Rkr. Voladri et al., STRUCTURE-FUNCTION-RELATIONSHIPS AMONG WILD-TYPE VARIANTS OF STAPHYLOCOCCUS-AUREUS BETA-LACTAMASE - IMPORTANCE OF AMINO-ACID-128 AND AMINO-ACID-216, Journal of bacteriology, 178(24), 1996, pp. 7248-7253
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.