Purification and properties of thiol beta-lactamase. A mutant of pBR322 beta-lactamase in which the active site serine has been replaced with cysteine

J Biol Chem. 1984 Apr 25;259(8):5327-32.


The specifically mutated enzyme thiol beta-lactamase has been expressed in Escherichia coli by means of the trp promoter and purified to homogeneity. The gene for this enzyme results from a single base change N410 A----T in the gene of pBR322 RTEM beta-lactamase (EC, penicillinase, penicillin amido-beta-lactamhydrolase) which alters the codon for the active site Ser 70 to that for Cys. Precursor thiol beta-lactamase is processed to give the same NH2-terminal sequence as that for wild type enzyme. In contrast to the wild type enzyme, thiol beta-lactamase contains one free titratable thiol group/molecule. Thiol beta-lactamase catalyzes the hydrolysis of beta-lactams with a substrate specificity that is distinct from that of wild type enzyme. For benzyl-penicillin and ampicillin, the Km values are similar to wild type values although the kcat values are 1-2% that of wild type enzyme. For the cephalosporin nitrocefin, the Km is greater than 10-fold that of the wild type and the kcat is at least as large as the kcat for the wild type enzyme. Thiol beta-lactamase is different from wild type beta-lactamase in that it is not competitively inhibited by boric acid although a small degree of noncompetitive inhibition does occur. Whereas the circular dichroism spectra of both enzymes are nearly identical, thiol beta-lactamase at 40 degrees C is 3-fold more resistant to trypsin than is the wild type enzyme.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Circular Dichroism
  • Cysteine*
  • Escherichia coli / enzymology*
  • Kinetics
  • Mutation*
  • Plasmids
  • Serine*
  • Substrate Specificity
  • beta-Lactamases / genetics
  • beta-Lactamases / isolation & purification*
  • beta-Lactamases / metabolism


  • Serine
  • thiol beta-lactamase
  • beta-Lactamases
  • Cysteine