Dependence of hydrolysis of β-lactams with a zinc(II)-β-lactamase produced from Serratia marcescens (IMP-1) on pH and concentration of zinc(II) ion: Dissociation of Zn(II) from IMP-1 in acidic medium

Abstract

The pH dependence for the hydrolysis of β-lactam antibiotics by a metallo-β-lactamase (IMP-1) produced from Serratia marcescens was investigated varying the concentration of Zn(II). The activity of IMP-1 for imipenem was decreased at pH less than pH 5.3 without external addition of Zn(II) ions but was recovered with addition of Zn(II). Varying the concentration of external Zn(II), the molar activity of the enzyme, kobs, that was defined by the velocity of hydrolysis of imipenem/concentration of IMP-1 was expressed by kobs=νinit/[E]T= k max[Zn]/(Kd+[Zn]) in which Kd stands for the dissociation constant between Zn(II) and IMP-1. The dissociation constants, Kd, vary with pH; Kd=840 × 10-6 M at pH 4.3 and Kd=0.19 × 10-6 M at pH 6.0. The plot of -log Kd against pH showed a straight line having a slope of 4.0 below pH 5.0, showing the existence of four functional groups which may be protonated upon dissociation of Zn(II) ion(s). The kcat, Km, and kcat/Km of hydrolysis of imipenem and cephalothin in the presence of sufficient concentration of Zn(NO3)2 for saturation of IMP-1 with Zn(II) showed similar dependency to each other on pH between pH 6.0 and 9.0.