o n s ta n t k 2 / K m i f k 2 > k g . P lo t s of k i s tS3 0 a re shown in
F ig u re s 4 and 5. The i n t e r c e p t s of such p l o t s a re g e n e r a l
ly zero w i t h in e x p erim e n tal e r r o r . The v a lu e s of k / [ S ] Q
determ ined from the a b s c i s s a i n t e r c e p t s of k i s k / [ S ] 0
p l o t s should be equal to k2 /Km and were c lo s e ly s i m i l a r in
each c ase . In a l l c ase s th e r a t e s of a c y l a t i o n of a lp h a -
chym otrypsin by N -a cy lim id a zo les a re much g r e a t e r than
th o se of d e a c y la ti o n . S u b tr a c tio n of k g from k employing
e q u a tio n s 2 and 4 and th e v a lu e s of kg determ ined p r e v io u s
ly^^ or in t h i s study f o r d e a c y la tio n of acyl alpha-chymo-
t r y p s i n , produced no change in th e se c o n d -o rd e r r a t e cons
t a n t s k 2 / K m , i . e . , p l o t s of k - k g vs [ S ] Q and k - k g .ys k / [ S ] 0
were i d e n t i c a l to u n c o rre c te d p l o t s . Thus k g i s not s i g n i
f i c a n t in comparison to k. The e x ac t slope of the p l o t of
k y_s [S ] Q from eq u atio n 3 i s ( k 2 + k g ) / K m , which w i l l only be
equal to k 2 / K m i f k 2 > > k g . Since the seco n d -o rd er r a t e
c o n s ta n ts a re i d e n t i c a l whether determ ined from p l o t s of k
vs [ S ] Q (e q u a tio n 3) or k-kg .ys [ S ] Q (e q u a tio n 2) i t i s
c l e a r t h a t k 2 > > k g . F u r th e r , sin c e k g i s not s i g n i f i c a n t in
comparison to k th en k g drops out of both e q u a tio n s 4 and 5
and th e s e then become e q u iv a le n t .
see r e f e r e n c e s 15 and 17.
26
F igu re 2: P l o t s of k .ys k / [ S ] g f o r a c y l a t i o n of a lp h a -
chym otrypsin a t pH 6.48 by N - is o v a le r y lim id a z o le
(G) and by N -b u ty ry lim id a z o le a t pH 5.98 ( A )
a t 30°C.
82
(t_oes t_ | A I ) e-OI-x (°S/f)
S'G 0'8 9'Z O'Z Q'l O'I S’ O
01-
F igu re 3: P l o t s of k i s k / [ S ] fi f o r the a c y l a t i o n of a lp h a -
chym otrypsin a t pH 7.0 by N-(p-methoxy)
b en zo y lim id az o le (O) and a t pH 8 . 0 by N-(p-
c h lo ro ) b e n zo y lim id az o le ( • ) a t 30°C.
29
k x 1 0 (sec-1)
60
4 5
3 0
1 5
I ...1 ------- T " 1 1 .......T""” 1 1 ....... 1...... I..... “ 1 ------- 1 ...... “ 1 ------- 1 !
•
•
C
»
•
e
D
- < 1
• >
•
D
•
0
i i i
i i i i ' i i i i 1 1
8 1 6 2 4 3 2 4 0 4 8 5 6
(k/S0)x 10-2 (sec"1 M~1)
30
F igu re 4: P l o ts of k jys CS]_ f o r the a c y l a t i o n of a lp h a -
chym otrypsin a t pH 6.48 by N - is o v a le r y lim id a z o le
( O ) and a t pH 5.98 by N -b u ty ry lim id a z o le ( A )
a t 30°C.
T—
I
o
CD
10
9
8
7
5
4
3
2
1
6 0 3 2 4 1 5
Sox103M
32
F igure 5 P lo t of k i s £S] 0 f o r a c y l a t i o n of a lp h a -
chym otrypsin a t pH 7.0 by N-(p-m ethoxy)
b e n zo y lim id az o le a t 30°C.
33
4 0
3 0
O
0
CO
o
T“
X 2 0
■o
a)
O
-X
10
2 8 3 2 2 0 2 4 1 6
SQ x 104 (M)
The l o g ( k 2 /Km)-pH p r o f i l e s fo r a c y l a t i o n of a lp h a -ch y -
m otrypsin by a l i p h a t i c N -a c y lim id a z o le s, N-benzoylimida-
z o le s , and N -c y c lo h e x y la c y lim id a z o le s appear in F ig u re s 6 ,
7, and 8 r e s p e c t i v e l y . ( T rim e th y la c e ty lim id a z o le ( I I I ) ap
p ears on F ig ure 8 r a t h e r than F ig ure 6 ) . The pH dependence
of the a c y l a t i o n r e a c t i o n i s not s tro n g ly in f lu e n c e d by the
s t r u c t u r e of the a l i p h a t i c a cy l groups (F ig u re s 6 and 8 ),
a c y l a t i o n being e s s e n t i a l l y independent of pH. The com
pounds w ith s t r a i g h t - c h a i n a cy l groups have a maximum in
t h e i r p r o f i l e s near pH 6 .0 . However, th e i n c r e a s e s in th e
r a t e c o n s ta n ts w ith i n c r e a s i n g pH a t v a lu e s below 6.0 are
sm all. N -3 ,3 -d im e th y lb u ty ry lim id a z o le (VI) shows a small
in c r e a s e in r a t e w ith i n c r e a s i n g pH to pH 8 .0 . The c y clo -
hexyl and hydrocinnamoy 1 a c y lim id a z o le s show a small in
c re a s e in th e r a t e c o n s ta n t with i n c r e a s i n g pH to pH 7 .5 ,
with th e e x c e p tio n of N -c y c lo h e x y la c y lim id a z o le (XV) which
shows a drop of approxim ately one lo g u n i t from pH 6.5 to
9 .0 . The v a lu e s of are ^he order
VII=XVII>XVIII>XVI>V>XV>II>IV>III>VI>I. The r a t e of a c y la
t io n g e n e r a lly i n c r e a s e s as th e le n g th of the acyl group
i n c r e a s e s . T rim e th y la c e ty lim id a z o le ( I I I ) (alpha carbon
branching) has a k2 ^ Km only s l i g h t l y l a r g e r than
N-3, 3 - d im e th y lb u ty r y lim id a z o le (VI) ( b e ta carbon branch
in g ) . N -c y clo h ex y lc arb o n y lim id az o le (XV), which has a six
35
carbon r i n g i s s l i g h t l y slower than N -hexanoylim idazole,
altho ug h t h i s d i f f e r e n c e i n c r e a s e s g r e a t l y above pH 6 .5 .
F i n a l l y , . N -c y c lo h e x y lb u ty ry lim id a z o le (XVIII) i s slower
than both N -c y c lo h e x y lp ro p io n y lim id a z o le (XVII) and N-hy-
drocinnam oylim idazole (VII) a lth o u g h i t i s one m ethylene
group lo n g e r than both of them. At pH 7.5 ^ ^ m i s
1.2x10 6 M"1 s e c " 1 ; XVII, 1 .28x10 6 M~1 s e c ” 1 ; XVIII, 4.64x105
r
M “ 1 s e c - 1 ( pH 8 . 0 ) ; 5 0 XVI, 1.01x10 5 M ” 1 se c ~ 1 ;V, 4.96x10 4
M ” 1 s e c ” 1 ; XV, 7 .73x10 3 M “ 1 s e c ” 1 ; I I , 1.12x103 M"1 s e c ” 1 ; IV,
870 M"1 se c ” 1 ; I I I , 350 M ” 1 se c ” 1 (pH 8 . 0 ) ; VI, 154 M ” 1 s e c ” 1 ;
I, 18 M “ 1 s e c “ 1 . N-3, 3 - d i m e th y l b u t y r y l- N '- m e t h y l- i m id a z o l i -
um ion (XX) shows an i n c r e a s i n g r a t e c o n s ta n t w ith i n c r e a s
ing pH below pH 7 .0 and a pH independent r e a c t i o n a t pH>7.
The l i m i t i n g r a t e c o n s ta n t (k£/Km) i s 2.4 x1 0 4 M “ 1 se c ” 1 , and
th e v alue of pKapp i s 6 . 6 . The l i m i t i n g r a t e c o n s ta n t f o r
the N’-m ethyl d e r i v a t i v e was measured in D20 a t pD v a lu e s
of 7.5 and 8 .0 . The r a t i o of (k£/Km) H/(k £ /K m) D i s 3 .1 .
F ig u re s 9 and 10 a re p l o t s of l o g ( k 2 /Km) x s th e Hansch
pi and T a f t Es c o n s t a n t s r e s p e c t i v e l y a t pH 8 .0 . The data
was a ls o computer f i t t e d to e q u a tio n 6 u sin g a n o n - l in e a r
l e a s t sq u a re s a n a l y s i s .
l o g ( k 2 /Km) = xEg + yPi + c ( 6 )
This compound was s u b j e c t to a 10-20% e r r o r in m easure
ments of a c y l a t i o n r a t e c o n s ta n ts .
36
F igu re 6 P l o t s of l o g ( k 2 /Km) x s pH fo r the a c y l a t i o n of
alp h a-ch y m o try p sin by N -a cy lim id a zo les a t 30°C.
