Isolation of a Staphylococcus aureus beta-lactamase-dicloxacillin complex and kinetic studies on the reactivation of the enzyme.
Exposure of the beta-lactamase from Staphylococcus aureus to the slowly reacting substrates cloxacillin or dicloxacillin results in time-dependent inactivation of the enzyme. Methods for the rapid separation of a beta-lactamase-dicloxacillin complex from excess inhibitor, using centrifuged columns of Sephadex G-25 or DEAE-Sephadex G-25, are described. The enzyme-dicloxacillin complex releases active enzyme, with specific activity identical to that of untreated enzyme, after storage at pH 7.5 at 15 degrees C. Full reactivation was accompanied by the release of 0.8 eq of hydrolyzed dicloxacillin. The complex is stable for up to 40 h when stored at pH 3 at 4 degrees C. The reactivation process, which occurs with first-order kinetics at 15 degrees C and pH values between 4 and 8, displays a pH dependence with apparent pKa's of 4.6 and 8.5, and a limiting value of the reactivation rate constant of 0.022 min-1. Deviation from first-order kinetics at pH 9 is consistent with a competing irreversible inactivation of the enzyme at that pH. This behavior differs substantially from that of the similarly inactivated beta-lactamase I from Bacillus cereus, whose rate of reactivation is independent of pH, but which undergoes irreversible denaturation at acidic pH [A. L. Fink, K. M. Behner, and A. K. Tan (1987) Biochemistry 26, 4248-4258]. Addition of hydroxylamine to the S. aureus beta-lactamase-dicloxacillin, complex stimulates the rate of reactivation by a maximum of 35%. This effect is hyperbolically dependent on the concentration of hydroxylamine with half-maximal stimulation at 2.8 mM. The Km for ampicillin hydrolysis catalyzed by the partially reactivated enzyme is identical to that measured for catalysis by the untreated enzyme. We discuss our observations in relation to models for the transient inhibition process.
Hardy
L W
LW
Department of Biochemistry, University of California, Berkeley 94720.
Kirsch
J F
JF
D004009:Dicloxacillin / Q000378:metabolism* / Q000494:pharmacology; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D013053:Spectrophotometry; D013211:Staphylococcus aureus / Q000201:enzymology*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000302:isolation & purification / Q000378:metabolism*
D006898:Hydroxylamines; D065093:beta-Lactamase Inhibitors; D019811:Hydroxylamine; D004009:Dicloxacillin; D001618:beta-Lactamases