April 20, 1961
ETHYLl-ACETYL-2-BENZYLCARBAZATEAND a-CHYMOTRYPSIN INTERACTION
1879
ORGANIC AND BIOLOGICAL CHEMISTRY [CONTRIBUTION No. 2623
FROM THE
GATESAND CRELLINLABORATORIES OF CHEMISTRY, CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA, CALIFORNIA]
The Interaction of Ethyl 1-Acetyl-2-benzylcarbazate with alpha-Chymotrypsin’ BY ABRAHAMN. KURTZAND CARLNIEMANN~ RECEIVED OCTOBER13, 1960 Ethyl l-acetyl-2-benzylcarbazate, an analog of ethyl acetyl-D and L-phenylalaninate, is not a substrate of a-chymotrypsin. Although the carbazate inhibits the a-chymotrypsin catalyzed hydrolysis of methyl chloroacetyl-L-valinate, it is less effective in this action than ethyl acetyl-D-phenyhianinate. The significance of these observations relative to the mechanism of action of a-chymotrypsin is discussed.
The purpose of this investigation was to determine the kinetic consequences of replacement of the a-methine group, in a “typical” substrate of a-chymotrypsin, by a nitrogen atom. The compound chosen for study was ethyl l-acetyl-2ben~ylcarbazate~ (I), an analog of ethyl acetylD and L-phenylalaninate (11). The former com-
pound possessed three of the four structural features of a “typical” substrate of a-chymotrypsin, i.e., an a-benzyl side chain, an a-acetamido group and a carbethoxy function, but with these groups disposed about a nitrogen atom instead of a methine group. An aqueous solution containing ethyl l-acetyl2-benzylcarbazate and a-chymotrypsin was maintained a t 25’ and p H 7.95 for one week. During this time the amount of base consumed, in maintaining the p H constant, was less than that required for a comparable system containing only enzyme. The intended substrate was recovered from the reaction mixture in 94% yield. Thus, replacement of the a-methine group by a nitrogen atom, in a molecule that possessed all of the other requisite structural features, led to a compound that was incapable of functioning as a substrate. The preceding observation raised the question as to whether ethyl 1-acetyl-2-benzylcarbazate is exceptionally resistant to hydrolysis. It may be recalled that whereas 2-methyl-4benzyl-5-oxazolone is hydrolyzed a t room temperat ~ r e the , ~ base catalyzed ethanolysis of the analoregous 2-methyl-4-benzyl-l,3,4-oxadiazolone-5 quires refluxing conditions. The lesser reactivity of the latter compound probably is due to a reduction in the electrophilic character of the carbon atom in position 5 by the unshared pair of electrons of the nitrogen atom in position 4. The same situation would prevail in the base catalyzed hydrolysis of ethyl 1-acetyl-2-benzylcarbazate, and it was anticipated that this compound would be hydrolyzed a t a slower rate than ethyl acetyl-D or L-phenylalaninate. The question was, how much slower? (1) Supported in p a r t by a grant from the National Institutes of Health, U. S. Public Health Service. (2) T o whom inquiries regarding this article should be sent. (3) A. N. Kurtz and C. Niemann, J . Org. Chcm., in press. (4) M. Bergmann, F. Stern and C. Witte, Ann., 449, 277 (1926).
