Relations between beta-lactamases and penicillin-binding proteins: beta-lactamase activity of penicillin-binding protein 5 from Escherichia coli.

Abstract

The molecular mechanism of the hydrolysis of penicillin by penicillin-binding protein (PBP) 5 was investigated. Cysteine-115 had been previously implicated in the hydrolysis of the acyl-enzyme intermediate in PBP5. In studies with oligonucleotide-directed mutagenesis, the role of cysteine-115 in the mechanism of PBP5 was investigated by the mutation of this residue to either a serine or an alanine residue. Genetic truncation of the studied proteins at their carboxyl termini resulted in the production of a soluble form of these proteins (sPBPs). The purified sPBPs were assayed for both their beta-lactamase and their D-alanine carboxypeptidase activities. The results of these assays indicate that the cysteine residue does not directly participate in the enzymatic mechanism. In a further investigation of the catalytic mechanism of beta-lactam hydrolysis, a soluble, naturally occurring mutant was purified in large amounts and crystallized from concentrated polyethylene glycol solutions. This mutant of PBP5 (sPBP5') has a near-normal acylation rate with respect to [14C]penicillin, but shows a drastic increase in the half-life of the acyl-enzyme complex. The crystals of sPBP5' diffract to greater than 3 A resolution and are suitable for detailed crystallographic analysis.