The effects of a single amino acid substitution on the substrate profile of Escherichia coli β-lactamase

Abstract

Site-specific mutagenesis is one of the most powerful tools for the study of protein structure and function to date. The introduction of methods using small chemically synthesized oligonucleotide primers to target mutations to individual nucleotides has made it possible to examine the effects of even single amino acid substitutions on the catalytic properties of an enzyme. Escherichia coli RTEM β-lactamase, an enzyme which hydrolyzes the cyclic amide bond in β-lactam antibiotics, provides the bacteria with resistance to both penicillins and cephalosporins. In this study, a single amino acid substitution was introduced at the active site of this enzyme. This procedure utilized oligonucleotide-directed mutagenesis to substitute adenine for thymine at the condon for amino acid 70 resulting in the replacement of the wild-type serine residue by cysteine. E. coli cells producing the new cysteine mutant β-lactamase showed decreased resistance to penicillin series antibiotics as compared to cells producing the wild-type enzyme. However, these same cells showed increased resistance to cephalosporins over the wild-type. These results suggested that the cysteine mutant β-lactamase was not simple decreased in activity but has unique catalytic properties which result in an altered substrate profile when compared to the while-type enzyme.