Efficient Inhibition of Class A and Class D β-Lactamases by Michaelis Complexes

Matthew Kalp,Anjaneyulu Sheri,Robert A Bonomo,Paul R Carey,John D Buynak,Christopher R Bethel

doi:10.1074/jbc.c700080200

Abstract

A 6-alkylidiene penam sulfone, SA-1-204, is an efficient inhibitor of both SHV-1 and OXA-1 beta-lactamases with K(I) = 42 +/- 4 nm and 1.0 +/- 0.1 microm, respectively. To gain insight into the reaction chemistry of SA-1-204, the reactions between this inhibitor and SHV-1 and OXA-1 were studied by Raman spectroscopy in single crystals and in solution. Raman signatures characteristic of the unreacted beta-lactam ring show that in both phases the inhibitor binds as a noncovalent Michaelis-like complex. This complex is present as the major population for periods of up to an hour. On longer time scales, the Raman data show that beta-lactam ring opening eventually leads to a complex mixture of reaction products. However, the data clearly demonstrate that the key species for inhibition on the time scale of bacterial half-lives is the noncovalent complex preceding acylation.

Highlights

A 6-alkylidiene penam sulfone, SA-1-204, is an efficient inhibitor of both SHV-1 and OXA-1 ␤-lactamases with KI ‫؍‬ 42 ؎ 4 nM and 1.0 ؎ 0.1 ␮M, respectively
To gain insight into the reaction chemistry of SA-1-204, the reactions between this inhibitor and SHV-1 and OXA-1 were studied by Raman spectroscopy in single crystals and in solution
Using two forms of Raman spectroscopy, we demonstrate that a 6-alkylidiene penam sulfone (Fig. 1) inhibits SHV-1 and OXA-1 without covalent modification of the active-site serine or cleavage of the four-member ␤-lactam ring

Summary

Introduction

A 6-alkylidiene penam sulfone, SA-1-204, is an efficient inhibitor of both SHV-1 and OXA-1 ␤-lactamases with KI ‫؍‬ 42 ؎ 4 nM and 1.0 ؎ 0.1 ␮M, respectively. The Raman data show that ␤-lactam ring opening eventually leads to a complex mixture of reaction products.

Results

Conclusion