hlay 20, 1961
C O M M U N I C A T I O N S T O THE
The great reactivity may well account for the fact that previous attempts to prepare and identify ruthenium hexafluoride have been unsuccessful. HOWARD H. CLAASSES .-~RGONNE SATIONAL LABORATORY HENRYSELIC ILLINOIS JOHN G. MALM ARGOXXE, CEDRICL. CHERNICK FORDMOTORCOMPANY BERNARD WEIXSTOCK SCIENTIFIC RESEARCH LABORATORY DEARBORX, MICHIGAN RECEIVED APRIL6, 1961
0.5
2391
EDITOR
c
THE FORMATION O F THE ACYL-ENZYME INTERMEDIATE, TRANS-CINNAMOYL-CPCHYMOTRYPSIN, IN THE HYDROLYSES OF NON-LABILE TRANSCINNAMIC ACID ESTERS’n2
Sir: The hydrolysis of labile acyl derivatives (e.g., nitrophenyl esters) is catalyzed by a-chymotrypsin in a three-step process: (i) adsorption of the substrate on the enzyme; (ii) acylation of the enzyme with the release of the phenol; and (iii) deacylation of the acyl-enzyme giving the carboxylic acid product and regenerating the e n ~ y m e . However, ~ it has been maintained on the basis of kinetic arguments that the cr-chymotrypsin-catalyzed hydrolysis of methyl hippurate4s5does not proceed through a hippuryl-enzyme intermediate and, by implication, that the a-chymotrypsin-catalyzed hydrolyses of other non-labile acyl derivatives also do not involve acyl-enzyme formation. We have investigated the possible existence of trans-cinnamoyl-cr-chymotrypsin (I) in the enzyme-catalyzed hydrolyses of a series of substrates, both labile and non-labile, derived from transcinnamic acid (see Table I ) . In each case, unequivocal evidence has been found for the involvement of I, both froin rate measurements and absorption spectral characteristics. Therefore, we conclude
I
0
I
I
200 400 600 Time in seconds.
800
Fig. 1.-The a-chymotrypsin-catalyzed hydrolysis of methyl cinnamate: [El0 = 1.33 X 10-8M, [ S ] O= 1.20 X lO-‘M, pH 7.80, 25.0°, 3.2% CHICN.
that the formation of an acyl-enzyme intermediate is not an artifact of a-chymotryptic catalyses resulting from the lability of the ester function, but is indeed part of the general mechanism of such catalyses . The rates of enzyme-catalyzed hydrolysis of substrates 1 to 5, inclusive, are followed conveniently spectrophotometrically a t 310 mp; in each case one observes a very rapid increase in absorbance followed by a slower, first-order decay whose rate constant is 13 f 0.5 X set.-'. Further, the appearance of cinnamate ion a t 260 mp has the same rate as the decrease of absorbance a t 310 mp (loss of acyl-enzyme). Since the hydrolysis of oTABLE I nitrophenyl cinnamate has been shown3 to proceed RATESOF O-CHUMOTRYPSIS CATALYZED HYDROLYSES through I (whose deacylation is rate-controlling), the present data can best be interpreted in terms kz X 108 ki X 103 (sec. -11 (sec. -!j of the formation of the common intermediate, I, Substrate pH acylation deacylation in the hydrolysis of all five substrates6 In each 1 N-Cinnamo?.lirriidazoleU 9.0 13 case, acylation is very fast compared with deacyla2 p-Nitrophenyl cinnamatea 9.0 ... 13 tion for these labile derivatives. 3 m-Nitrophen5-1 cinnnrriatea 9.0 ... 13 However, if the intermediacy of I is general, 4 o-Nitrophenyl cinnaniai ea 9.0 ... 13 reactions in which either acylation or deacylation 5 p-Cresyl cinnamatea 9.0 ... 13 is rate-controlling may be expected to occur,’ for 6 Methyl cinnamateb i.80 2.66 (11.1) example, in the hydrolysis of alkyl esters. Sub7 Benzyl cinnamatec 6.95 ... 5.31 strates 6 and 7 are two such esters. In order to 8 ~-Cinnamo)-limidazolec 6.95 ... 