Site-saturation studies of beta-lactamase: production and characterization of mutant beta-lactamases with all possible amino acid substitutions at residue 71.

Abstract

A mutagenic technique that "saturates" a particular site in a protein with all possible amino acid substitutions was used to study the role of residue 71 in .beta.-lactamase (EC 3.5.2.6). Threonine is conserved at residue 71 in all class A .beta.-lactamases and is adjacent to the active site Ser-70. All 19 mutants of the enzyme were characterized by the penam and cephem antibiotic resistance they provided to Escherichia coli LS1 cells. Surprisingly, cells producing any of 14 of the mutant .beta.-lactamases displayed appreciable resistance to ampicillin; only cells with mutants having Tyr, Trp, Asp, Lys, or Arg at residue 71 had no observable resistance to ampicillin. However, the mutants are less stable to cellular proteases than wild-type enzyme is. These results suggest that Thr-71 is not essential for binding or catalysis but is important for stability of the .beta.-lactamase protein. An apparent change in specificity indicates that residue 71 influences the region of the protein that accommodates the side chain attached to the .beta.-lactam ring of the substrate.