Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.
The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.
Christensen
H
H
Sir William Dunn School of Pathology, University of Oxford, U.K.
Martin
M T
MT
Waley
S G
SG
D000215:Acylation; D001409:Bacillus cereus / Q000201:enzymology; D002511:Cephalosporins / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D010397:Penicillanic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D010400:Penicillin G / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology; D013379:Substrate Specificity; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
D002511:Cephalosporins; D065093:beta-Lactamase Inhibitors; D010397:Penicillanic Acid; D003903:Deuterium; C031528:6-iodopenicillanic acid; D001618:beta-Lactamases; C021720:nitrocefin; D010400:Penicillin G