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.
Département de Biologie Médicale, Pavillon Marchand, Université Laval, Ste-Foy, Québec, Canada.
S
S
Trépanier
J R
JR
Knox
N
N
Clairoux
F
F
Sanschagrin
R C
RC
Levesque
A
A
Huletsky
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