In aqueous solutions a t 25.0 f 0.1” and 0.1 M i n sodium chloride and over a PH range of 7.5 to 9.0, the base catalyzed hydrolysis of ethyl l-acetyl-2benzylcarbazate was described by the relation v = k B [RC02C2Hs][OH-] and a value of kg = 3.42 A 0.04 1. mole-’ min.-’ was obtained. For ethyl acetyl-DL-phenylalaninate kg = 17.7 f 1.6 1. mole-’ min.-’. If steric factors are excluded and if the electronic factors encountered in the base catalyzed hydrolysis are similar to those operative in the enzyme catalyzed hydrolysis, the five-fold difference in the rates of the base catalyzed hydrolysis of ethyl l-acetyl-2-benzylcarbazate and ethyl acetyl-mphenylalaninate does not afford a satisfactory explanation as to why ethyl acetyl-L-phenylalaninate can function as a substrate of a-chymotrypsin and ethyl 1-acetyl-2-benzylcarbazate cannot. While the unshared pair of electrons on the anitrogen atom will lead to depolarization of the adjacent carbonyl group, it does not appear reasonable that this effect can account for the loss in substrate activity, particularly when the rate of the base catalyzed hydrolysis of ethyl l-acetyl-2benzylcarbazate is only one-fifth as rapid as that of ethyl acetyl-DL-phenylalaninate. The hydrolysis of /3-N-benzyl-6-N-carbethoxyN‘-benzyl-N’-carbethoxycarbamylhydrazine with concd. hydrochloric acid to give benzylurethan and a 98% yield of ethyl 2-ben~ylcarbazate~ provides an instructive, but biased, example of the hydrolytic stability of a carbalkoxy group in an alkyl 1acyl-2-alkylcarbazate under conditions where the acid catalyzed hydrolysis of the l-acyl group may proceed in high yield. The example cited is particularly favorable to the point being made because of the nature of the l-acyl group, ie., i t is an N-alkyl-N-acyl carbamyl group. However] with any simple alkyl l-acyl-2-alkylcarbazate one would expect the rate of an acid catalyzed deesterification to be slow because protonation of the nitrogen atom alpha to the carbethoxy group would cause a marked decrease in the apparent basicity of the adjacent carboxyl oxygen atom. If the mechanism of enzyme catalysis is analogous to that of acid catalysis and, in addition, is such as to permit protonation of the a-nitrogen atom, in ethyl l-acetyl-2-benzylcarbazate,it is possible that the cation so formed would not be able to function as a substrate even though all other factors were favorable for substrate activity. It is probable that the bond between the a-
1880
ABRAHAM N. KURTZAND CARLN I E M A N N
nitrogen and adjacent carbonyl carbon atoms in ethyl 1-acetyl-2-benzylcarbazate has a higher internal barrier of rotation than the bond between the a-carbon and adjacent carbonyl carbon atoms in ethyl acetyl-D- or 1,-phenylalaninate. This singular structural feature and the inability of ethyl 1-acetyl-2-benzylcarbazate to function as a substrate led us to examine the latter compound as an inhibitor of the a-chymotrypsin catalyzed hydrolysis of chloroacetyl-L-valine methyl ester. The inhibition studies were conducted in aqueous solutions a t 25.0 + O . l o , pH 7.90 A 0.01 and 0.10 M in sodium chloride. The experimental procedure was that described by Applewhite, Martin and Niemann.6 Chloroacetyl-L-valine methyl ester6,' was used a t substrate concentrations which varied from 4.50 to 56.0 X M , ethyl l-acetyl2-benzylcarbazate a t inhibitor concentrations of 0, 4.41, 8.83 and 18.1 X M and a-chymotrypsin a t an enzyme concentration of 0.1464 mg. protein-nitrogen per ml. For each inhibitor concentration 9 to 12 experiments were performed a t 8 or 9 different substrate concentrations. The initial velocities were determined by the method of Booman and Niemann,s using in this instance a program written for the Datatron 20.5 digital c ~ m p u t e r and , ~ were corrected for enzyme and substrate blank reactions by the method of Martin and Niemann.1° The constants Ks' and k3 were then evaluated, with a Datatron 205, using a program written for a least squares fit to the cqu at'ion
Vol. 83
TABLE I ISHIBITION 017 THE ~ ~ H Y Y O T R Y P S I N CATALYZED HYDROLYSIS OF CHLOROACETYL-L-VALINE METHYL ESTERBY ETHYL ~-ACETYL-~-BENZYLCARBAZATE' [IIO3
Ks'b, c
0 4.41 8.83 18.1
46.9 f 2 . 9 55.3 i4 . 8 56.5 f 5 . 5 83.0k7.7
Kj"b,d
hbe
KI''3,f
43.2 i 2 . 9 1 . 6 5 f 0.09 53.8 i 5 . 5 1.55 f .11 60.0 zk 6 . 8 1 . 4 2 k . I 3 85.0k9.21 . 4 9 3 ~ .13 Mean value 1.54 =t0.14 20 f 6 a In aqueous solutions at 25.0", pH 7.90 f 0.01 and 0.1 M in sodium chloride. In units of M. Value based Value based upon value of R 3 determined simultaneously. upon mean value of ka. e In units of M/min./mg. protein-nitrogen per ml. f Derived from values of KQ" assuniing competitive inhibition.