37
6.0
5 . 5
5 .0
4 . 5
N'-Methyl XX
4 .0
^ 3.5
5* 3 . 0
2 .5
2.0
1.5
1.0
5 .0 5 .5 6 .0 6 .5 7 .0 7 .5 8 .0 8 . 5 9 .0
pH
38
F igu re 7
P l o t s of l o g ( k 2/Km) _ys pH f o r th e a c y l a t i o n of
alp h a-ch y m o try p sin by N -b enzoylim idazoles a t
30 C.
5.0
4.5
2 4.0
C M
5 3.5
0
Q 3.0 h
2.5
2.0
t ---------- r i ~ r
5.0 5.5
/
N - M e t h y l XIX
m
x n i
J_ _ _ _ _ _ _ _ L J_ _ _ _ _ _ _ _ L
6.0 6.5 7.0 7.5 8.0 8.5
pH
9.0
F igure 8 P l o t s of l o g ( k 2 /Km) \rs pH f o r th e a c y l a t i o n of
alp h a-ch y m o try p sin by N -c y c lo h e x y la c y lim id a z o le s
a t 30°C.
6.0
5 . 5
5 . 0
O
4 . 5
4 . 0
^ 3 . 5
CM
CD 3 - 0
X 2
2 . 5
2.0
1 .5
5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 8 . 5 9 . 0
42
The computer f i t t e d p aram eters were x = 1 .01±.0.25,
y=1.88±0.20, and c=0.34±0.23. The p aram eters were a ls o
f i t t e d in d ep e n d en tly to l o g ( k 2/Km). These v a lu e s were
x=-2.1 0±0.65, y=1.57±0.13. The N -a c y lim id a z o le s and t h e i r
r e s p e c t i v e l o g ( k 2/Km), Es ’ s , and p i ’ s a re l i s t e d in Table
2. F igu re 11 shows a p l o t of l o g ( k 2/Km) fo r a c y lim id a z o le s
vs the c o r r e s p o n d i n g . r a t e c o n s t a n t s f o r th e p - n itr o p h e n y l
e s t e r s . The r e g r e s s i o n l i n e shown has a slope of 1.15
(R=0.84). The same l i n e w ith the branched a cy l groups r e
moved g iv es a slope of 1.66 (R=0.91).
The l o g ( k 2/Km)-pH p r o f i l e s f o r a c y l a t i o n of a lp h a -c h y -
m otrypsin by N -benzoylim idazoles (F ig u re 7) show th e pH de
pendence of the a c y l a t i o n r e a c t i o n i s in f lu e n c e d by the na
t u r e of the s u b s t i t u e n t . A c y latio n i s e s s e n t i a l l y pH
independent when th e s u b s t i t u e n t i s e l e c t r o n d o natin g
( V III, IX, XII, X I I I ) . E le c tro n w ithdraw ing s u b s t i t u e n t s
(X, XI, XIV) produce an i n c r e a s e i n k2/Km from pH 5.0 to
ap pro xim ately pH 7 .0 . T h e r e a f t e r , th e r a t e c o n s ta n ts a re
n e arly pH in d ep en d en t. The p r o f i l e f o r N -(p -d im e th y l-
a m in o J-ben zoy l-N '-m eth yl-im idazo liu m ion fo llo w s a t h e o r e t
i c a l curve f o r d i s s o c i a t i o n of a group w ith a pKa of 6 .5 .
51 Tobias, P .; Heidema, J . H . ; Lo, K.W.; K a iser, E . T . ; Kez-
dy, F .J . J. Am. Chem. Soc. 1969, £1, 2 0 2 .Also r e f e r e n c
es 12, 14, and 18 (b).
43
TABLE 2
The Log(k2/K ) a t pH 8.0 of S everal N-A cylim idazoles With
tne Corresponding Pi and Es V alues.
Compound Log(kp/Km) Pi fi
a t pH 8
I 1 .25 0.52 0
I I
2.95 1 .43 -0 .3 6
I I I 2.52 1 .68 -1 .54
IV
2.87 1 .82 -0 .9 3
V 4.65 2.43 -0 .4 0
VI 2.25 2.18 -1 .74
VII 6.05 2.89 -0 .3 8
XV
3.70 2.51 -0 .7 9
XVI 5 .00 3.01 -0 .9 8
XVII 6.10 3.51 -0 .9 8
The l i m i t i n g value of k 2 /Km a t high pH i s 6.3x10** M “ ^ se c “ ^.
The l i m i t i n g r a t e c o n s ta n t f o r th e N’-m ethyl d e r i v a t i v e was
measured in D20 a t a pD of 7 . 5 . The r a t i o of
(^k k / K m ) t i / ( k h / K m ) D i s 2 - 1 *
As seen in F igu re 7 the v a lu e s of k2/Km follow the
order X>XIV>XI>XII>IX>VIII>XIII. The r a t e of a c y l a t i o n in
g e n eral i n c r e a s e s as e l e c t r o n w ithdraw al in th e acy l group
i n c r e a s e s , though d i f f e r e n c e s a re not l a r g e . At pH 7.5
44
F igure 9 P lo t of l o g ( k 2/Km) pi c o n s t a n t s l i s t e d in
Table 2.
45
7.0
r* 6.0
g 4 . 0
* 3 . 0
C M
C D 2.0
O
1.0
0
1.0 2.0 4.0 3.0
F igure 10: P lo t of log(kp/K m) vs Es c o n s t a n t s l i s t e d in
Table 2,
47
log k cat /Km (M 1se c 1 )
^ IO Q ^ O l CD
Figu re 11
: Log(k2/Km) ( N -acy lim idazo le) x s l o g ( k 2/Km)
( n i tr o p h e n y l e s t e r s ) .
49
7.0
C O
C D
O
N
6.0
C tf
"D
I
5.0
>
O
cd 4.0
w
E
N -
3.0
C M
O)
2.0
0
- Q ^ c h 2ch2-
( c h 2 )4 c h 3
CH.
c h 3 - c - c h 2 - «
CH
C H 3 -C -
c h 3 f t
■d / / • - c h 2 c h 2 c h 3
-C H 2CH(CH3 )2
• - c h 2 c h 3
1.0 2.0 3.0 4.0 5.0 6.0
log 42/Km (esters)
7.0
V JI
o
k2/Km i s X, 1.7x104 M"1s e c ”1 ; XIV, 1.2x104 M"1s e c “ 1 ; XI,
5.6x103 M “ 1se c ” 1 ; XII, 3.3x103 M “ 1s e c “ 1 ; IX, 1 .7x103
M"1sec~ 1 ; V I I I , 960 M"1s e c " 1 ; X III, 420 M - 1 s e c - 1 . P l o t s of
l o g ( k 2 /Kra) a t pH 5.0 and 7.5 _ys sigma, th e Hammett s u b s t i
t u e n t c o n s ta n t, a re shown in F ig ure 12. The slo p e s of
th ese p l o t s (rho) a re 0.9 (R=0.93) f o r r a t e c o n s ta n ts a t pH
7.5 and near zero f o r r a t e c o n s ta n ts a t pH 5 .0 . Rate con
s t a n t s a t pH 4.01 were a ls o determ ined f o r some of these
compounds. The r a t e v a lu e s were X III, 100 M “ ^ se c “ 1 ; IX,
240 M ” 1s e c " 1 ; XI, 180 M"1se c _ l ; X, 250 M"1s e c " 1 . The Ham
m ett rho v a lu e , as a t pH 5 .0 , i s near z e ro .
The r a t e s of d e a c y la tio n of the a cy l enzymes formed
from the N -a c y lim id a z o le s were measured as d e sc rib e d in th e
prev iou s c h a p te r. A t y p i c a l p l o t of absorbance a t 400 nm
vs time a f t e r i n j e c t i o n of an a l i q u o t of p - n itr o p h e n y l e s
t e r i n t o a s o l u t i o n of acyl enzyme i s shown in F igure 13,
where [E ]o=4x10“ ^M, [ N - i s o v a l e r y l i m i d a z o l e ] 0=3.1 x10“ ^M, and
[ p - n itr o p h e n y l i s o v a l e r a t e ] Q=1 .8x10” 4M. Only a small i n i
t i a l r i s e in absorbance f o r the s o l u t i o n to which th e N-a-
c y lim id a z o le had been p re v io u s ly added, but a much l a r g e r
i n i t i a l i n c r e a s e due to r a p id a c y l a t i o n when th e same a-
mount of p - n itr o p h e n y l e s t e r i s added to th e enzyme a lo n e.
The d i f f e r e n c e in absorbance a t time z ero in th e two case s,
o b tain ed by e x t r a p o l a t i o n , corresponded to a c o n c e n tr a tio n
51
F igu re 12 : P l o t s of l o g ( k 2 /Km) a t 30°C f o r a c y l a t i o n of
a lp h a -ch y m o try p sin by s u b s t i t u t e d N-benzoyl-
im id a zo les its the Hammett c o n s ta n t (sigma) a t
pH 5.0 ( O ) and a t pH 7.5 ( • ) .
C M
3.0
( D
O
- j
2.0
-0 .6 -0 .4 -0 .2 0 0.2 0.4 0.6 0.8
U 1
u>
Figure 13: P lo t of p - n i tr o p h e n y l i s o v a l e r a t e h y d r o l y s i s by
alp h a-ch y m o try p sin w ith (low er l i n e ) and w i t h
out (upper l i n e ) N -is o v a le r y lim id a z o le .
