Relations between beta-lactamases and penicillin-binding proteins: beta-lactamase activity of penicillin-binding protein 5 from Escherichia coli.
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.
Nicholas
R A
RA
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts.
Strominger
J L
JL
D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003460:Crystallization; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006602:Hexosyltransferases; D006868:Hydrolysis; D007700:Kinetics; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D010957:Plasmids; D011401:Promoter Regions, Genetic; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases; D003545:Cysteine