Mass spectral kinetic study of acylation and deacylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant

Biochemistry. 1995 Sep 19;34(37):11660-7. doi: 10.1021/bi00037a003.


The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the acylation and deacylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deacylation for penicillin hydrolysis, but acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deacylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better Km and an increased acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the omega-loop conformation in the active site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acylation
  • Cefotaxime / chemistry
  • Cefotaxime / metabolism*
  • Cloxacillin / chemistry
  • Cloxacillin / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Kinetics
  • Mass Spectrometry
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Oxacillin / chemistry
  • Oxacillin / metabolism
  • Penicillin G / chemistry
  • Penicillin G / metabolism
  • Penicillins / chemistry
  • Penicillins / metabolism*
  • Spectrophotometry, Ultraviolet
  • beta-Lactamases / chemistry
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism*


  • Penicillins
  • beta-Lactamases
  • Cefotaxime
  • Cloxacillin
  • Penicillin G
  • Oxacillin