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American Chemical Society
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In an effort to probe the structure of the reaction intermediate of metallo-β-lactamase L1 when reacted with nitrocefin and other β-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 β-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 Zn2-binding site of L1 in the hydrolytic reaction.
Garrity, James D.; Bennett, Brian; and Crowder, Michael W., "Direct Evidence That the Reaction Intermediate of Metallo-β-lactamase L1 Is Metal Bound" (2005). Physics Faculty Research and Publications. 30.
ADA Accessible Version
Accepted version. Biochemistry, Vol. 44, No. 3 (January 25, 2005): 1078-1087. DOI. © 2005 American Chemical Society Publications. Used with permission.
Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.