Dec. 5, 1962 [CONTRIBUTION No. 2830
a-CHYMOTRYPSIN-CATALYZED REACTIONS FROM THE
4487
GATESAND CRELLINLABORATORIES OB CHEMISTRY, CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA, CALIF.]
Steric Course and Specificity of a-Chymotrypsin-catalyzed Reactions. I' BY GEORGE E. HE IN^
AND
CARLNIEMA"~
RECEIVED APRIL 16, 1962 The kinetics of the a-chymotrypsin-catalyzed hydrolysis of formyl-D- and &phenylalanine methyl ester, benzoyl-D- and L-alanine methyl ester, D- and L-3-carbornethoxydihydroisocarbostyriland 3-carbomethoxyisocarbostyril have been determined. The inhibition of the hydrolysis of b e n z o y h - and Lalanine methyl ester and of D- and L-3-carbomethoxydihydroisocarbostyril by indole has also been examined. From these data it has been demonstrated: (a) that a-chymotrypsincatalyzed reactions proceed with relative rather than absolute stereospecificity; (b) that the degree of relative stereospecificity is determined, as 3 first approximation, by the size of the groups attached to the asymmetric carbon atom; and (c) that with D- and L-3-carbomethoxydihydroisocarbostyrilan inversion of antipodal specificity is observed.
Introduction The stereospecificity encountered in enzymecatalyzed reactions has long been associated with the formation of a diastereoisomeric enzyme substrate c o m p l e ~ . ~ -In~ particular cases the greater, if not overwhelming, reactivity of one member of an enantiomorphic pair of substrates has been accounted for by reference to the predicted different molecular properties of the two possible diastereoisomeric enzyme-substrate complexes. Nevertheless, comprehension of reactions catalyzed by enzymes requiring no coenzyme has progressed relatively little beyond the apt, but vague, lock and key analogy proposed by Fischer.6 This is particularly true for the proteinases, where the almost universal use of conformationally indeterminate model substrates has given practically no information about the conformation of the active site of the enzyme other than to confirm its asymmetry. I n contrast to the above, notable progress has been made, for example, in understanding reactions catalyzed by DPN and T P N dependent dehydrog e n a s e ~by ~ use of conformationally constrained substrates to elucidate the steric course of these enzyme-catalyzed reactions. The recent discovery of a conformationally constrained substrate of a-chymotrypsinlUhas opened a path to an interpretation of the steric course and specificity of achymotrypsin-catalyzed reactions which we shall develop in this and subsequent communications. Results The substrates selected for study were three pairs of structurally related compounds, all derivatives of alanine methyl ester: formyl-D- and Lphenylalanine methyl ester (I), benzoyl-o- and Lalanine methyl ester (11) and D- and L-3-carbomethoxydihydroisocarbostyril (111); cf. Fig. 1. For each pair the hydrolysis of both enantiomers (1) Supported in part by a grant from the Sational Institutes of Health, U. S. Public Health Service. (2) National Science Foundation Postdoctoral PeIlow 1961-1962: present address, Dept. of Chemistry, Boston Univ., Boston 15, Mass. (3) To whom inquiries regarding this article should be sent. (4) L. Pasteur, Compl. rend., 46, G15 (1858). ( 5 ) E. Fischer and H. Thierfelder, Bel., 27, 2036 (1894). ( 8 ) E. Fischer, ibid., 27, 2985 (1894). (7) E. Fischer, Z . physiol. Chcm., 2 6 , 60 (1898). (8)A. G. Ogston, Nolure, 162, 963 (1948). (9) For a recent review see G. W. Wolstenholme and C. XI. O'Connor. "Steric Course of Microbiological Reactions." Ciba Foundation Study Group No. 2, Little, Brown and Co., Boston, Mass., 1989. (10) G. E. Hein, R. B. XlcGriff and C. h-iernann, J. Am. Chcm. Soc., 62, 1830 (1900).
