It is pointed out that translational and (overall) rotational motions provide the important entropic driving force for enzymic and intramolecular rate accelerations and the chelate effect; internal rotations and unusually severe orientational requirements are generally of secondary importance. desolvation. Unequivocal evidence that theories giving rate accelerations on the order of 55 AM from entropic factors are wrong or incomplete is provided by several intramolecular reactions, such as ring closures of succinate derivatives, in which the presence of one or more free rotations ensures that a reacting group can move out of an unfavorable conformation so that the fraction of the starting material in a high-energy, strained or desolvated form will be negligible. The comparison between intra-and intermolecular reactions may be based on the free energy of either the transition state, from rate measurements, or the product, from equilibrium measurements, relative to the starting material; the latter comparison has the advantage that the structure of the product is known. Thus, the equilibrium constant for succinic anhydride formation (Eq. 1) is3 X 105(1) more favorable than that for acetic anhydride formation from the free acids, which implies an "effective concentration" of 3 X 105 M of the carboxylic acid groups of succinic acid relative to each other (2, 9), and the "effective concentration" of the neighboring carboxylate group that determines the rate of anhydride formation from substituted monophenyl succinate anions (Eq. 2) is about 105 M (4,10,11

The plot of kcat/Km against pH for the Bacillus cereus 569/H beta-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pKa 5.6+/-0.2 and one of pKa 9.5+/-0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pKa of 5.6+/-0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a Ki of 70 microM. The pH-dependence of Ki shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of Ki between pH 6 and 9 indicates that the pKa of zinc(II)-bound water must be 5.6 and not the higher pKa of 9.5. The kinetic solvent isotope effect on kcat/Km is 1.3+/-0.5 and that on kcat is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B beta-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

Experimental evidence for a [2 + 2] mechanism in the Lewis acid-promoted formation of a,b-unsaturated esters from ethoxyacetylene and aldehydes. Synthesis and characterisation of 4-ethoxyoxetes.

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