In an effort to understand the reaction mechanism of a B2 metallo-β-lactamase, steady-state and presteady state kinetic and rapid-freeze quench EPR studies were conducted on ImiS and its reaction with imipenem and meropenem. pH Dependence studies revealed no inflection points in the pH range of 5.0 -8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Sitedirected mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s −1 and a slower rate of fluorescence regain of 98 s −1 . Stopped-flow UV-Vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s −1 . These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C-N bond cleavage. Rapid-freeze quench EPR studies on Co(II)-substituted ImiS demonstrated the appearance of a rhombic signal after 10 milliseconds that is assigned to a reaction intermediate that has a 5-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 milliseconds. Taken together, these results allow for a reaction mechanism to be offered for the B2 metallo-β-lactamases and demonstrates that the mononuclear Zn(II)-and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.Bacterial resistance to antibiotics is a growing clinical concern (1,2). Zn(II)-containing β-lactamases (metallo-β-lactamases, MβL's) contain 1-2 moles of Zn(II) per mole of enzyme, hydrolyze all known cephalosporins, carbapenems and penicillins, are not inhibited by clavulanic acid and other classical β-lactamase inhibitors, and have no known clinically-useful inhibitor towards them (3,4). Previous studies have shown that there is significant structural and mechanistic diversity among the MβL's, leading to the grouping of the enzymes into three distinct subclasses: B1, B2, and B3 (5,6). Sequence identity ranges from 25-40% between members in one subclass and from 10-20% between members in different subclasses. Subclass B1 enzymes have been found in strains of Bacillus, Bacteroides, Pseudomonas, Serratia, and Chryseobacterium, and subclass B3 enzymes have been found in strains of Stenotrophomonas, Legionella, Fluoribacter, Janthinobacterium and Caulobacter (3,4). Enzymes from the B1 and B3 subclasses have broad substrate profiles and require two Zn(II) † This work was supported by the National Institutes of Health (GM40052 to MWC; AI056231 to BB, and EB001980 to the Medical College of Wisconsin).*To whom correspondence should be addressed: M. W. Crowder, e-mail: crowdemw@muohio.edu, phone: (513) 529-7274, fax: (513) 529-5715.
NIH Public Access
Author ManuscriptBiochemistry. A...