8335 may be obtained by using the k,,,, (40 s-]) for phenyl benzoate under the same conditions. This calculation gives K, = 9.8 X 10-5 M. The pH dependence of kcat./K, is not pure sigmoidal, but shows clear evidence for the involvement of a group, active in the free base form whose pKa’ = 5.0.15 Thus this work also provides the first example of the catalysis of carbon-carbon bond cleavage by the carboxylesterases.I6
case of a t least one reaction, the hydrogenation of a-methylstyrene by hydridopentacarbonylmanganese( I) (eq 1). This evidence encompasses one of the only definitive applications to date of the C I D N P technique to the elucidation of the mechanism of a reaction involving a transition metal complex.
Acknowledgments. It is a pleasure to acknowledge discussions with Dr. R. L. Blakeley. This work was supported in part by the Australian Research Grants Committee.
Reaction 1 was found to proceed quantitatively (confirmed by N M R , UV spectroscopy, and GLC) in benzene solution at rates conveniently measurable in the temperature range 40-75 OC. Kinetic measurements, in which the concentrations of C ~ H S C ( C H ) ~ = C HC6HsCH(CH3)2, ~, and H M n ( C 0 ) 5 all were monitored by N M R , yielded the second-order rate law, eq 2, with values of (2.65 f 0.12) X (9.0 f 1.0) X and (20.0 f 1.4) X M-’ s-l for k’at 45.0,56.2, and 64.5 O C , respectively, corresponding to AH* = 21.4 f 0.3 kcal mol-] and AS* = -12 f 1 cal mol-’ K-1.3 The kinetic results are consistent with the following mechanism (eq 3-5) for which more direct evidence is presented below and according to which k‘ = k3k13/(k-3 k’3).4
References and Notes E. Bamann and M. Schmeller, Hoppe-Seyler’s 2.Physiol. Chem., 188,251 (1930). M. C. Berndt, J. de Jersey, and B. Zerner. J. Am. Chem. Soc.. previous communication in this issue. See footnote 2 in ref 2. kOH for ethyl mandelate at 25 O C is -0.1 1 M-’ s-’, while LoH for ethyl phenylglyoxylate is 427 M-’ s-l at 25 OC, the temperature of the enzymatic experiments. See footnotes 4 and 10 in ref 2. C. A. Grob and P. W. Schiess, Angew. Chem., lnt. Ed. Engl., 6, 1 (1967). in 0.05 M phosphate buffer, pH 7.50, at 25 O C , kcat = 103 s-’ and K,,, = 2.4 x 10-5 M. Spectral data ootained wltn [E],, > [I],,:c252 for ethyl phenylglyoxylate IS 6600: in tne presence of an excess of enzyme, t252. extrapolated to zero time. s 1560: the absorbance increases ,n a flrst-order reaction to a final c252 of 13 100. in goo0 agreement witn ( 2 5 2 for phenylglyoxylate ion (13 200j. We have previously shown that hydroxyurea is stmukaneously an inhibitor and SJostrate of the metalloenzyme, jack bean urease (EC 3 5.1.5j: R. L. Blakeley. -. A. Hinds, H E. Kunze, E. C. Webb, and B. Zerner, Blochemistry, 8, 1991 (1969);N E. Dixon. C. Gazzola. J. J. Watters. R. L. Blakeley. an0 B. Zerner. J. Am. Chem. Soc.. 97,4130 (1975); N. E. Dixon. C. Gazzola, R. L. Blakeley, and 8. Zerner. ibrd.. 97, 4131 (1975). The kea: (6.2 f 1.7 s - ’ ) and K, (9.1 f 3.0 X M) for pnitrophenyl phenylglyoxylateat pH 6.25 are also consistent with reversible aDOrtiVe hemiketal formation. I