54
0.7
0.6
0.5
o
o
0.4
<
0.3
0.2
0.1
20 10 30 40
Time (min)
v_n
ui
of p - n itr o p h e n o l equal to th e enzyme c o n c e n tr a ti o n . The
N -a c y lim id a z o le s have t h e r e f o r e a c y l a t e d th e a c t i v e s i t e
alm ost com p letely . The r a t e s of d e a c y la ti o n f o r th e two
s o l u t i o n s were, however, i d e n t i c a l . The l i m i t i n g v a lu e of
k:^ fo r a c y la te d alpha-chym otrypsin. p rep ared w ith and w i t h
out N -a cy lim id a zo le r e s p e c t i v e l y a re as f o llo w s : h y d ro c in -
namoyl (pH 7.05) 0.142 sec"'' and 0.144 s e c " ' ; p-cyanoben-
zo y l (pH 8 .0 1 ) 3.89x10"3 s e c ” 1 and 3 .9 2 x 1 0 “^ s e c ” 1 ;
» *
p-dim ethylam inobenzoyl (pH 8.01) 9.98x10"^ s e c ” 1 and
9.14x10“ ^ s e c ” 1 . The v a lu e s of a t pH 8.00 fo r th e f o l
lowing cyclohexylacylenzym es were a ls o measured by. a c y la -
t i n g w ith the p - n itr o p h e n y l e s t e r : c y c lo h e x y lc a rb o n y l,
3 .28x10"2 s e c " 1 ; c y c l o h e x y l a c e ty l , 1.29x10"2 s e c " 1 ; c y c lo -
1 1
h e x y lp ro p io n y l, 5.15x10" sec" , c y c lo h e x y lb u ty r y l,
2 .4 4 x 1 0 "' s e c " 1 .
In F ig u re 7 th e v a lu e s of ^ 7*5 ^or ^ I I I anc*
the analogous N1- m e th y la te d d e r i v a t i v e (XIX) d i f f e r only by
a f a c t o r of 150. This i s a ls o t r u e in th e non-enzym atic
OH" c a ta l y z e d h y d r o l y s i s r e a c t i o n s . P l o t s of lo g kobsd a t
30°C, 0 .1 1 , x s pH are shown in F igu re 14. Rate c o n s t a n t s
are determ ined in HC1 s o l u t i o n s or 0.01M b u f f e r s (fo rm ate,
a c e t a t e , c a c o d y la te , phosphate, T r is , b o r a te , c arb o n a te ,
d i e t h y l a m i n e ) . There i s b u f f e r c a t a l y s i s . I t was shown,
however, t h a t such low c o n c e n tr a ti o n s of b u f f e r do not have
56
an e x p e rim e n ta lly s i g n i f i c a n t e f f e c t on th e observed r a t e
c o n s t a n t s . The e q u a tio n f o r kobsd in h y d r o l y s i s of N-(p-
d im e th y lam in oJ-b enzo yl-N ’-m eth y l-im id azo liu m ion (XIX) i s
given in e q u a tio n 7 w hile f o r the h y d r o l y s i s of the unmeth-
y l a t e d d e r i v a t i v e (X III) i s given in e q u a tio n 8.
^ ^ k-,ag + k2Ka aH + kQHKa Kw
° bS aH + Ka aH
k 1a§ + k2Ka aH + kOHKwKa aH + ^6HK wKa Ka
obs = ------- 5-----------5------------------------------------ (8)
aH + KaaH + Ka Ka aH
The r a t e c o n s t a n t s k^ and k2 are f o r a t t a c k of H20 on th e
s p e c ie s w ith the p-dimethylamino' group p ro to n a te d and un-
p ro to n a te d , r e s p e c t i v e l y . The se co n d -o rd e r r a t e c o n s ta n t
k0H : i -s f o r a t t a c k 0H“ on th e N 1-m ethylated or p ro to n a te d
s p e c i e s of XIII having th e p-dim ethylam ino group in th e
n e u tr a l f r e e base form, w hile k ^ i s f o r OH” a t t a c k on th e
n e u tr a l s p e c ie s of X III. Ka and are d i s s o c i a t i o n con
s t a n t s f o r the c o n ju g a te a c id s of the N’-m e th y la te d and un-
m ethy lated s p e c i e s , r e s p e c t i v e l y . The se co n d -o rd e r r a t e
c o n s ta n ts k^H and k0H. a r e 21.5 and 2150 M ” 1s e c “ 1 . A pH-in-
dependent r e a c t i o n i s e n co u n tere d a t pH 4-7 in h y d r o l y s i s
of the N’-m ethyl d e r i v a t i v e w ith k2=7.1x10“ ^ s e c ” 1 . At low
57
pH hydronium ion (H^O*) c a t a l y s i s i s observed w ith both
compounds which must be due to p r o t o n a t io n of th e p -d im eth
ylamino group, sec"^ (k^=k^/Ka ), Rate measurements
were not o b ta in e d a t s u f f i c i e n t l y high a c i d i t i e s to perm it
d e te r m in a tio n of Ka , i . e . , t h i s c o n s ta n t i s l a r g e r than
1CT1 M.
58
F igure 14: P lo t s of log k . vs pH f o r h y d r o l y s i s of
N -(p-dim ethylaSiB oJbenzoyl-N *-m ethy1-im ida-
zolium ion ( O ) and N-( p-dim ethylam ino) benzoyl-
im idazole ( # ) in H50 a t 30 C, 0.01M b u f f e r ,
0 . 11 . *
59
09
LOG 4 0bsd (SEC-1)
I 1 I I I
Ol W N 3 -
r o
X3
I
CO
Chapter IV
DISCUSSION
In th e r e a c t i o n s of enzymes such as a lp h a -ch y m o try p sin
the k i n e t i c s may be d e s c r ib e d by in d iv i d u a l r a t e c o n s ta n ts
as in e q u a tio n 1. T y p ic a lly , however, k i n e t i c measurements
of enzyme r e a c t i o n s have been determ ined in term s of kc a ^,
the tu rn o v e r c o n s ta n t, and Km(a p p ), th e a p p aren t bin ding
c o n s t a n t . These a re complex c o n s t a n t s and th e r e l a t i o n s h i p
between kcafc, Km(app) and th e r a t e c o n s t a n t s in e q u a tio n 1
w i l l vary w ith the n a tu r e of th e s u b s t r a t e . Furtherm ore,
the e x is te n c e of m u l t i p l e forms of b in d in g , i . e . , nonpro
d u c tiv e b in d in g , may f u r t h e r confuse th e comparison of k i
n e ti c c o n s t a n t s along th e r e a c t i o n p a t h w a y . ^ I t has been
shown, however, t h a t kc a ^/Km(app) i s u n a f f e c te d by th e va
g a r i e s of s u b s t r a t e b in d in g and i s undoubtedly the only r e
l i a b l e c o n s ta n t in s t u d i e s of s t r u c t u r e a c t i v i t y r e l a t i o n
s h ip s . 53 The c o n s ta n t k c a ^/Km i s th e se co n d -o rd e r r a t e
c o n s ta n t f o r r e a c t i o n s between f r e e enzyme and th e sub-
52 Bender, M.L. ; Kezdy, F . J . Ann. Rev. Biochem. 1965, 3JJL ,
49.
53 (a) Brot, F .E .; Bender, M.L. J_t Am. Chem. Soc. 1969,
2JL, 87. (b) F e r s h t, A., "Enzyme S t r u c t u r e and Mechan
ism"; W.H. Freeman and Co.: San F ra n c is c o , 1977, p . 96.
61
s t r a t e . A s e p a r a t io n of c o n s t a n t s in th e p re s e n t study,
then, i s not only u n f e a s i b l e but h ig h ly u n d e s i r a b l e .
I f , in a c y l a t i o n r e a c t i o n s , Km i s much l a r g e r than th e
i n i t i a l s u b s t r a t e c o n c e n tr a ti o n , then th e c o n c e n tr a ti o n of
ES w i l l be so small as to be u n d e te c ta b le , and th e r e a c t i o n
w i l l be e x p e r im e n ta lly second o r d e r . The r a t e c o n s ta n t
determ ined in t h i s case from p l o t s of k - k g JLS [ S ] G, or k vs
[ S ] Q i f k g i s n e g l i g i b l e , i s th e r a t i o k 2 / K m . ^ ^ I t was
found t h a t the r a t e s of a c y l a t i o n of alp h a-ch y m p try p sin by
the N -a c y lim id a z o le s were much g r e a t e r than th o se of dea-
c y l a t i o n . In a l l c ase s, w ith the e x c e p tio n of N -a c e ty l,
s u b t r a c t i o n of kg from k (employing e q u a tio n 3) produced no
change in th e se c o n d -o rd e r r a t e c o n s ta n t, i . e . , p l o t s of
k - k g .vs ( k - k g ) / [ S ] 0 and p l o t s of k - k g vs [ SD0 were i d e n t i
c a l w ith u n c o rr e c te d p l o t s . This i s because k g i s not s i g
n i f i c a n t w ith r e s p e c t to k. The e x ac t slo p e of th e p l o t of
k y_s [ S ] 0 from e q u a tio n 3 i s , of co u rse, (k 2 +kg)/Km, which
w i l l only be equal to k 2/ K m i f k 2 > > k g . Since the second-
ord er r a t e c o n s t a n t s a re i d e n t i c a l whether determ ined from
p l o t s of k y s CS 30 ( e q u a tio n 3) or k - k g x s CS ] 0 (e q u a tio n
2 ) , i t i s c l e a r t h a t k 2 > > k g and a c c o rd in g ly t h a t
( k 2 + k g ) [ S ] 0 > k g K m i s v e r y l i k e l y .
see r e f e r e n c e 48.