5.24 [Elo G l.05[S]od; [S]a = 2-4 X 10-5M; 25.6’; 1.6 make these systems experimentally accessible, to 397, CHIC?;. For exact experimental conditions, see high concentrations of the enzyme and ratios of Fig. 1. [E]/[S] > 1 have been used. The hydrolysis of [Elo = 8.68 X 10-‘M; [SI0 = 7.12 X lO+M; 25.0’; 3.2% C H L S . [El0 = initial enzyme concentramethyl cinnamate is shown in Fig. 1. The absorbtion; [Sla = initial substrate concentration. ance increases to a maximum in about 100 seconds and then decreases according to a strict first-order (1) This research was supported by Grant H-5726 of t h e National Institutes of Health, rate law (after approximately 450 sec.). The rate (2) Paper VI1 in t h e series “The Mechanism of Action of Proteolytic constant for this latter process is 2.66 X Enzymes,” previous paper, M. L. Bender, G. R. Schonbaurn, G. A. sec.-l. Deacylation of I under identical condiHamilton and B. Zerner, J. A m . Chem. SOL,83, 1255 (1961). ( 3 ) For references, see G. R. Schonbaum, K. Nakamura and M , I,. tions (see Fig. 1) has a rate constant of 11.1 X Bender, ibid., 81, 4746 (1959). sec.-1.8 Consequently the decay process for (4) S. A. Bernhard, W. C. Coles and J. F. N o w e l l , ibid., 82, 3043 (1960). ( 5 ) See al-o 11.I,. Bender and W. A. Glasson, ;bid., 82, 3336 (19GO),
for kinetic results in the hydrolysis of N-acetyl-L-phenylalanine methyl ester which cannot be explained on the basis of acyl-enzyme formation.
(6) Suhstrates 1 , 3 and 3 have different rates o f acylation: unpub lished work with G. R. Schonbaum. (7) H. Gutfreund and B. R. Hammond, Biochem. J . , 73,526 (1959). (8) Since these reactions were carried out a t p H 7 . 8 instead of fiH 9.0, t h e deacylation is somewhat slower here than with substrates 1-5.
COMMUNICATIONS TO THE EDITOR
2392
methyl cinnamate is a direct measure of the acylation r e a ~ t i o n . ~Application of the classical kinetic
Vol. 83
lo4 (in 2 minutes). Hydride abstraction was proved by recovery of triphenylmethane from formulation for two consecutive first-order reac- the pentane extracts. tionsl0 to this system leads to a quantitative fit of It became desirable to attempt the preparation the absorbance vs. time curve. Using the inde- of 1,2,4,4-tetramethyl-3,5-dimethylenecyclopentene pendently determined rate constants for acylation (11) so as to determine whether protonation of I1 (2.66 X sec.-l) and deacylation of the acyl- also leads to the magenta species. sec.-l), together with the enzyme, I (11.1 X I with N-bromosuccinimide in carbon tetraindependently determined molar absorptivities a t chloride a t 0' gave an unstable bromination prod310 mp of methyl cinnamate (e = 3.27 X lo3), uct, which was dehydrobrominated readily with I (E = 1.096 X lo4) and cinnamate ion (e = 1.00 trimethylamine in 12 hours a t 25'. Preparative X l o 3 under experimentul conditions), one calcu- vapor phase chromatography of the product lates tmax for the absorbance as. time curve as 105 yielded a new hydrocarbon, presumably 1,2,4,4seconds (observed, 100 10 see.), and A m a x = tetramethyl-3,5-dimethylenecyclopentene,(11) y., 0.496 (observed, 0.470). Thus, both qualitatively 29%, n 2 0 D 1.5181, mass spec. mol. wt., 148. Anal. and quantitatively the increase in absorbance in the Calcd. for CllHI,,: C, 89.10; H, 10.90, Found: enzyme-catalyzed hydrolysis of methyl cinnamate C, 88.96; H, 10.79. The structure assignment is can be accounted for on the basis of the formation based on these data: (a) infrared bands: B (>C of the highly absorbing intermediate, trans-cin- =CH2) 3090 cm.-l (m); 1726 cm.-l (w) (overtorie namoyl-a-chymotrypsin. l1 of 860); 1395 cm.-l (m); 860 em.-' (vs); fi For benzyl cinnamate, the absorbance vs. time (H2C=C-&C-C=CH2) 1618 crn.