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

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 w...

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Published in:Biological & pharmaceutical bulletin 2003, Vol.26(5), pp.589-594
Main Authors: Goto, Masafumi, Yasuzawa, Hisami, Higashi, Toshihiro, Yamaguchi, Yoshihiro, Kawanami, Akiko, Mifune, Shiho, Mori, Hiromasa, Nakayama, Hitoshi, Harada, Kumiko, Arakawa, Yoshichika
Format: Article
Language:English
Subjects:
Anti-Bacterial Agents - chemistry
beta-Lactams - chemistry
Biological and medical sciences
Catalysis
Cephalothin - pharmacology
enzyme
Hydrogen-Ion Concentration
Hydrolysis
Imipenem - pharmacology
Kinetics
Medical sciences
mtallo-β-lactamase
Serratia marcescens - chemistry
zinc
Zinc - chemistry
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Summary: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=vinit/[E]T=kmax[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.
ISSN:0918-6158
1347-5215
DOI:10.1248/bpb.26.589