62
A c y latio n of alp h a -ch y m o try p sin by th e p r e s e n t s e r i e s
of N -a cy lim id a zo les i s a seco n d -o rd e r r e a c t i o n even a t very
high r a t i o s of [ S ] 0/ [ E ] Q (> 5 0 0 - fo ld ) , as. seen in F ig u re s 2
and 3 where k, th e p s e u d o - f i r s t - o r d e r r a t e c o n s ta n t ob
ta i n e d from th e p r o f l a v i n d isplacem en t measurements c o r r e s
ponding to a c y l a t i o n , i s p l o t t e d n_s k / [ S ] Q. (Since* k^ i s
much sm a lle r than k in th e s e r e a c t i o n s i t can be ig n o red
except in th e case of N -a c e ty lim id a z o le w ith which k^ must
be s u b t r a c t e d from k j . Such a p l o t should be a s t r a i g h t
l i n e w ith slope equal to -Km and o r d i n a t e i n t e r c e p t equal
to k2 .55 However, in a l l c ase s such p l o t s y i e l d e d i n f i n i t e
s lo p e s showing t h a t i f b in d in g i s o c c u r r in g , then Km i s
much l a r g e r than th e h i g h e s t s u b s t r a t e c o n c e n tr a ti o n s t u
died ( 10“ 3-10” 2M) s o t h a t ES i s e x p e r im e n ta lly u n d e te c t a
b le . The h i g h e s t s u b s t r a t e c o n c e n tr a ti o n i s , of course,
determ ined by the v e l o c i t y l i m i t s of th e stop ped -flow in
stru m en t. P l o t s of k i s CS]0 were n ic e ly l i n e a r as shown
in F ig u re s 4 and 5, g iv in g k2/Km as th e slo p e . The r a t i o
k2/Km i s not a f f e c t e d by any n o np rod uctiv e b in d in g of the
s u b s t r a t e .
The n e a rly pH-independent a c y l a t i o n of alpha-chymo
t r y p s i n by N -a c y lim id a z o le s could r e f l e c t s e v e r a l k i n e t i c -
a l l y e q u iv a le n t p o s s i b i l i t i e s . Any i n t e r p r e t a t i o n i s , of
55 see r e f e r e n c e 47.
63
co urse, dependent on Km being l i t t l e a f f e c t e d by changes in
pH, but t h i s i s a r e a s o n a b le assum ption sin c e Km for non
i o n iz i n g s u b s t r a t e s has been shown to be e s s e n t i a l l y pH i n
dependent in th e range 5-8.56 There i s no doubt t h a t s e r
ine-195 i s being a c y l a t e d in view of the i d e n t i c a l r a t e s of
d e a c y la ti o n of acyl chym otrypsins p repared from N -acy lim i
d a z o le s and co rre sp o n d in g p - n i tr o p h e n y l e s t e r s . The sim
p l e s t k i n e t i c i n t e r p r e t a t i o n i s t h a t N -a c y lim id a z o le s acy-
l a t e alp h a-ch y m o try p sin v ia both the p ro to n a te d and n e u t r a l
s p e c ie s or k i n e t i c e q u i v a l e n t s . Thus, the scheme of equa
t io n 9, which ta k e s i n t o account i o n i z a t i o n of both th e en
zyme and th e s u b s t r a t e , i s very l i k e l y being f o l lo w e d .57
K .
m ^
H'
E + S vS
\
+
K1 H+ k2 h+
\ \ \
EH* SH+ ESH
p roduct
K- (9)
k ’
r
^ p r o d u c t
56 (a) Bender, M.L.; Clement, G.E.; Kezdy, F . J . ; Heck, H.
J_t Am. Chem. Soc. 1964, 8(5, 3680. (b )S te w a rt, J .A .;
Lee, H .S .; Dobson, J.E . J_ * . Am. Chem. Soc. 1963, 8£L,
1537. (c) L a i d le r , K .J .; B arnard, M.L. T rans. Faradav
Soc. 1956, 52., 497. (d) Hammond, B .R .; G utfreund, H.
Biochem. J . 1955, fLl> 187. (e) Cunningham, L.W .; Brown,
C.S. J . B i o l. Chem. 1956, 221 f 2 8 7 .
57 see r e f e r e n c e 41.
64
In view of the pKg v a lu e s of N -a cy lim id a zo les (approx. 4)
and h i s t i d i n e - 5 7 (approx. 7 ) , t h i s scheme would e x p la in th e
near pH independence of e x p erim en tal r a t e c o n s t a n t s in th e
range 5 -9 , i f the r e a c t i o n s of the p ro to n a te d s p e c ie s were
of major s i g n i f i c a n c e below pH 6 - 6 .5 , and th e r e a c t i o n s of
the n e u t r a l s p e c ie s were of g r e a t e r im portance a t h ig h e r pH
v a lu e s . The e x p re s s io n f o r k d e riv e d from the scheme of
e q u a tio n 9 i s given in e q u a tio n 10 c o n s id e r in g t h a t %2>aH
and Km> [S ]0 .
k kr K1K3 + k«LK.,aH
" s t ‘ '
At a^>K^ e q u a tio n 10 reduces t o e q u a tio n 11, whereas a t
K^>aH e q u a tio n 12 i s o b ta in e d .
kr K1 k ’ K -,
T K maH KmK3
k
_*r_ _frfH_
s T
+ ~ v 7
Thus, a t pH<6 k^./Km could be th e predominant i n f lu e n c e on k
and a t pH>7 kr /Km would be of g r e a t e s t im portance i f th ese
term s a re not g r e a t l y d i f f e r e n t . In th e case of the
65
3 , 3 -d im e th y lb u ty r y l d e r i v a t i v e , k/./Km i s 1 0 0 -fo ld l a r g e r
than kr /Km (assuming correspondence of k^./Km to th e
N1-m e th y la te d compound). In th e case of th e p-dim ethylam i-
no d e r i v a t i v e k^./Km i s 1 5 0 -fo ld l a r g e r than kr /Km. There
f o r e th e r e a c t i o n through the two s p e c ie s w i l l be a p p ro x i
mately equal 2 pH u n i t s above th e pKa of the
N -a cy lim id a zo le, i . e . , a t pH 5 . 5 - 6 . Above t h i s pH the
r e a c t i o n of the n e u tr a l s p e c ie s w i l l then predom inate, and
r a t e c o n s t a n t s w i l l in c r e a s e s l i g h t l y w ith i n c r e a s in g pH
through th e pKa of h i s t i d i n e - 5 7 . This m ight vary with the
o th er compounds, depending on th e r e l a t i v e v a lu e s of kr and
k^.. For example, i f the r a t i o of th e s e r a t e c o n s t a n t s
(k^./kr ) i s 103 or g r e a t e r , then th e e x p erim e n tal r a t e con
s t a n t s w i l l d e c lin e as pH i s r a i s e d beyond 7, as was ob
served w ith N - b u ty ry l- and N - a c e ty lim id a z o le . N -cyclohex-
y lc a r b o n y lim id a z o le (XV) i s no doubt a d ram atic example of
t h i s happening. I t may be noted t h a t th e r a t i o s of the
r a t e c o n s t a n t s a re a ls o s i m i l a r in nonenzymatic 0H“ c a t a l y
s i s of the h y d r o l y s i s of the unm ethylated and N' - m e th y la te d
N -b en zo y lim id azo les, X III and XIX (F ig u re 14). Thus i t i s
probable t h a t th e same f a c t o r s a re i n f l u e n c i n g th e enzymat
ic and nonezymatic r e a c t i o n s of th ese compounds.
Mechanism of A c y la tio n . A method f o r d e te rm in in g th e
p o s i t i o n of a p roton in th e t r a n s i t i o n s t a t e in r e a c t i o n s
66
of N -a cy lim id a zo les i s t o compare r a t e c o n s t a n t s w ith those
of the c o rre sp o n d in g N -m ethylated d e r i v a t i v e s . i t can be
seen in F ig u re s 6 and 8 in th e l o g ( k 2 /Km) vs pH p r o f i l e f o r
a c y l a t i o n of a lp h a -ch y m o try p sin by N-3, 3 - d im e th y l b u t y r y l-
N*-m ethyl-im idazolium (XX)and N -(p -d im eth y lam in o )- ben zoy l-
N '-m eth yl-im idazo liu m (XIX) ions t h a t th e r a t e i n c r e a s e s
w ith i n c r e a s i n g pH u n t i l a maximum i s rea ch e d . The a p p a r
ent pKg's a re 6.6 and 6.5 r e s p e c t i v e l y which are c lo s e to
t h a t exp ected f o r h i s t i d i n e - 5 7 . ^ The maximum r a t e c o n s ta n t
a t pH 7-5 i s 2 o rd e rs of magnitude g r e a t e r than f o r th e un-
m eth y lated d e r i v a t i v e , whereas th e l o g a r ith m s of r a t e cons
t a n t s f o r th e s e compounds e x t r a p o l a t e to th e same v alue
near pH 4. This pH must correspond w ith the pKa of the
N-3, 3 - d im e th y lb u ty ry l and N -(p-dim eth ylam ino )ben zoy l im ida-
zolium io n s ( th e pKa of the N -acety lim id azo liu m ion i s
3 . 6 ) . ^ Thus a t low pH th e r e a c t i o n in v o lv e s th e N -a cy lim i-
dazolium ion r a t h e r than a k i n e t i c e q u i v a l e n t . I t i s appa
r e n t , however, t h a t a t pH>5 the N -m ethylated compound i s
a c y l a t i n g th e enzyme v ia a m e c h a n is tic pathway not a v a i l a
b le to th e unm ethylated d e r i v a t i v e .
O ak enfu ll, D.G.; S alv esen , K. ; Jen cks, W.P. J_s. Am.
Chem. Soc. 1971 > S3., 188. Also r e f e r e n c e 32.
59 see r e f e r e n c e 7 ( a ) .
Jencks, W.P.; C a r riu o lo , J. J3 B i o l . Chem. 1959, 234 f
1272 , 128 0 .
