Acetylketene was produced by flash photolysis of 2,2,6-trimethyl-4H-1,3-dioxin-4-one in aqueous solution, and rates of hydration of the ketene to acetoacetic acid enol and subsequent ketonization of the enol were measured in this solvent across the acidity range [H+] = 1−10-13 M. Acetylketene proved to be a remarkably reactive substance, undergoing uncatalyzed hydration with the rate constant k = 1.5 × 106 s-1, some 104 times more rapidly than ketene itself; the acetylketene hydration reaction was also catalyzed weakly by hydroxide ion but not by hydrogen ion. Ketonization of acetoacetic acid enol was much slower with rates in the millisecond to second range. The reaction showed a complex rate profile that could be interpreted in terms of rate-determining carbon protonaton of the carboxylate-ionized form of the enol in the acid region and rate-determining carbon protonation of the doubly ionized carboxylate−enolate form in the basic region. Analysis of the data provided the acidity constant pQ a = 4.05 for the carboxylic acid group of the enol and pQ a E = 13.18 for its enolic hydroxyl group. (These acidity constants are concentration quotients referring to an ionic strength of 0.10 M). Combination of the present results with information on the enolization of acetoacetic acid available from the literature gave K E = 5.6 × 10-3, pK E = 2.25, as an estimate of the keto−enol equilibrium constant.

Rates of hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide catalyzed by subtilisin and its N155G mutant were measured in H2O, D2O, and “HDO” (1:1 H2O:D2O). The solvent isotope effects (proton inventories) determined by these data showed no differences between the wild-type and mutant enzymes, despite the fact that the mutation removes a hydrogen-bonding interaction in the oxyanion hole of the enzyme worth two orders of magnitude in reaction rate. This suggests that curvature previously observed (ref ) in the proton inventory for a reaction catalyzed by methyl chymotrypsin is also not due to oxyanion hole interactions, and this curvature can in fact be accounted for by a medium effect. Proton inventory analysis of the isotope effects for subtilisin and its mutant also indicate the presence of strong medium effects in those systems.

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Deuterium-protium fractionation factors were determined for benzylamine and benzylammonium ion by the traditional 1H N M R method and for the benzylammonium ion by a newly devised 13C N M R method. The results, 4phCH2NL2 = 0.958 f 0.070 and 4phCH2NL,+ = 1.081 f 0.019, when combined with the solvent isotope effect on the ionization of benzylammonium ion, also determined here, K H / K D = 3.36 f 0.13, give @ P~C H~N L , += 0.80 f 0.13 as the medium effect for transfer of this ion from H20 to D20. These results show that introduction of a positive charge into an N-L bond does not decrease its fractionation factor as is the case for 0-L bonds, where ~O L + /~O L = 0.7. This difference is attributed to the tetrahedral structure of ammonium ions and the consequent stiffness of their bondbending vibrations. Elsevier:Amsterdam, 1976; Chapter 1.(5) The special symbol "8' is generally used for the fractionation factor of the hydronium ion.(6) (a) Kresge, A. J.; Allred, A. L.