plex involving enzyme, substrate and inhibitor was formed,ll or, as Blum has suggested,12 one in which two or more conformational forms of the enzyme were being generated by interaction of the enzyme with the inhibitor (modifier). The constants given in Table I were evaluated from initial velocities computed by the orthogonal polynomial procedurea using a to.lo significance level to determine the degree of polynomial to be used for representation of the dP/dt relationships, which in 38 out of 43 experiments required polynomials of order substantially greater than unity for the 1 to 9 min. reaction period. Since the total extent of reaction rarely exceeded 10% one might expect more linear relationships. Therefore, several alternative procedures for testing the reliability of individual initial velocities were examined. The first method was based upon the proposition that as [SIo approaches zero, Michaelis-Menten It will be seen from the data summarized in Table I that the values of k3 exhibit a small and kinetics approach first order kinetics; hence, possibly significant drift toward lower values with values of the initial velocities observed a t low subincreasing inhibitor concentration. If this drift strate concentrations are less reliable for the evaluis ignored and the mean value of k3 used to cal- ation of K s and k3 than those observed a t high substrate concentrations. For each individual culate a set of Ks" values, a value of KI" = 20 6X M is obtained. This value may be taken point a new slope was computed between the limits as the enzyme-inhibitor dissociation constant of Ks'! k3 and that point. From this new slope a new a-chymotrypsin-ethyl l-acetyl-2-benzylcarbazate,value, kai, was calculated and compared with the under the conditions specified, if the latter com- k3 value derived from all of the points. The aspound is a totally competitive inhibitor and the sembled k3i values were then compared by the analysis of variances a t a 99% significance level drift in ka values is ignored. for rejection. By selecting a t test value for a 99% An attempt mas made to determine whether the confidence limit only those runs that were in serious drift in k3 values noted above was or was not signifi- error were made evident. Following this step cant, Our interest in this matter arose from the and k 3 were recalculated using the acceptable possibility that a-chymotrypsin, chloroacetyl-L- Ks' data. The values so obtained are given in the valine methyl ester and ethyl I -acetyl-2-benzyl- first and second columns Table 11. In three out carbazate were not functioning as a totally competitive system but as one in which a ternary com- of four cases data derived from experiments conducted a t the lowest level of [SI0 were rejected. ( 5 ) T. H. Applewhite, R . B. Martin and C. Xiemann, J . A m . Chem. The second method assumed that all inherent Soc., 80, 1457 (1958). errors were associated with the ordinate [S]O. (6) T. H. Applewhite, H. Waite and C. h-iemann, i b i d . , 80, 1405 [E\/vo and that the basis of rejection should be a (1958). ( 7 ) In ref. 6 a value of Xg = 47.1 i 0.8 X 10-' M and ka = 1.755 + difference between the observed ordinate value 0.028 X 10-0 M/min./mg. protein-nitrogen per nl.is given for chloroand the ordinate value calculated from a least acetyl-L-valine methyl ester in aqueous solutions a t 25.0 k O . l ' , squares solution of the slope and intercept. The p H 7.90 =t0.01 and 0.1 A4 in sodium chloride. Re-evaluation of the differences between the measured [ S ] O[ E ] o / ~ o original d a t a using a more reasonable estimate of t h e extent of t h e enzyme blank reaction gave a value of K s = 39.3 + 0.7 X 10-8 M . and values and those calculated from the slope of the k3 = 1.61 + 0.03 X 10-2 M/min./mg. protein-nitrogen per ml. least squares line based on all of the data were (1956).
*
(8) K. A . Booman and C. Niemann, ibid., 7 8 , 3642 (9) H. I. Abrasb. A . PI'. Kurtz and C. Niemann, Biocheni. et Biop h y s . A c l a , 45, 378 (1960). (10) R . B. Martin and C. Xiemann, i b i d . , 26, 634 (1967).
(11) H. T. Huang and C. Niemann, J . A m . Ckem. Soc., 76, 1395 (1953). (12) J. J. Blum, Arch. Biochem. B i o p h y s , 66, 486 (1955).