67
The downward bend in th e l o g ( k 2 /Km)-pH p r o f i l e s f o r
a c y l a t i o n of the enzyme by XIX and XX a t a p p ro x im ately pH 7
i s most l i k e l y r e f l e c t i n g a c r i t i c a l i o n i z a t i o n . A change
i n r a t e - d e t e r m i n i n g s te p , which would a ls o produce a down
ward bend in th e p r o f i l e , would not be ex pected in view of
the N -m ethylated le a v in g group. A change in r a t e - d e t e r m i n
ing s te p o bv iou sly r e q u i r e s an i n te r m e d i a t e s te p , i . e . , a
t e t r a h e d r a l in te r m e d i a t e , which i f formed would be expected
to break down more r a p i d l y to p ro d u cts th an to r e a c t a n t s a t
a l l pH v a lu e s . Thus th e p r o f i l e s i n d i c a t e p a r t i c i p a t i o n by
the base form of h i s t i d i n e - 5 7 . The scheme of e q u a tio n 13
i s being follo w ed from which e q u a tio n 14 i s o b ta in e d , where
K.| i s th e d i s s o c i a t i o n c o n s ta n t of h i s t i d i n e - 5 7 .
H+ k !
EH+— —7 E + SCH^f—; Z^ESCH^ ^ p r o d u c t s (13)
K '1
k k*. K1
ST Km (K1 + aH)
H is ti d in e - 5 7 could a s s i s t a c y l a t i o n of ser-19 5 by p a r
t i a l l y a b s t r a c t i n g a p roton from th e s e r i n e hydroxyl in th e
t r a n s i t i o n s t a t e or i t could be a c t i n g as a n u c le o p h ile
with subsequent r a p id t r a n s f e r of th e acyl group to s e r -
68
in e.^ ^ I t has been su g g ested t h a t h is - 5 7 might f u n c t i o n as
a n u c le o p h ile in a c y l a t i o n by r e a c t i v e n itro p h e n y l e s
t e r s , ^ and th e le a v in g group pKg of N -m ethy l-im id azole i s
approx im ately 7, i . e . , comparable to p - n i t r o p h e n o l . A
c l a s s i c a l method f o r d i s t i n g u i s h i n g n u c l e o p h i l i c and g en er
al base mechanisms in sim ple chemical r e a c t i o n s i s th e D2 O
so lv e n t i s o to p e e f f e c t . A g e n eral base c a ta ly z e d r e a c t i o n
w ith proton 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 proceed
more slowly in D2 O than in H2 O, w hile a n u c le o p h il i c r e a c
t io n w ith o u t proton t r a n s f e r w i l l occur a t n e a rly the same
r a t e . When a p p lie d to enzyme r e a c t i o n s t h i s method i s am
biguous. ^ However, i t has been argued t h a t th e s o lv e n t
i s o to p e e f f e c t in both a c y l a t i o n and d e a c y la ti o n r e a c t i o n s
of alp h a -ch y m o try p sin a llo w s a r e l i a b l e m e c h a n istic i n
t e r p r e t a t i o n . ^ ^ This view point i s s t r o n g l y sup po rted by th e
^ As m entioned in th e f i r s t c h a p te r , a s p a r t a te - 1 0 2 i s
w i t h in hydrogen bonding d i s t a n c e of h i s t i d i n e - 5 7 . A
chemical model study ( r e f e r e n c e t h i s f o o t n o te ) has shown
t h a t asp-102 should have l i t t l e e f f e c t on th e observed
k i n e t i c s i f the pK^ v a lu e s a re normal. In view of t h i s
and th e a f o r e m entioned d is c u s s i o n on th e l a c k of e v i
dence f o r a 1c h a r g e - r e l a y ’ mechanism, t h i s d is c u s s i o n i s
p r e s e n te d in term s of c a t a l y s i s by h i s t i d i n e f u n c t i o n in g
w ith o u t p a r t i c i p a t i o n by asp-102. (Rogers, G.A.;
B ru ice , T.C. J . Am. Chem. Soc. 197*1, 9At 2473.)
^ see r e f e r e n c e 1 8 (b ).
^3 Bender, M.L.; P o llo ck , E. J. ; , Neveu, M.C. J_i. Am. Chem.
Soc. 1962, ilii., 595, and r e f e r e n c e 7 ( a ) .
^ Jencks, W.P. Ann. Rev. Biochem. 1963, 3Z, 603.
69
d e a c y la tio n of [ (p -n itro p h e n o x y )c a rb o n y l]c h y m o try p sin which
proceeds in l a r g e p a r t v ia n u c le o p h il i c a t t a c k by h is-5 7
with a D20 s o lv e n t i s o to p e e f f e c t clo se to u n ity as p r e d i c
ted f o r a n u c le o p h il i c r e a c t i o n . ^ The p l a t e a u in th e
l o g ( k 2/Km) .vs pH p r o f i l e f o r a c y l a t i o n by XIX and XX in D20
o ccurs w ith a l i m i t i n g r a t e c o n s ta n t t h a t i s 3*1- and
2 . 1 - f o l d l e s s th an in H20, r e s p e c t i v e l y , i n d i c a t i n g t h a t
proton t r a n s f e r i s o c c u r rin g in th e t r a n s i t i o n s t a t e . I t
would rea so n a b ly be exp ected t h a t Km(D20) would be somewhat
l e s s than Km(H20 ) .^7 in t h i s r e a c t i o n the h i s t i d i n e co nju
g ate a cid cannot a s s i s t le a v in g group d e p a r tu r e . There
fo re , the most l i k e l y mechanism i s XXI, in which h i s t i d i n e
p a r t i a l l y a b s t r a c t s a p roto n from the s e r i n e hydroxyl in
the t r a n s i t i o n s t a t e . A mechanism in which h i s t i d i n e - 5 7
a b s t r a c t s a proton from a t e t r a h e d r a l i n te r m e d ia te formed
in a c y l a t i o n of the n e u t r a l s e r i n e hydroxyl i s u n l i k e l y .
For breakdown of th e i n te r m e d i a t e to be r a t e d e te rm in in g ,
i t would n e c e s s a r i l y have to decompose to r e a c t a n t s much
f a s t e r than to p ro d u c ts . Mechanisms in v o lv in g r a t e - d e t e r
mining breakdown of a t e t r a h e d r a l i n te r m e d ia te a re not in
( a ) Bender, M.L.; Schonbaum, G.R.; Hamilton, G .A .; Zer-
ner, B. Jj. Am. Chem. Soc. 1961, 83., 1255. (b) Bender,
M.L.; Hamilton, G.A. Am. Chem. S o c. 1962, 8 4 ,
2 5 7 0 .Also r e f e r e n c e 5 6 ( a ) .
f\ fi
see r e f e r e n c e 4 0 ( c ) .
^7 see r e f e r e n c e s 65 (b) and ( c ) .
70
1 o *
accord w ith th e la c k of ' 0 exchange in h y d r o l y s i s of pro-
fi ft
to n a te d N -a c y lim id a z o le s ° ° or w ith the la c k of evidence fo r
s t a b l e t e t r a h e d r a l i n te r m e d i a t e s in t h e i r b im o le cu lar r e a c
t i o n s with a l c o h o l s ; * ^ i n nonenzymatic a l c o h o l y s i s r e a c
t i o n s of th e s e compounds th e t r a n s i t i o n s t a t e must resem ble
r e a c t a n t s .
XXI
Since h i s t i d i n e must p a r t i c i p a t e in th e base form in
a c y l a t i o n of a lp h a -ch y m o try p sin by N1- m e th y la te d N-acylim-
id a z o le s (XIX and XX), i t must a ls o do so in an analogous
manner in r e a c t i o n s of the c o n ju g a te a c id s of the unmethyl-
r e f e r e n c e 11 .
r e fe r e n c e 13
71
a te d d e r i v a t i v e s . However, i f t h a t were th e only pathway
a v a i l a b l e , then th e pH-log ( r a t e c o n s t a n t ) p r o f i l e s would be
pH independent a t low pH ( 5 - 7 ) , but th e slo pe would be - 1 .0
a t pH v a lu e s g r e a t e r than th e pKa of h i s t i d i n e - 5 7 . There
f o r e , a d i f f e r e n t m e c h a n is tic pathway i s being u t i l i z e d a t
pH>7, which must be a r e a c t i o n of n e u t r a l N -a cy lim id a zo le,
as in scheme XXII, or a k i n e t i c e q u iv a le n t such as scheme
XXIII.
R
0
ch2-
x x ii
Mechanism XXIII in v o lv e s r e a c t i o n of n e u t r a l N -acylim ida-
z o le w ith a z w i t t e r i o n i c a c t i v e s i t e , i . e . , s e r i n e anion
and h i s t i d i n y l c a t i o n . A ttack of u n - io n iz e d s e r i n e would
a ls o be pH in dep end en t w ith o u t involvem ent of h i s t i d i n e - 5 7 ,
72
1
\ = J
OCH
HN
NH
XXIII
but such a r e a c t i o n does not occur in a c y l a t i o n by the
N1-m e th y la te d a c y lim id a z o le ( th e r e a c t i o n would be pH in d e
pendent a t a l l pH v a l u e s ) . Since h is - 5 7 i s involved in
r e a c t i o n s of the m eth y lated d e r i v a t i v e (and by analogy N-a-
c y lim idazolium i o n s ) , i t would a ls o be exp ected to p a r t i c i
p a te in r e a c t i o n s of the n e u t r a l s p e c i e s . Likewise, a t t a c k
of the s e r i n e anion on th e N-m ethylated compound does not
o c c u r , t h e r e b y a llo w in g XXIV to be r u le d o u t. Mechanism
XXII would r e q u i r e e x p u ls io n of the u n s ta b le im idazo le an
ion, whereas XXIII would p ro v id e p a r t i a l p r o to n a tio n of the
7° A slope of 1.0 in th e l o g ( k 2/K ) vs pH p r o f i l e would be
o b tain ed f o r such a r e a c t i o n of the N -m ethylated d e r i v a
t i v e . This might perhaps be d e te c te d a t high pH where a
s u f f i c i e n t c o n c e n tr a tio n of s e r i n e anion i s p re s e n t
(pKa =13.6) i f such o b s e r v a tio n s were e x p e rim e n ta lly
f e a s i b l e .
