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

Biochemistry. 2009 Oct 20;48(41):9912-20. doi: 10.1021/bi9012098.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Catalytic Domain
  • Cephalosporins / metabolism
  • Crystallography, X-Ray
  • Kinetics
  • Microbial Sensitivity Tests
  • Mutagenesis, Site-Directed
  • Plasmids
  • Serine / chemistry
  • Serine / metabolism
  • Spectrum Analysis, Raman
  • Sulbactam / metabolism
  • beta-Lactamase Inhibitors
  • beta-Lactamases / chemistry*
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism*


  • Cephalosporins
  • beta-Lactamase Inhibitors
  • Serine
  • beta-lactamase SHV-2
  • beta-lactamase SHV-5
  • beta-Lactamases
  • Sulbactam