ETHYL1-ACETYL-2-BENZYLCARBAZATE AND 0-CHYMOTRYPSIN INTERACTION
April 20, 1961
TABLE I1 RE-EVALUATION OF VALUESOF Ks' I Ks '0
50.6 + 2 . 9 59.2 f 4 . 8 63.1 i 5 . i 82.0 f 9 . 9 a In units of
AND
kl 111
I1
ka b
1.74 + 0 . 0 9 1 . 6 1 i .12 1.53 .13 1 . 4 5 f .16 M. In units of
+
Ks'a
50.6 f 2 . 9 59.2 + 4 . 8 58.6 f 2 . 4 82.0 f 9 . 9 M/min./mg.
k3 b
Ks'a
1.74 i 0 . 0 9 56.7 f 2.4 1.61 i .12 60.0 f 1 . 1 65.1 f 2 . 2 1 . 4 8 f .05 1 . 4 5 f .16 83.8 + 3 . 4 protein-nitrogen per ml.
ka b
-
1 . 8 5 f 0.07 1.59 i .03 1 . 5 1 f .05 1.37 + .05
1881
[IIP"
0 4.41 8.83 18.1
computed and compared by the analysis of vari- zate, ;.e., 20 f 6 X l P 3M i t follows that the active ances to a 99.57, confidence level. After rejection site of a-chymotrypsin is more accessible to ethyl of the data not passing the test, Ks' and k3 were re- acetyl-D-phenylalaninate than it is to the analogous calculated to give the values listed in the third and carbazate. fourth columns of Table 11. A system involving a-chymotrypsin and ethyl The third method used for re-evaluation assumed acetyl-L-phenylalaninate has been reportedI6 to that the orthogonal polynomial procedure was have a Ks value of 1.1-1.2 X M , which is not biased in favor of a non-linear relationship during markedly different from the value of Ks = 1.80 X the first one or two minutes of an experiment. M recently given for methyl acetyl-L-phenylThe data obtained during the 1 to 9 minute in- alaninate.I7 The fact that the Ks value for ethyl terval were used to calculate an initial velocity acetyl-L-phenylalaninate is smaller than the K1 by fitting the data to a first order equation by the value of ethyl acetyl-D-phenylalaninateand the method of least squares and then using this value latter value is smaller than the K I value for ethyl to evaluate Ks' and k3. These values are given in 1-acetyl-2-benzylcarbazatesuggests that accesfifth and sixth columns of Table 11. sibility to the active site of the enzyme is in the The values of Ks' and k3 given in Table I1 are order ethyl acetyl-L-phenylalaninate> ethyl acetylcharacterized by a uniform increase in values of D-phenylalaninate > ethyl 1-acetyl-2-benzylcarbaKs' and a uniform decrease in values of ka with in- zate. creasing inhibitor concentration and thus confirm The implication that the active site of a-chymothe trend in values noted in Table I. The sig- trypsin is less accessible to ethyl 1-acetyl-2-benzyl nificance in the downward trend in k3 values listed carbazate than to ethyl acetyl-L-phenylalaninate in Table I1 was tested a t a 95% chi square level does not in itself provide an explanation as to why from which it was concluded that no differences the latter compound is a substrate and the former in precision existed amongst the values obtained is not. However, it does appear that there is a a t the different inhibitor concentrations. Follow- singular structural feature of the carbazate that ing this analysis a 95y0 F test for homogeneity impedes its access to the active site of the enzyme of the four k3 values of each set was performed irrespective of whether the comparison is made with the result that to a 9570 confidence limit the with ethyl acetyl-L-phenylalaninate, which is a different inhibitor concentrations gave signifi- substrate, or with ethyl acetyl-D-phenylalaninate, cantly different ka values. which is not. The preceding results suggest that the system The inability of ethyl 1-acetyl-2-benzylcarbaa-chymotrypsin-chloroacetyl-L-valinemethyl ester-ethyl 1-acetyl-2-benzylcarbazate may be other zate to function as a substrate could arise as a than totally ~ o m p e t i t i v e . ~However, ~ for the consequence of several limiting situations. If the carbazate is present in the enzyme-subpurpose a t hand it is reasonable to regard this system as one approximating totally competitive strate complex as the base, rather than as the conjugate acid arising from protonation of the ainhibition. Ethyl acetyl-D-phenylalaninate has not been nitrogen atom, it is clear that the lack of reactivity evaluated as a competitive inhibitor. However, cannot be explained solely in terms of a greater the corresponding methyl ester is known to have degree of depolarization of the carbethoxy carbonyl a K Tvalue of 2.4 f 0.4 X l o w 3M . 