73
le a v in g group in th e t r a n s i t i o n s t a t e f o r product forma
t i o n .
0
o c h 2-
xxiv
The i m i d a z o le - c a ta ly z e d a l c o h o l y s i s of N - a c e ty lim id a z o le
was c o n sid e re d to proceed by a mechanism s i m i l a r to XXIII
in Which the le a v in g group i s p ro to n a te d by th e c o n ju g a te
a c id of the c a t a l y s t . 71 Whether th e t r a n s i t i o n s t a t e w i l l
more c lo s e ly resem ble XXII or XXIII w i l l depend on th e r e
l a t i v e im portance of i n c r e a s e d b a s i c i t y of the n u c le o p h ile
and s t a b i l i z a t i o n of the l e a v in g group (XXIII) and i n
c re a s e d c o n c e n tr a ti o n of th e r e a c t i v e s p e c ie s (XXII). The
z w i t t e r i o n i c a c t i v e s i t e of XXIII would be p r e s e n t only a t
71 see reference 58(b).
74
very low c o n c e n tr a ti o n . By ta k in g th e pKa of the s e r i n e
hydroxyl to be 1 3.6^ 2 and th e pKa of h is -5 7 to be 6.6 i t
fo llo w s from e q u a tio n 15 t h a t the r a t i o of z w i t t e r i o n i c to
n e u t r a l a c t i v e s i t e i s 10“^.
K .-r E-CHpO” ] [HisH+ ] 2.51 x 1.0 M
,_SSE. =---i— ----— =------------= io-7 (1
Kh i s ECH2 0H][His] 2.51 x 10” r M
The value of k2/Km fo r N -hexanoylim idazole a t high pH i s
4 — 1 — 1
5x10 M 's e c . Thus r e a c t i o n v ia mechanism XXIII would
r e q u i r e a t r u e se c o n d -o rd e r r a t e c o n s ta n t of 5 x 1 0 ^
— 1 —1
M sec which i s g r e a t e r than t h a t f o r a d i f f u s i o n - c o n
t r o l l e d r e a c t i o n ( 1 0 ^ M~^sec” ^ ). Thus mechanism XXIII de
mands an improbably high r a t e c o n s ta n t and can t h e r e f o r e be
r u le d o u t. This le a v e s XXI± as th e most l i k e l y mechanism
f o r the a c y l a t i o n r e a c t i o n . I t i s c l e a r t h a t c o n c e n tr a tio n
f a c t o r s outweigh the m e c h a n is tic ad van tag es t h a t would be
o b ta in e d w ith XXIII or XXIV.
The c o n c lu sio n can be drawn t h a t h i s t i d i n e - 5 7 p a r t i c i
p a te s in th e a c y l a t i o n r e a c t i o n s of N -a ey lim id a zo les and
N -acylim idazolium ions by p a r t i a l l y a b s t r a c t i n g a proton
from the s e r i n e hydroxyl as i t a t t a c k s th e carbonyl carbon.
This i s a mechanism t h a t has been g e n e r a lly c o n sid e re d in
B ruice, T .C .; F if e , T.H.; Bruno, J . J . ; Brandon, N.E.
Biochem istry 1962, 1, 7.
75
a c y l a t i o n r e a c t i o n s of chym otrypsin.73 However, in a c y l a
tio n r e a c t i o n s of s p e c i f i c amides a t e t r a h e d r a l in te r m e d i
a te i s probably form ed.7^ a n i tr o g e n i s o to p e e f f e c t in th e
a lp h a -c h y m o try p s in -c a ta ly z e d h y d r o l y s i s fo N - a c e t y l - L - t r y p -
tophanamide r e q u i r e s t h a t th e C-N bond of the amide i s bro
ken in th e r a t e - d e t e r m i n i n g s t e p . I f the r a t e - d e t e r m i n i n g
s t e p in a c y l a t i o n by s p e c i f i c amide s u b s t r a t e s i s breakdown
of a t e t r a h e d r a l i n te r m e d i a t e , then, s in c e n u c l e o p h i l i c a t
tac k would be a p r e e q u ilib riu m s te p and t h e r e f o r e path i n
dependent, a m e c h a n is tic r o l e f o r h is-5 7 could not be spe
c i f i e d in t h a t s t e p . However, th e p r e s e n t r e s u l t s w ith
N -a cy lim id a zo les and N -acylim idazolium io ns show t h a t i n
deed g e n e ra l base a b s t r a c t i o n of a p roto n from the s e r i n e
hydroxyl can be an e f f i c i e n t mechanism in a c y l a t i o n of the
enzyme by i t s s u b s t r a t e s and i s in f a c t r e q u i r e d in r e a c
t i o n s of th e l a t t e r compounds.
E l e c t r o n i c E f f e c t s in A c y la tio n . The rho value ob
ta in e d from th e p l o t in F ig u re 12 a t pH 7.5 i s only s l i g h t
ly p o s i t i v e ( 0 . 9 ) . I t should be noted t h a t in view of th e
near pH independence of ^ ^ m ’ rho value w i l l s t i l l be
73 r e f e r e n c e s 7 (a) and (b)
7^ (a) F e r s h t , A.R.; Requena, Y. J _ jl Am. Chem. Soc. 1971,
H , 7079. (b) F e r s h t, A. R. J^. Am, _ _ Chem. Soc. 1972, 9 4 .
293.
75 see r e f e r e n c e 22.
76
app rox im ately 0.9 a t h ig h e r pH where th e r e can be no
c o n t r i b u t i o n from th e r e a c t i o n of the p ro to n a te d s p e c i e s .
The se c o n d -o rd e r r a t e c o n s ta n t fo r a c y l a t i o n i s a composite
c o n s ta n t, and s u b s t i t u e n t groups m ight t h e r e f o r e produce
changes in e i t h e r 1<2 or Km. A study of th e b in d in g of sub
s t i t u t e d benzamides to chym otrypsin u sin g p r o f l a v i n d i s
placement y i e l d e d a rho f o r th e d i s s o c i a t i o n c o n s t a n t s (Ks )
t h a t was near z e r o . T h u s th e pH -rate c o n s ta n t p r o f i l e s of
F igu re 7 p r i m a r il y r e f l e c t th e i n f lu e n c e of pH on k2 * Rate
c o n s t a n t s (k 2/Km) fo r a c y l a t i o n of th e enzyme by s u b s t i t u t
ed benzoate e s t e r s having p o la r meta and para s u b s t i t u e n t s
were found t o give re a so n a b ly good r e g r e s s i o n l i n e s in Ham
mett p l o t s , a ltho ug h th e r e was marked p o s i t i v e d e v ia ti o n
produced by nonpolar s u b s t i t u e n t s , presumably due to nonpo
l a r bind in g e f f e c t s . 77 With the p r e s e n t s e r i e s of s u b s t i
t u t e d N -ben zoy lim id azoles p-CH^ i s th e only nonpolar sub
s t i t u e n t employed, and i t w i l l be noted in F ig u re 12 t h a t
i t g iv es only a sm all d e v ia ti o n on th e p l o t s . The r a t e
c o n s t a n t s fo r th e u n s u b s t i t u t e d d e r i v a t i v e (V III) d e v ia te
s l i g h t l y in a n e g a tiv e manner, which su g g e sts t h a t any
s t e r i c i n t e r a c t i o n s of the s u b s t i t u e n t groups w ith the ac
t i v e s i t e a re not a p p r e c ia b ly i n c r e a s in g Km in comparison
76 M arini, J . L . ; Caplow, M. Am. Chem. Soc. 1971, S3.,
5560.
77 r € fe r e n c e 33
77
w ith V III. Even m-N02 g iv es a good f i t on th e p l o t s e s t a b
l i s h e d w ith th e para s u b s t i t u e n t s . Thus, i t i s probable
t h a t v a r i a t i o n s in Km a re not s i g n i f i c a n t l y a l t e r i n g th e
s lo p e s . The rho value a t pH 7.5 very l i k e l y i n d i c a t e s t h a t
i n c r e a s e d e l e c t r o n w ithdraw al in th e a cy l group of th e N-
b e n z o y lim id a z o le s i n c r e a s e s th e r a t e c o n s ta n t k2 fo r a c y l a
t i o n . However, t h i s rho i s c o n s id e ra b ly l e s s p o s i t i v e than
t h a t in hydroxide ion c a ta ly z e d h y d r o l y s i s (+1.4).^® The
sm all value of rho i n d i c a t e s only l i t t l e charge development
i n th e t r a n s i t i o n s t a t e (XXV); i t i s pro bab le t h a t n e i t h e r
bond making nor b rea k in g has p ro g re s se d to a g r e a t e x te n t.
