STRUCTURE-FUNCTION-RELATIONSHIPS AMONG WILD-TYPE VARIANTS OF STAPHYLOCOCCUS-AUREUS BETA-LACTAMASE - IMPORTANCE OF AMINO-ACID-128 AND AMINO-ACID-216

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of St aphylococcus aurcus beta-lactamase, designed A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in prim ary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences loca ted close to the active site, we hypothesized that these differences m ight explain the kinetic heterogeneity of the wild-type beta-lactamase s. To test this hypothesis, genes encoding the type A, C, and D beta-l actamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of aspa ragine for serine at residue 216 of type A beta-lactamase resulted in a kinetic profile indistinguishable from that of type C beta-lactamase , whereas the substitution of serine for asparagine at the same site i n the type C enzyme produced a kinetic type A mutant. Similar bidirect ional substitutions identified the threonine-to-alanine difference at residue 128 as being responsible for the kinetic differences between t he type A and D enzymes. Neither residue 216 nor 128 has previously be en shown to be kinetically important among serine-active-site beta-lac tamases.