-l (vs). (b) curve also shows a maximum, again indicating the Xmax isoiictane 285.4 (sh), 275.0, 265.6, emax 16750, formation of the acyl-enzyme intermediate. How- 25150, 21400. This agrees well with the spectrum ever, in this case, one obtains the rate of deacyla- of 3,6-dimethylenecyclohexene, (c) n .m.r. spection of I from the first-order portion of the curve. trum (40 M.c.) in C.P.S. rel. to Si(CH3)d solvent: For example, the rate constant obtained under the -43.5 rel. a rea 6 (gem-dimethyl); -72.3, iel. area conditions speafied in Table I is 5.31 X see.-'. 6 (two vinyl methyl groups); - 185.8 and - 190.0 The rate constant for the deacylation of I under rel. area 2 each (two exocyclic methylene groups). identical conditions is 5.24 X sec.-l. Deacyla- The doublet is ascribed to unequal diamagnetic tion is therefore the slow step in the hydrolysis of shielding of the a and b vinyl hydrogen atoms,' this ester under the experimental conditions. which do not split each others resonances further Thus the acyl-enzyme hypothesis achieves gen- due to the low spinspin coupling constants5 erality for a-chymotrypsin-catalyzed hydrolyses of An intensely magenta solution (Amax 552 mk) both labile and non-labile substrates. However, a could be obtained instantaneously: (1) upon addidiscrepancy still exists between these results and tion of methanesulfonic acid (to conc. 5 X 11) those of Bernhard, et aE.,4for the specific spbstrate, to an initially colorless acetic acid solution of I1 methyl hippurate and those of Bender and Glas- (6.4 X M), emax 23,500; (2) upon addition of sons for the specific substrate, N-acetyl-L-phenyl- a drop of the bromination solution of I to an ionizalanine methyl ester. ing solvent, such as acetic acid, formic acid or ay. HC1 (> 3 M ) . I n both cases the color is discharged (9) The values of the acylation and deacylation rate constants for upon addition of sodium acetate in acetic acid, methyl cinnamate are several orders of magnitude smaller than those of specific substrates of a-chymotrypsin, but there is no reason to regenerated upon addition of methane sulfonic believe that the reactions reported here are qualitatively different acid. mp, emax
*
from those of specific substrates. (10) The acylation may be considered to be a pseudo first-order process since the enzyme is in great excess. (11) Preliminary experiments with 8-phenyletbyl and cyclohexyl cinnamates show behavior very similar to that found for the methyl ester. (12) Alfred P. Sloan Foundation Research Fellow.
H ~ C HC~H J ~ H
H3C CH3
)* c " -Hf , p C H ~f i C H ~ -11+
Hb
CHJ
HjC
I1
DEPARTMENT OF CHEMISTRY NORTHWESTERN UNIVERSITY MYRON L. BENDER'^ EVANSTON, ILLINOIS BURTZERNER RECEIVED MARCH 22, 1961
H3C
TTT
CH3
111 a
c
PREPARATION OF 1,2,4,4-TETRAMETHYL-3,5-DIMETHYLENECYCLOPENTENE; THE MAGENTA SPECIES DERIVED FROM IT BY PROTONATION AND FROM HEXAMETHYLCYCLOPENTADIENE BY HYProtonation of I1 gives undoubtedly 111 (111, DRIDE ABSTRACTION I I I b ) . 111 is also the expected product from
Sir :
An intensely magenta solution is obtained from hexamethylcyclopentadienel (I) upon treatment with trityl perchlorate2 in acetonitrile; A,, 552 (1) L. de Vries, J . A m . Chcm. SOL.,Sa, 5242 (1960); J . Org. Chcm.,
as, 1838 (leeo).
(2) H . J. Dauben, J . Am. Chem. SOC., 19,7557 (1957); J . O w . Chem., as, 1442 (1960).
-
I by hydride abstraction. Since dehydrobromination of the bromination product of I gives 11, it (3) R . E. Denson and V. E. Lindsey, J. A m . Chem. SOL.,81, 4250 (1959); W. J. Bailey and R. Barclay, i b i d . , 81, 6393 (1959). (4) See n.m.r of longifolene, S Dev, Tcfrahcdron,9, 1 (1960). (5) J. A. Pople, W. G. Schneider and H. J. Bernstein, "High Resolution N.m.r.," McCraw-Hill Book Co. Inc., New York, N. Y., 1059. PP. 238-240.
