Why the extended-spectrum beta-lactamases SHV-2 and SHV-5 are "hypersusceptible" to mechanism-based inhibitors.

Extended-spectrum beta-lactamases (ESBLs) are derivatives of enzymes such as SHV-1 and TEM-1 that have undergone site-specific mutations that enable them to hydrolyze, and thus inactivate, oxyimino-cephalosporins, such as cefotaxime and ceftazidime. X-ray crystallographic data provide an explanation for this in that the mutations bring about an expansion of the binding pocket by moving a beta-strand that forms part of the active site wall. Another characteristic of ESBLs that has remained enigmatic is the fact that they are "hypersusceptible" to inhibition by the mechanism-based inactivators tazobactam, sulbactam, and clavulanic acid. Here, we provide a rationale for this "hypersusceptibility" based on a comparative analysis of the intermediates formed by these compounds with wild-type (WT) SHV-1 beta-lactamase and its ESBL variants SHV-2 and SHV-5, which carry the G238S and G238S/E240K substitutions, respectively. A Raman spectroscopic analysis of the reactions in single crystals shows that, compared to WT, the SHV-2 and SHV-5 variants have relatively higher populations of the stable trans-enamine intermediate over the less stable and more easily hydrolyzable cis-enamine and imine co-intermediates. In solution, SHV-2 and SHV-5 also form larger populations of an enamine species compared to SHV-1 as detected by stopped-flow kinetic experiments under single-turnover conditions. Moreover, a simple Raman band shape analysis predicts that the trans-enamine intermediates themselves in SHV-2 and SHV-5 are held in more stable, rigid conformations compared to their trans-enamine analogues in WT SHV-1. As a result of this stabilization, more of the trans-enamine intermediate is formed, which subsequently lowers the K(I) values of the mechanism-based inhibitors up to 50-fold in SHV-2 and SHV-5.