Structure-function studies of Ser-289 in the class C beta-lactamase from Enterobacter cloacae P99.
Site-directed mutagenesis of Ser-289 of the class C beta-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in beta-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant beta-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC beta-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the kcat of four of the five beta-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant beta-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.
Trépanier
S
S
Département de Biologie Médicale, Pavillon Marchand, Université Laval, Ste-Foy, Québec, Canada.
Knox
J R
JR
Clairoux
N
N
Sanschagrin
F
F
Levesque
R C
RC
Huletsky
A
A
D001426:Bacterial Proteins; D002384:Catalysis; D002433:Cefaclor / Q000494:pharmacology; D002437:Cefazolin / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D016972:Enterobacter cloacae / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry / Q000235:genetics*; D013329:Structure-Activity Relationship; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D002511:Cephalosporins; D012694:Serine; D002433:Cefaclor; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D002437:Cefazolin