XXV
reference 9(b)
78
In a c y l a t i o n r e a c t i o n s of s u b s t i t u t e d phenyl a c e t a t e s ^ r ho
i s +1.8 fo r k2/Km, but th e c o r r e l a t i o n was b e t t e r w ith s i g
ma" than sigma. Hubbard and Kirsch®0 determ ined a rho of
0.96 ^or a ° y l a t io n of the enzyme by a s e r i e s of
acyl s u b s t i t u t e d p - n i tr o p h e n y l b enzo ates and 1.66 f o r the
analogous 2 ,4 - d i n i t r o p h e n y l b en zo a te s. Thus, th e rho fo r
a c y l a t i o n by the N -ben zoy lim idazo les i s l e s s th an t h a t ob
ta i n e d w ith analogous c a rb o x y la te e s t e r s which have le a v in g
groups w ith pKa ' s a t l e a s t 7 pK u n i t s low er.
The rho f o r OH" c a ta ly z e d h y d r o l y s i s of e th y l ben-
Q -I
z o a t e s ° 1 i s l a r g e and p o s i t i v e ( 2 .4 8 ) , p o s s ib ly r e f l e c t i n g
the i n f lu e n c e of e l e c t r o n w ithdraw al on n u c le o p h il i c a t t a c k
by OH" a t th e d e a c ti v a t e d carbonyl of the benzoate e s t e r s .
As th e le a v in g group i s improved th e rho value d e c l in e s ;
rho f o r OH" c a ta ly z e d h y d r o l y s i s of p - n itr o p h e n y l ben-
O p
z o a te s i s 2 .0 . The rho fo r a l k a l i n e h y d r o l y s i s of sub
s t i t u t e d N -ben zoy lim idazo les i s only 1 .4 even though the
im id azole anion le a v in g group has a pKa of 14.5,®^ i . e . ,
Q j!
reaso nab ly s i m i l a r to t h a t of e th a n o l (1 6 ). Likewise, the
79 r e f e r e n c e 18(a)
r e f e r e n c e 33
re f e r e n c e 7 (a)
r e f e r e n c e 33
Walba, H. ; I s e n s e e , R.W. J . Ore. Chem. 1960, £6., 2789.
79
Oc .
rho v alu e f o r w ater c a t a l y s i s i s 1.3 5. The small rho va
l u e s f o r h y d r o l y s i s of N -b enzoylim idazoles must r e f l e c t
t r a n s i t i o n s t a t e s t r u c t u r e s t h a t a re q u i t e d i f f e r e n t than
in comparable r e a c t i o n s of e s t e r s . Thus th e resonance s t a
b i l i z e d le a v in g group of N -b enzoylim idazoles i s profoundly
in f lu e n c i n g th e r e a c t i o n s . F u r th e r , the rho f o r the enzyme
c a ta ly z e d r e a c t i o n i s much l e s s than th e non-enzym atic
r e a c t i o n i n d i c a t i n g a much e a r l i e r t r a n s i t i o n s t a t e f o r the
enzyme r e a c t i o n . An e a r l y t r a n s i t i o n s t a t e im p lie s t h a t
th e n u c l e o p h i l i c a t t a c k i s being g r e a t l y f a c i l i t a t e d by the
enzyme. Thus, n u c le o p h il i c a t t a c k by the hydroxyl group of
s e rin e -1 9 5 i s more f a v o r a b le than a t t a c k by hydroxide ion
even though the pKa of s e r i n e i s only 13-6.®^ Hydrogen
bonding of th e so lv e n t w ater to th e l e a v in g group (XXVI)
may be improving th e ease of bond b reak ing beyond t h a t ex
pected f o r an e s t e r or amide w ith a r e l a t i v e l y poor l e a v in g
group. I t should be noted t h a t th e OH” c a ta ly z e d r e a c t i o n s
of N -benzoylim idazoles have abnormal D20 s o lv e n t is o to p e
e f f e c t s ^ which cn be e x p la in e d by proton t r a n s f e r from H2 0
in th e t r a n s i t i o n s t a t e .
O h
r e f e r e n c e 9(a)
Choi, M. ; Thornton, E.R. i*. Am. Chem. Soc. 1974, Q6 f
1428.
r e f e r e n c e 72
see r e f e r e n c e 85
80
o- - - -H N 6 >
I \= J
H
I
I
O H
XXVI
On th e o th e r hand, th e normal in v e rs e D20 so lv e n t iso to p e
e f f e c t (k0D/kQH=1.2-1 .4) i s found in h y d r o l y s i s of the
N’-m e th y la te d d e r i v a t i v e s w ith which hydrogen bonding of
water to th e le a v in g group cannot occur. T r a n s i t i o n s t a t e
XXVI im p lie s g e n eral a cid c a t a l y s i s in r e a c t i o n s w ith h ig h
ly b a sic n u c le o p h il e s . A s i m i l a r type of mechanism has
been su gg ested in im id a zo le c a ta ly z e d a l c o h o l y s i s of N-ace-
O O
t y li m id a z o le .
The s t e r i c a c c e l e r a t i o n of the r a t e of OH" c a ta ly z e d
h y d r o l y s i s of N -a cy lim id a zo les w ith a lk y l group branching
a t th e a lp h a -c a rb o n of th e a cy l group and th e la c k of ^®0
exchange imply a c o n c e rte d mechanism. This along w ith the
O Q
see reference 58(b).
81
a l c o h o l y s i s s t u d i e s ^ Gf N -a c e ty lim id a z o le showing an ab
sence of a k i n e t i c a l l y s i g n i f i c a n t t e t r a h e d r a l in te r m e d ia te
i n d i c a t e t h a t improvement of le a v in g group a b i l i t y by hy
drogen bonding to s o lv e n t, as in XXVI, could be an impor
t a n t f a c t o r le a d in g to a c o n c e rte d r e a c t i o n . Concerted
mechanisms would e x p la in th e sm all rho v a lu e s in th e r e a c
t i o n s of N -b enzoylim idazoles sin c e i n c r e a s e d e l e c t r o n
w ithdraw al in th e acy l group would enhance n u c le o p h il i c a t
tac k but would h in d e r le a v in g group d e p a r tu r e .
The v a lu e s of k2 /Km f o r the s e r i e s of N -benzoylim ida
z o le s a re c lo s e ly s i m i l a r a t pH 5.0 g iv in g a rho near z e ro .
The rho v a lu e s near zero t h a t have been found p r e v io u s ly in
hydronium ion c a ta ly z e d e s t e r and amide h y d r o l y s i s ^ ® are
due to compensating e f f e c t s of e l e c t r o n w ithdraw al on pro-
tonatiO n and n u c l e o p h i l i c a t t a c k by w ater. The rho value
fo r hydronium ion c a ta ly z e d h y d r o l y s i s of s u b s t i t u t e d N-
b e n z o y lim id a z o le s a t pH 4 .7 -6 i s 0 . 9 9 , ^ 1 which i n d i c a t e s
t h a t the ease of n u c l e o p h i l i c a t t a c k outweighs b a s i c i t y
c o n s i d e r a t i o n s in th e h y d r o l y s i s of th ese compounds. At pH
5 th e N -b enzo ylim id azoles w i l l be s i g n i f i c a n t l y p ro to n a te d
(ap p ro x im ately 10%). Less e x te n s iv e but c o n c lu siv e k i n e t i c
r e f e r e n c e 13
9® r e f e r e n c e 7 (a) .
r e f e r e n c e 83
82
data were a ls o o b ta in e d a t pH 4. As a consequence of th e
n e arly pH-independent r a t e c o n s ta n ts a f u r t h e r r e d u c t io n in
pH has only a small e f f e c t on th e r a t e c o n s t a n t s . Thus, a
f u r t h e r r e d u c tio n in pH w ith a concom itant i n c r e a s e in th e
f r a c t i o n of p ro to n a te d s u b s t r a t e and p ro to n a te d h is -5 7
le a d s to a rho near the pKg (pH 4) t h a t i s a ls o z e r o . ^ In
c o n t r a s t , th e rho f o r h y d r o l y s i s of f u l l y p ro to n a te d N-ben
z o y lim id a z o le s i s 1 .7.93 The rho near z e ro a t pH<5 in th e
enzymatic a c y l a t i o n r e a c t i o n can be c o n sid e re d a c o n tin u a
t io n of th e tre n d noted above in which th e le a v in g group i s
f u r t h e r improved by p r o t o n a t io n , thereb y redu cing rho by
s h i f t i n g th e p o s i t i o n of th e t r a n s i t i o n s t a t e towards r e a c
t a n t s . 9^ The rho near z e ro i n d i c a t e s a t r a n s i t i o n s t a t e in
which th e r e i s l i t t l e n e t change in charge in comparison
92 S u b s t i t u e n t e f f e c t s on th e pKa should be rea so n a b ly
small ( r e f . 8 3 ). I t can be seen in F ig u re 14 t h a t th e
pKa fo r the dimethylamino s u b s t i t u t e d compound i s ap-
pro xim atley 4. That f o r the m - n itr o s u b s t i t u t e d d e r i v a
t i v e should be somewhat l e s s (pKa = 3 .4 ) .
93 r e f e r e n c e 30
91 * A rho of z e ro can f l s o r e s u l t i f t h e r e i s a l i n e a r r e l a
t i o n s h i p between S and H and one i s f o r t u i t o u s l y work
ing a t th e i s o k i n e t i c te m p e ra tu re ( th e slope of such a
p l o t ) . I s o k i n e t i c te m p e r a tu re s a re u s u a l ly o u ts id e the
range of e x p e r im e n ta lly a c c e s s i b l e te m p e ra tu re s (L ef-
f l e r , J.E . J_ * . Org. Chem. 1955, 2 0 T 1202). A p o s s ib l e
i s o k i n e t i c r e l a t i o n s h i p in d e a c y la ti o n of acyl chymo-
t r y p s i n s was found to have B=435°K ( r e f . 15). In th e
p r e s e n t case, even i f B were 303°K th e rho value should
s t i l l be small in th e tem p era tu re range a t which th e en
zyme can be s tu d i e d .