was catalyzed to a measurable extent by a-chymotrypsin; cf. Table I. The hydrolysis of both enantiomers of each of the preceding three pairs confirms and extends previous observationsl1-13 that the stereospecificity of achymotrypsin-catalyzed reactions is relative rather than absolute. I n contrast to the earlier examples, which were esters of a-halo or a-hydroxy acids,I1-la those described here demonstrate that relative stereospecificity may also be observed with certain a-amino acid derivatives. l 4 Although the stereospecificity may be relative rather than absolute, i t is usually assumed that for different pairs of enantiomorphic compounds, a particular enzyme will either show no stereospecificity or will always favor those enantiomers possessing related absolute configurations. I n fact, the enzymatic determination of the absolute configuration of a-amino acids depends upon the validity of this assumption.l5 All previous experience with a-chymotrypsincatalyzed reactions would lead one to expect a relative stereospecificity in favor of substrates possessing the L- or S16 configuration, The data summarized in Table I does not support this expectation. For two of the pairs, formyl-D- and L-phenylalanine methyl ester and benzoyl D- and L-alanine methyl ester, the more rapidly hydrolyzed enantiomer belongs to the L-series, the behavior usually observed with a-chymotrypsin. However, for the third pair, D- and L-3-carbomethoxydihydroisocarbostyril, the more rapidly hydrolyzed compound is the D-antipode! In order to assert that an inversion of antipodal specificity has occurred, it is necessary to ascertain that the more rapidly hydrolyzed members of the pairs in question are indeed of opposite absolute configuration. Formyl-L-phenylalanine methyl ester and benzoyl-L-alanine methyl ester were both prepared in an unambiguous way from the corresponding L-a-amino acids. These acids have been related and are of the S-series.16 D-&Carbomethoxydihydroisocarbostyril was prepared from (11) J. E. Snoke and H. Neurath, J. B i d . Chcm., 182, 577 (1950). (12) J. E. Snoke and H. Neurath, Arch. Biochcm., 21, 351 (1949). (13) S. ICaufman and H. Neurath, ibid.. 21, 437 (1949). (14) In unpublished experiments conducted in these laboratories Dr. R. M. Bock found that acetyl-D-tryptophan methyl ester is also hydrolyzed by a-chymotrypsin. (15) J. P. Greenstein and Xl. Winitz, "Chemistry of t h e Amino Acids,'' John Wiley and Sons, Inc., New York, N. Y.,1961, Vol. 1, PP. 130-152, 728-750; VOI. 2, pp. 1753-1812. (16) R. S. Cahn, C. K. Ingold and V. Prelog, Expcrienfio, 12, 81 (1956).
4488
GEORGE E. HEINAND CARLNIEMANN
Vol. 84
TABLE I a-CHYhIOTRTPSIN-CATALYZEDHYDROLYSIS O F S O M E ACYLATED
islo,
Substrate Methyl formyl-L-phenylalaninate Methyl formyl-D-phenylalaninate
Expkb
19-3
Methyl benzoyl-L-alaninate
20-0
Methyl benzoyl-D-alaninate
26-7
L-3-Carbomethoxydihydroisocarbostyril
17-1
D-3-Carbomethoxydihydroisocarbostyril Carbomethoxyisocarbostyril
31-3 14-0
WAMINO ACID ESTERS’
koOd mM Set.-’ Hydrolyzed too rapidly t o evaluate K Oand ka 0.189-15.9 3.54 X 10-1 0 . 2 4 8 =k 0.232 0.00343 0,00006 2.50 -15.9 3.54 X 10-8 9.75 -C 0.86 0.261 z t 0.011 0.98 -18.9 3.54 X 10-i 3 . 2 9 k 0 . 1 8 0,0107 i 0.0002 ,406- 3.63 2.01 X 11.69 & 1.53 0 . 1 2 4 -C 0.014 .216- 2.14 1.05 X 10-7I 0 . 5 2 7 zt 0.80 2 2 . 7 i 1 . 2 .127- 0.509 1.66 X 10-6 1 . 4 1 i 0.41 0.134 i 0.052
mM
[ELC M
K0,d
*
ko/Ko, M-1 seC.-l -105g
(ko/Ko)I, (ko/Ko)D -10‘
13.83 26.77 8.23
3.23 10.61 2 . 4 G X 10-4
4 . 3 1 X 104 95.04
li In aqueous solutions a t 25.0°, fiH 7.90 i 0.05 and 0.20 M in sodium chloride. * Number of experiments performed for evaluation of K Oand k o ; second number refers to those rejected by statistical reiterative procedure used for evaluation of K Oand ko, Based upon a molecular weight of 25,000 and a nitrogen content of 16.5%. Evaluated by a least squares fit t o the equation ( [ E ] [ S ] / V O=) (Ko/ko) ( [ S ] / k o )as described in text. e Mean value of range from 1.94 X to 2.09 X Mean value of range from 0.94 X 10-7 to 1.15 X lo-’. Estimated from the rate of hydrolysis a t low values of
+
1SIo.