1 4 On the basis group of the carbazate relative to that of the reof the K I values of methyl and ethyl acetyl-D- active a-acylamino acid ester. However, the lack tryptophanate, 0.09 and 0.25 X i1P and those of reactivity could arise indirectly from the greater degree of constraint in the bond between the CYof ethyl acetyl-D-tyrosinate, 5.0 f 1.0 X M,I4 acetyl-D-tyrosinamide, 12 f 2 X i\P4 and nitrogen and adjacent carbonyl carbon atoms, relative to that in the analogous bond of the a-acylacetyl-D-phenylalaninamide, 12 + 3 X i t is unlikely that the K I value of ethyl acetyl- amino acid ester, and directly from an inability M of the carbazate to assume conformations in the D-phenylalaninate is greater than 10 X and is probably ca. 5 f 2 X M . From this enzyme-substrate complex that are accessible to K I value and that of ethyl 1-acetyl-2-benzylcarba- ethyl acetyl-L-phenylalaninate. In this instance the inertness of the carbazate would arise largely (13) A treatment of this system as one involving ternary complex from its unfavorable orientation in the enzymeformation is given in t h e Ph.D. Thesis of A. N. Kurtz, Calif. Inst. Tech., Pasadena (1960). I t is not reproduced here because of the substrate complex.
limitations arising from t h e lack of an unambiguous value of K I for ethyl 1-acetyl-2-henzylcarbazatein a totally competitive system. (14) R. 5. Foster and C . Niemann, J . A m . Chem. Soc., 77, 3370 (1955). (15) H. T. H u a n g and C. Niemann, ibid., 74, 101 (1952).
(16) B. R. Hammond and H . Gutfreund, Biochem. J . , 61, 187 (1955). (17) M.L. Bender and W.A. Glasson, J . A m . Chem. Soc., 83,3336 (1960).
1882
HANSHELMUT BAER
The factors leading to unfavorable orientation of tlie carbazate in the enzyme-substrate complex niight be eliniinated by protonation of tlie (Ynitrogcn atom of the carbazate. However, in the conjugate acid o f the carbazate the apparent basicity of the carbethoxg carbonyl oxygen atom would be diininished to such a degree that even if the conjugate acid were able to assume those conformations accessible to ethyl acetyl-L-phenylalaninate in its enzyme-substrate complex, it is probable that it would still be unable to function as a substrate. If the conjugate acid of the carbazate is able to assume those conformations accessible to ethyl acetyl-L-phenylalaninate in its complex with the enzyme, it should also be able to assume those conformations available to ethyl acetyl-rl-phenylalaninate in its complex with the enzyme. This situation in itself would not prevent the carbazate from functioning as a substrate. However, the fact that the KI value of the carbazate is substantially greater than that of ethyl acetyl-~-phenylalaninate does not support the view that the carbazate is present in its complex with the enzyme in the form of the conjugate acid unless one invokes the added hypothesis that protonation is possible only in those conformations that are analogous to those assumed by ethyl acetyl-L-phenylalaninate. The most direct explanation of the inability o f ethyl 1-acetyl-2-benzylcarbazateto function as a substrate of a-chymotrypsin is that i t is unable to assume an orientation in the enzyme substrate complex that can lead to reaction products. Experimental Attempted Hydrolysis of Ethyl 1-Acetyl-2-benzylcarbazate by a-Chymotrypsin.-A solution of 3.06 g. of ethyl 1-acetyl-2-benzylcarbazate3and 0.275 g. of a-chymotrypsin in 230 nil. of carbon dioxide free water was adjusted t o pH 7.95 with 0.1 N aqueous sodium hydroxide, the resulting solution made up to 250 ml., thermostatted a t 25” and the jH held a t 7.95 by the addition of 0.1 N aqueous sodium hydroxide. After 70 min. 2.35 mi. of base had been added, after 275 min. a total of 3.50 ml., after 16.5 hr. a total of 4.00 ml., after which time no further addition of base was required to maintain the p H a t 7.95 for a period of one week. The total amount of base added corresponded to a maximum extent of hydrolysis of 1.3%, a value less than that observed for a comparable system containing only enzyme.
