Hardy L W, Kirsch J F
Department of Biochemistry, University of California, Berkeley 94720.
Arch Biochem Biophys. 1989 Jan;268(1):338-48. doi: 10.1016/0003-9861(89)90595-x.
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.
金黄色葡萄球菌的β-内酰胺酶与反应缓慢的底物氯唑西林或双氯西林接触会导致该酶发生时间依赖性失活。本文描述了使用Sephadex G-25或DEAE-Sephadex G-25离心柱从过量抑制剂中快速分离β-内酰胺酶 - 双氯西林复合物的方法。酶 - 双氯西林复合物在15℃、pH 7.5下储存后会释放出活性酶，其比活性与未处理的酶相同。完全重新激活伴随着0.8当量水解双氯西林的释放。该复合物在4℃、pH 3下储存长达40小时仍保持稳定。在15℃以及pH值在4至8之间时，重新激活过程符合一级动力学，呈现出pH依赖性，表观pKa值分别为4.6和8.5，重新激活速率常数的极限值为0.022 min⁻¹。在pH 9时偏离一级动力学与该酶在该pH下发生竞争性不可逆失活一致。这种行为与蜡状芽孢杆菌中类似失活的β-内酰胺酶I有很大不同，后者的重新激活速率与pH无关，但在酸性pH下会发生不可逆变性[A. L. Fink, K. M. Behner, and A. K. Tan (1987) Biochemistry 26, 4248 - 4258]。向金黄色葡萄球菌β-内酰胺酶 - 双氯西林复合物中添加羟胺最多可使重新激活速率提高35%。这种效应呈双曲线依赖于羟胺浓度，在2.8 mM时达到最大刺激的一半。部分重新激活的酶催化氨苄西林水解的Km与未处理的酶催化时测得的Km相同。我们结合瞬时抑制过程的模型讨论了我们的观察结果。