D-phenylalanine and, barring any odd number of p r e v i o ~ s l y . ‘ ~With this procedure it was possible steps in the synthesis which inverted configuration, 220 digital computer programmed as described should belong to the D-phenylalanine, or R, series. to follow reactions whose initial velocities did not The nature of the synthetic route from D-phenyl- exceed 10-4M,/min. and to evaluate the constants ko alanine to D-3-carbomethoxydihydroisocarbostyril and KOof the rate equation for all substrates exmakes any inversion improbable, since a t no time cept formyl-L-phenylalanine methyl ester. With were any of the bonds between the asymmetric this very reactive substrate, instrumental limitacarbon atom and its adjacent four atoms broken, tions were encountered. Any attempt to slow nor was there any valence change in any atom down the reaction by decreasing the concentration attached to the asymmetric carbon atom. Some of substrate or enzyme resulted in conditions unracemization was encountered in the conversion favorable for the determination of the kinetic constants in the former instance or in non-reproducof D-phenylalanine to ~-3-carboxy-1,2,3,4-tetrahydroisoquinoline. However, the preparation of ible results in the latter. I t had been found both enantiomers of this intermediate and of earlier20 that reactions conducted a t enzyme the final product, D- and L-3-carbomethoxydi- concentrations of less than lo--’Af are complihydroisocarbostyril, with experimentally equal cated by adsorption of enzyme on the surfaces of and opposite rotations affords ample evidence for the glass reaction vessels and electrodes. At these the optical integrity and purity of these substrates. concentrations only a part of the enzyme is molecuThus, an inversion of the usual antipodal specificity larly dispersed, the amount being markedly deobserved in a-chymotrypsin-catalyzed reactions pendent upon the prior history of the glass surfaces. Thus, the lack of reproducibility noted above is has been demonstrated. understandable. The kinetic properties of the systems containing D-3-carbomethoxydihydroisocarbostyril were uncomfortably close to the limits of experimental capability and consequently a relatively large number of experiments was reO = d quired to obtain significant values for the two ‘NH-C ‘NH-C ‘NH-C kinetic constants of this substrate. Empirical ti’ ‘C02CH3 H’ ‘COzCH, H’ ‘COzCH, standardization of the experimental procedure I II m which could have led to more consistent, but not necessarily more accurate, results a t low enzyme Fig. 1.-Substrates of a-chymotrypsin. concentrations was considered and rejected beAll kinetic experiments were conducted in cause there is no assurance that molecularly disaqueous solutions a t 25.0”, FH 7.90 ==I 0.05 and persed and surface adsorbed enzyme have the same 0.20 M in sodium chloride with the rates being kinetic properties. determined with the aid of a pH-stat.17t18The While i t was not possible to evaluate ko and KO primary data, which consisted of recorder traces for formyl-L-phenylalanine methyl ester, it was of the amount of base added to the reaction system possible to arrive a t an estimate of kolKo for this us. time, were first reduced to obtain values of the so-called initial velocities, which in turn were used substrate. Since the rate of an enzynie-catalyzed to compute values of KO and ko for the rate equation reaction described by ey. 1 is proportional t o ko and, when [S10 I-%
e---*$
:/
-
8.0
*\ -
y 6.0
4.0
Y
>I-e-
D
-
-
t
8.06.0 -
I
I
7.0
8.0
The results obtained with D- and ~-3-carbometh- Fig. z.--pH-activity lelationships for a-chymotrypsino x ~ d i h ~ d r o i s o c a r b o s tare ~il so catalyzed hydrolysis of D- and L-3-carbomethoxydihydroisothat i t may be questioned whether these com- carbostvril, (21) R. J. Foster and C. Niemann, J . A m . Chem. Soc., 77, 1886 (1955). (22) R. J. Foster, H.J. Shine and C. Niemann, ibid., 77, 2378 (1955). (23) E. S. Awad, 11. 1-curath aud i3.S . IIartley. J . Uiol. Chein., 236, 1’C 35 (1960).
activity curv-es similar to those observed for other neutral ester type sub~trates.17~18ivhile the pre(21) G . I€& (19G1).
