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.
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.
Laboratory of Pharmaceutical Physical Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, Honmachi, Japan. gmphiwin@gpo.kumamoto-u.ac.jp
Masafumi
M
Goto
Hisami
H
Yasuzawa
Toshihiro
T
Higashi
Yoshihiro
Y
Yamaguchi
Akiko
A
Kawanami
Shiho
S
Mifune
Hiromasa
H
Mori
Hitoshi
H
Nakayama
Kumiko
K
Harada
Yoshichika
Y
Arakawa
D000900:Anti-Bacterial Agents / Q000737:chemistry*; D002384:Catalysis; D002512:Cephalothin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015378:Imipenem / Q000494:pharmacology; D007700:Kinetics; D012706:Serratia marcescens / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*; D047090:beta-Lactams / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D047090:beta-Lactams; D015378:Imipenem; D015032:Zinc; D002512:Cephalothin