83
w ith the r e a c t a n t . Thus, rho im p lie s a t r a n s i t i o n s t a t e
w ith very l i t t l e bond making w ith the n u c le o p h ile and l i t
t l e bond b rea k in g , w ith the t r a n s i t i o n s t a t e more c lo s e l y
resem bling r e a c t a n t s th an t h a t f o r the r e a c t i o n a t pH 7 . 5 .
F u r th e r , a rho v alue a t pH 7.5 comparable to th e rho found
f o r e s t e r s w ith much b e t t e r l e a v in g groups i n d i c a t e s t h a t
N -ben zoy lim id azoles a c y l a t e alp h a -ch y m o try p sin through a
t r a n s i t i o n s t a t e t h a t resem b les r e a c t a n t s more c lo s e l y than
do th e t r a n s i t i o n s t a t e s of e s t e r s in a c y l a t i o n r e a c t i o n s
of alp h a -ch y m o try p sin .
S t e r i c and Hydrophobic E f f e c t s in A c y la tio n . S t e r i c
e f f e c t s in a c y l a t i o n of a lp h a -ch y m o try p sin by p - n i tr o p h e n y l
e s t e r s have been th oro u g h ly s t u d i e d ^ as have the su bse
quent d e a c y la ti o n r e a c t i o n s . ^ P l o t s of th e lo g a r ith m s of
the r a t e c o n s t a n t s .ys Es , the T a ft s t e r i c c o n s t a n t s ( p i ) , 97
are l i n e a r ( s l o p e = 1 .0 ) , except t h a t th e p o in t f o r n -hexa-
noyl l i e s c o n s id e r a b ly above the l i n e . Thus, with the ex
c e p tio n of th e n-hexanoyl acyl group, s t e r i c e f f e c t s in th e
enzymatic r e a c t i o n s correspond w ith those in OH“ - c a t a l y z e d
e s t e r h y d r o l y s i s . The s t e r i c e f f e c t s in th e d e a c y la ti o n
r e a c t i o n a re a ls o in accord w ith those found in im id a zo le
95 r e f e r e n c e 14
95 r e f e r e n c e 15
97 r e f e r e n c e 24
84
c a ta ly z e d h y d r o l y s i s of e s t e r s of N -a c e ty ls e rin e a m id e ,
where i n c r e a s i n g s t e r i c bulk in th e acyl group d e c r e a s e s
th e r a t e . However, the observed o rd e r of r e a c t i v i t y fo r
a c y l a t i o n of a lp h a -ch y m o try p sin by N -a c y lim id a z o le s i s not
in accord w ith t h a t in th e g e n eral base or hydroxide ion
c a ta ly z e d h y d r o l y s i s . of th e s e compounds.99 N-hexanoyl- and
N -b u ty ry lim id a z o le a c y l a t e the enzyme f a s t e r than th e o th e r
compounds in th e s e r i e s , whereas N - a c e ty lim id a z o le r e a c t s
more slowly than th e o th e r compounds. The d e r i v a t i v e s w ith
b ran c h ed -c h ain acy l groups occupy in te r m e d i a t e p o s i t i o n s in
the s e r i e s . Thus, i n c r e a s i n g chain le n g t h a c c e l e r a t e s th e
r e a c t i o n g r e a t l y , w hile chain bran chin g r e t a r d s th e r a te ..
Accordingly,- N - is o v a le r y lim id a z o le i s more r e a c t i v e than
N - 3 ,3 - d im e th y lb u ty r y lim id a z o le but i s l e s s r e a c t i v e than
N - b u ty ry lim id a z o le . Alpha branching a p p e a rs to have no a c
c e l e r a t i n g e f f e c t i n th e enzym atic r a t e s comparable to th e
nonenzymatic r e a c t i o n . This i s seen in th e very sm all
d i f f e r e n c e between th e p r o f i l e s o f N - t r i m e th y l a c e ty l i m i d a -
zole and N-3, 3 - d im e th y lb u ty ry lim id a z o le ( F ig u re s 6 and 8 ).
The c o r r e l a t i o n of l o g ( k 2 /Km) with th e Es c o n s t a n t s i s poor
f o r the a c y l a t i o n of th e enzyme by th e N -a c y lim id a z o le s
(F ig u re 10). A computer f i t t e d l i n e has a slope of
M i l s t e i n , J . B . ; F i f e , T.H. J . Am. Chem. Soc. 1968, 9 0 ,
2164.
99 r e f e r e n c e 9 (a)
85
-2 .1 0 ± 0 .6 5 (F ig u re 10). However, th e same d ata c o r r e l a t e s
very well w ith the Hansch pi c o n s t a n t s (F ig u re 9 ) . A com
p u ter f i t t e d l i n e f o r F ig u re 9 has a slope of 1.57± 0.13.
When a l l th e data i s computer f i t t e d to e q u a tio n 6 t h e r e i s
a change in th e a c t u a l v a lu e s computed but th e q u a l i t y of
the c o r r e l a t i o n , i . e . , th e p e rc e n t d e v ia ti o n , rem ains e s
s e n t i a l l y the same. I t would appear then t h a t the
a c y l a t i o n r a t e c o n s t a n t s a r e e f f e c t e d to a f a r g r e a t e r ex
t e n t by the hydrophobic n a tu r e of the acy l group th an by
s t e r i c f a c t o r s . The r e v e r s e i s t r u e f o r p - n i tr o p h e n y l e s
t e r s (x=1.51 and y = 0.63 a t pH 7 . 9 9 ) . 100 The a c y l a t i o n of
a lp h a -ch y m o try p sin by N -a cy lim id a zo les i s much more g r e a t l y
enhanced by th e h y d ro p h o b ic ity of the a cy l group th an a re
n itr o p h e n y l e s t e r s , as evidenced by the g r e a t d i f f e r e n c e in
t h e i r r e s p e c t i v e y v a lu e s . F u r th e r , the s t e r i c e f f e c t s ap
pear to be much l e s s im p o rta n t, whereas they a re of much
g r e a t e r im portance f o r the n itr o p h e n y l e s t e r s . The p l o t in
F igu re 11 has a slope of 1.15. When th e branched a cy l
groups a re removed, thus e l i m i n a t i n g th e compounds w ith the
g r e a t e s t s t e r i c bulk, th e slo p e i n c r e a s e s to 1.66. There
f o r e , th e l a r g e p o s i t i v e e f f e c t of h y d ro p h o b icity on th e
a c y l a t i o n r e a c t i o n of N -a cy lim id a zo les r e l a t i v e to n i t r o
phenyl e s t e r s g iv es su p p o rt to th e model of a t r a n s i t i o n
100 Hansch, C. ; Coats, E. J . Pharm. S c i. 1970, 5jL, 731.
86
s t a t e which resem bles r e a c t a n t s , i . e . , l i t t l e charge
fo rm atio n r e l a t i v e to th e c o rre sp o n d in g n i tr o p h e n y l e s t e r
t r a n s i t i o n s t a t e .
Summary. N -a cy lim id a zo les a re an i d e a l s u b s t r a t e w ith
which to study the a c y l a t i o n r e a c t i o n of alpha-chym otryp
s in . T heir unique h y d r o l y t i c behavior has allow ed th e de
t a i l e d a n a l y s i s of the a c y l a t i o n s te p f o r alpha-chym otryp
s i n . F u r th e r , because of th e a b i l i t y to m e th y la te the
l e a v in g group, t h i s i s th e f i r s t study in which k i n e t i c
e q u i v a l e n t s i n an enzym atic study could be unambiguosly e-
lim in a te d . These s u b s t r a t e s have o f f e r e d more d e t a i l e d i n
form atio n on th e t r a n s i t i o n s t a t e in an enzym atic r e a c t i o n
than has p r e v io u s ly been a v a i l a b l e through more c o n v en tio n
al s u b s t r a t e s . Thus i t would appear t h a t the t r a n s i t i o n
s t a t e i n th e a c y l a t i o n r e a c t i o n of chymotrypsin by N -acyl
im id a zo les resem bles r e a c t a n t s more than p ro d u cts as e v i
denced by the very low Hammett rho v a lu e s . The high c o r r e
l a t i o n T his shows th e enzyme i s g r e a t l y f a c i l i t a t i n g s e r i n e
n u c l e o p h i l i c a t t a c k , making s e r i n e a t t a c k even more f a v o r
a b le than hydroxide ion a t t a c k , d e s p ite a pKa fo r s e r i n e of
only 1 3 .6 . The high c o r r e l a t i o n w ith the Hansch pi con
s t a n t s seems to su g g e st t h a t very l i t t l e charge i s being
g e n e ra te d in th e t r a n s i t i o n s t a t e , an o b s e r v a tio n which
adds f u r t h e r su p p o rt to th e view of a t r a n s i t i o n s t a t e
87
which resem b les r e a c t a n t s . I t i s c l e a r from the p r e s e n t
study t h a t f o r r e a c t i o n s of alp h a-ch y m o try p sin th e mecha
nism w i l l change as a f u n c t i o n of s u b s t r a t e s t r u c t u r e ,
mechanism being a d e s c r i p t i o n of the t r a n s i t i o n s t a t e .
_ 8 l 8_
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Asset Metadata

Core Title The mechanism of alpha-chymotrypsin acylation by N-acylimidazoles

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1983-09

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)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-613321

Unique identifier UC11354984

Identifier DP21613.pdf (filename),usctheses-c17-613321 (legacy record id)

Legacy Identifier DP21613.pdf

Dmrecord 613321

Document Type Dissertation

Rights Kogan, Robert Linn

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...

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