Dependence of Hydrolysis of .BETA.-Lactams with a Zinc(II)-.BETA.-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 beta-lactam antibiotics by a metallo-beta-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, k(obs), that was defined by the velocity of hydrolysis of imipenem/concentration of IMP-1 was expressed by k(obs)=v(init)/[E](T)=k(max)[Zn]/(K(d)+[Zn]) in which K(d) stands for the dissociation constant between Zn(II) and IMP-1. The dissociation constants, K(d), vary with pH; K(d)=840 x 10(-6) M at pH 4.3 and K(d)=0.19 x 10(-6) M at pH 6.0. The plot of -log K(d) 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 k(cat), K(m), and k(cat)/K(m) of hydrolysis of imipenem and cephalothin in the presence of sufficient concentration of Zn(NO(3))(2) for saturation of IMP-1 with Zn(II) showed similar dependency to each other on pH between pH 6.0 and 9.0.

Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography

“…Therefore, prior to the examination of pH dependence of the IMP-1 mutants, the K d was obtained as reported previously, and these values are similar to those reported for WT (Table II) (34). The pH dependence of the hydrolysis for the IMP-1 mutants was then measured under conditions of excess Zn(II) by the method reported previously (34). The K m and k cat values for WT and D120(81)A were almost constant between pH 4.6 and 9.0, but the k cat for D120(81)E increased by 10 2 -fold when the pH was increased from 5.2 to 9.0 (Fig.…”

“…We reported previously that IMP-1 is inactivated in acidic medium as the result of the dissociation of Zn(II) from the holoenzyme (34). Therefore, prior to the examination of pH dependence of the IMP-1 mutants, the K d was obtained as reported previously, and these values are similar to those reported for WT (Table II) (34). The pH dependence of the hydrolysis for the IMP-1 mutants was then measured under conditions of excess Zn(II) by the method reported previously (34).…”

“…Enzyme Preparation-WT was purified as described previously (4,34). The mutagenic plasmids were transformed into E. coli JM109.…”

“…Ϫ1 (34). CD spectra were recorded on a Jasco J-720 circular dichrograph (Tokyo, Japan) at 25°C over 200 -250 nm.…”

“…Zn(II) Ion Dissociation from the IMP-1 Mutants-The rate of hydrolysis of nitrocefin by the IMP-1 mutants was monitored in varying concentrations of Zn(NO 3 ) 2 (0 -6000 M) in acetic acid/sodium acetate buffers (0.2 and 0.5 M NaCl) adjusted to pH 4.4 -5.6 as described previously (34). Buffer solutions were prepared using ultrapure water from Wako (Osaka, Japan).…”