Vol. 83
At the end of the reaction period the system was extracted with three 100 tnl. portions of carbon tetrarhloride and three 100 ml. portions of ethyl ether. The cumbined extracts were dried over anhydrous sodium sulfate and tlie solverit removed in zrucuo a t 25’3 t o give 2.88 g . (93%) crf a viscous, non-crystallizxhle oil, X ~ ~1.5131. D Tlie iiifrared spectrum iii carbon tetrachloride was identical with t h a t of ethyl 1-acetyl-2-benzyl carbazate.3 Base Catalyzed Hydrolyses of Ethyl 1-Acetyl-2-benzyl Carbazate and Ethyl Acetyl-~~-phenyla!aninate.-Stock solutions 0.040 M in substrate were prepared from carbon dioxide free water. i\liquots of these solutions were used to prepare reaction systems 0.1 Af in sodium chloride and with a total volume of 10.0 ml. All hydro!yses were conducted a t 25.0 f 0.1” in an at.niosphere of nitrogen a t a constant pH maintained with a pH-stat. Aqueous sodium hydroxide, 0.0107 N, was used as the titrant and each hydrolysis was followed for a period of 8 min. The schedule used for each substrate is given in Table 111. TABLE 111 SCHEDULEOR EXPERIMENTS FOR DETERMISATIOS OF
kR
Molarity of substrate
M
0.024 .024 ,024 ,036 .004 ,016 .024 .028
ihH
7.50 7.90 8.50 8,. 50 9.00 9.00 9.00 ‘3.00
The recorder traces of extent of reaction ws. time were linear and the velocities were computed from the chart coordinates. The data so ohtailled were described by the relation v = k B [RCO1R’][OH-] to within f. 10% and the second order constant k~ evaluated by a least squares fit of w us. the product [RCOQR’][OH-] using a program written for the Datatron 205 digital computer. Inhibition Studies.-The inhibition of the a-chymotrypsin catalyzed hydrolysis of chloroacetyl-L-valine methyl ester in aqueous solutions at 25.0 f. 0.1”, PH 7-90 i: 0.01 and 0.1 iM in sodium chloride by ethyl 1-acetyl-2-benzylcarbazatea was studied with the aid of a pH-stat as described previously.6 The only departure from the previous procedure was in the use of equal time intervals of one min. from t = 1 min. to t = 9 min. instead of t = n to t = 8 niin. In this particular study some diRculty was experienced in adjusting the p H of the enzyme solution so 3s t o miuimize “hunting” during the initial minute of reaction. Crystalline a-chymotrypsin, Arniour lot No. 283, i v a s iisecl throughout.
[CONTRIBUTION FROM THE NATIOXAL INSTITUTE O F ARTHRITIS AND METABOLIC DISEASES,NATIONAL INSTITUTES O F 1-IEALTH, BETHESDA 14, MD.]
Synthetic Kanosarnine BY HANSHELMUT BAER RECEIVEDDECEMBER 17, 1960 A niethyl 3-arnino-3-deoxy-p-~-hexopyranoside that has recently become readily available by a new synthesis was proved t o belong to the gluco series, ;.e., t o be the methyl 0-glycopyranoside of kanosamine. Acid hydrolysis and subsequent ISacetylation afforded kanosamine hydrochloride and N-acetylkanosarnine, respectively. The latter was degraded by means and N-acetyl-D-arabinosamine. of periodate t o give 2-acetamido-2-deoxy-4-~-formyl-~-arabinose
Recently we have described a synthesis that afforded, via a nitromethane condensation of periodate-oxidized methyl P-D-glycoGdes and subsequent hydrogenation, a methyl 3-nitro-3-deoxy-,RD-hexopyranoside (I, m.p. 204-205’, [ ( Y ] ~ ~-D12’) and the corresponding methyl 3-amino-3-deoxy-p-
D-hexopyranoside (11, m.p. 207-208’ dcc., [a]*”D Since the course of the synthesis was -34’).’ non-specific with regard to the stereochemistry a t carbon atoms 2, 3 and 4, the configuration of the products remained unsettled. Three of the eight (1) II. H. Baer. B ~ , . 9, 3 , 2 s t x (1960).
