Pre-steady state beta-lactamase kinetics. The trapping of a covalent intermediate and the interpretation of pH rate profiles.
The hydrolysis of sodium 3-dansylamidomethyl-7-beta (thienyl-2')-acetamido-ceph-3-em-4-oate, catalyzed by the beta-lactamase of Staphylococcus aureus PC1, has previously been shown (Anderson, E. G., and Pratt, R. F. (1981) J. Biol. Chem. 256, 11401-11404) to follow the reaction scheme Formula; see text. where ES' is an enzyme-substrate complex in which the substrate has undergone nucleophilic attack at the beta-lactam carbonyl group and P is product. Acid quenching of the reaction mixture has now been shown to yield, in amounts predicted by the rate constants, a covalent enzyme-substrate complex. The liability of this complex in alkaline solution is suggestive of that of an ester. Together, all of these results prove that the turnover of this apparently normal substrate by a class A beta-lactamase involves an acyl-enzyme intermediate. In the case of another fluorescent substrate, dansylcephalexin, no intermediate analogous to ES' accumulated during catalysis; presumably here, acylation of the enzyme is rate-determining. The pH profiles (pH 4-9) of the pre-steady state rate constants for hydrolysis of the former substrate have also been determined. Binding (1/K8) is pH invariant except at low pH where it weakens, probably because of substrate protonation and/or a protein conformational change. The rate constants, k2, k-2, and k3, are pH invariant at low pH but decrease at higher pH in a way which can be described by ionization of an essential acid of pKa around 7.7. This may be the same acid for each constant, being either an active participant at the active site, or a more distant acid which controls an essential conformational change.
D002511:Cephalosporins; D003619:Dansyl Compounds; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus / Q000201:enzymology*; D001618:beta-Lactamases / Q000378:metabolism*
D002511:Cephalosporins; D003619:Dansyl Compounds; D007202:Indicators and Reagents; C032510:3-dansylamidomethyl-7-beta(thienyl-2')-acetamidoceph-3-em-4-oate; D001618:beta-Lactamases