Pre-steady state beta-lactamase kinetics. Observation of a covalent intermediate during turnover of a fluorescent cephalosporin by the beta-lactamase of STaphylococcus aureus PC1.
The pre-steady state kinetics of the hydrolysis of sodium 3-dansylamidomethyl-7-beta (thienyl-2')-acetamidoceph-3-em-4-oate, catalyzed by the beta-lactamase of Staphylococcus aureus PC1, has been studied by the stopped flow method. Fluorescence measurements indicated the presence of two intermediate enzyme-substrate complexes, i.e. at least a three-step mechanism. The disappearance of the characteristic cephem chromophore, which reflects nucleophilic attack at the beta-lactam carbonyl group, correlated with the second step. These results show that an intermediate species with a covalently altered substrate occurs on the reaction path prior to the last step which includes release of product. This intermediate is most likely an acyl-enzyme. The data have been analyzed numerically and the derived rate constants are in accord with the steady state parameters.
D002511:Cephalosporins; D003619:Dansyl Compounds; D007202:Indicators and Reagents; D007700:Kinetics; D008433:Mathematics; D013050:Spectrometry, Fluorescence; 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