and C. S i e t n a n n , Pioc. S i l l l . i l r e i l . Sci., 47, 1311
4490
GEORGE
E. EIEIN
.4KD
ceding characteristics suggest that the dihydroisocarbostyril derivatives and the more conventional substrates interact with a-chymotrypsin a t a common site, the evidence is not rigorous. Therefore, a series of inhibition studies capable of more direct interpretation was performed. These studies are summarized in Table 11. The inhibition of the a-chymotrypsin-catalyzed hydrolysis of benzoyl-D- and L-alanine methyl ester by indole was examined because earlier studiesl7sZ5 had shown that this inhibitor could be used to distinguish between so-called bi- and trifunctional substrates of a-chymotrypsin.26 With the former, represented by benzoylglycine methyl ester, fully competitive inhibition is not observed, but instead simultaneous combination of enzyme, substrate and inhibitor to give a ternary complex capable of yielding reaction products. With the latter, represented by a-nicotinyl-L-tryptophanamide, fully competitive inhibition results, Le., only binary complexes of enzyme and substrate or enzyme and inhibitor are formed. The low order of relative stereospecificity, in favor of the Lantipode, observed for benzoyl-D- and L-alanine methyl ester raises the question whether these substrates function as bi- or trifunctional substrates in their interaction with a-chymotrypsin. The data given in Table I1 clearly demonstrate that inhibition of the a-chymotrypsin-catalyzed hydrolysis of both benzoyl-D- and L-alanine methyl ester by indole is fully competitive. The values of K I obtained with the D- and L-antipodes, ;.e., 0.60 f 0.30 and 0.58 f 0.43 mM, respectively, are in reasonable agreement with the earlier value of 0.80 f 0.30 mM.25,28 The inhibition of the achymotrypsin-catalyzed hydrolysis of D-%carbomethoxydihydroisocarbostyril by indole is also fully competitive. The value-of K~"sodetermined, 0.76 f 0.35 mM, is consistent with the values noted above. The conclusion that D-3-carbomethoxydihydroisocarbostyril and the more conventional substrates of a-chymotrypsin are hydrolyzed a t a common active site is inescapable. In contrast to the behavior observed with the Dantipode, inhibition of the a-chymotrypsin-catalyzed hydrolysis of L-3-carbomethoxydihydroisocarbostyril by indole is not fully competitive. In fact, i t was possible to calculate values of K I in good agreement with those obtained previously for cases of fully competitive inhibition by assuming in this instance fully non-competitive inhibition. The data summarized in Table I1 demonstrates the essential independence of KO and the dependence of ko upon [I] required for fully noncompetitive inhibition. The types of inhibition exhibited by indole in the preceding studies, along with the previously observed mixed type for this inhibitor, l 7 v z 5 demonstrate that the type of inhibition observed in an enzyme-substrateinhibitor system is a function of the substrate as well as of the enzyme and inhibitor. For indole and a-chymotrypsin there are now available examples of fully competitive, (25) H. T. Huanp and C. Siemann, J . A m . Cham. Soc., 76, 13Q5 (195'3). ( 2 0 ) R . J. Foster and C. Niemann, ibid., 77, 3370 (1955).
CARL
Vol. 84
I\;IEMANN
fully non-competitive and mixed, depending upon the structure of the substrate. In earlier s t ~ d i e s ~ a-N-acetyl-D-tryptophan~m~~ amide was found to be a fully competitive inhibitor of the a-chymotrypsin-catalyzed hydrolysis of eight representative substrates of this enzyme including two a-N-acylated-L-tryptophanamides, four a-N-acylated-L-tyrosinamides, benzoyl-Lvaline methyl ester and benzoylglycine methyl e ~ t e r . *A~mean ~ ~ ~value of Kr = 2.3 i: 0.4 m M was obtained for the amide type substrates22 and values of 2.6 f 0.4 and 2.1 + 1.2 m M for the two ester type substrates. U'hen the same inhibitor was evaluated against L-3-carbomethoxydihydroisocarbostyril the inhibition was fully noncompetitive with a value of K I = 1.96 0.56 m M ; cf. Table 11. These results confirm and extend those obtained with indole and demonstrate that L-3-carbomethoxydihydroisocarbostyril and a-N-acetyl-D-tryptophanamide can form a ternary complex with a-chymotrypsin. However, this ternary complex, in contrast to that formed from cu-chymotrypsin, benzoylglycine methyl ester and indole, l7nZ5apparently is incapable of decomposing to give reaction products.
Discussion Substrates represented by the formula R1CHR2CORa, where Rz # H I may combine with a-chymotrypsin through interaction of R1 = R1'CONH, Rz and COR3 with their complementary loci, p1, p2 and p3, present a t the asymmetric active site of the enzyme. All three interactions are presumed to be important for orientation of the substrate, but all three are not necessarily involved in determining the magnitude of the enzymesubstrate dissociation constant.24 IVhen any one of the three groups responsible for orientation of the substrate in the complex fails to function efficiently, because it is too small or is lacking in critical structural features, 2 4 then the compound may assume alternate orientations which for the L-antipode are either non-productive or less reactive than the single optimum orientation, but for the D-antipode may be productive. Thus, for formyl-D- and 1-phenylalanine methyl ester, with R1' = H, alternative orientations become more favored than when R1' = CH3or a group of greater steric requirement, with the result that with substrates of this type, where the enzyme-substrate dissociation constant is largely determined by RYp2 and COKa-ps interaction^,^^ the rate of forrnation of products from the L-antipode is depressed and from the D-antipode enhanced relative to t h e situation where R 1is more effective in its orienting role, as for example in acetyl-D- and L-phenylalanine inethyl ester. Although benzoyl-L-alanine methyl ester prohably approximates the S%,R,limit type, where the enzyme-substrate dissociation constant is largely determined by RI-pl or R1-p2 and C0R3-p:~intera c t i o n ~ the , ~ ~ expected behavior of the enantiomorphic pair is the same as that noted above arid in this instance arises from the iiiability of I
