Faculty Collaboration Database

Direct evidence that the reaction intermediate of metallo-beta-lactamase L1 is metal bound. Biochemistry 2005 Jan 25;44(3):1078-87

Date

01/19/2005

Pubmed ID

15654764

DOI

10.1021/bi048385b

Scopus ID

2-s2.0-12344323483 (requires institutional sign-in at Scopus site)   73 Citations

Abstract

In an effort to probe the structure of the reaction intermediate of metallo-beta-lactamase L1 when reacted with nitrocefin and other beta-lactams, time-dependent absorption and rapid-freeze-quench (RFQ) EPR spectra were obtained using the Co(II)-substituted form of the enzyme. When using nitrocefin as the substrate, time-dependent absorption spectra demonstrate that Co(II)-substituted L1 utilizes a reaction mechanism, similar to that of the native Zn(II) enzyme, in which a short-lived intermediate forms. RFQ-EPR spectra of this intermediate demonstrate that the binding of substrate results in a change in the electronic properties of one or both of the Co(II)'s in the enzyme that is consistent with a change in the coordination sphere of this metal ion. This observation provides evidence that the reaction intermediate is a metal-bound species. RFQ-EPR studies also demonstrate that other beta-lactams, such as cephalothin, meropenem, and penicillin G, proceed through an electronically similar complex and that the role of metal is similar in all cases. EPR spectroscopy has also identified distinct product-bound species of L1, indicating that reversible product binding must be considered in all future kinetic mechanisms. Consideration of the time-dependent optical and EPR studies in light of available crystallographic information indicates the intimate involvement of the metal ion in the Zn(2)-binding site of L1 in the hydrolytic reaction.

Author List

Garrity JD, Bennett B, Crowder MW

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University

MESH terms used to index this publication - Major topics in bold

Bacterial Proteins
Electron Spin Resonance Spectroscopy
Electrophoresis, Polyacrylamide Gel
Kinetics
Metals
Protein Binding
Substrate Specificity
beta-Lactamases