title: Gly-238-Ser substitution changes the substrate specificity of the SHV class A beta-lactamases.
abstract: The SHV-type beta-lactamase SHV-2A is related to SHV-1 by a Gly-238-Ser replacement. Strains carrying SHV-2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV-1 are sensitive to these drugs. We present a kinetic analysis of a SHV-1 and SHV-2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV-2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7-aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV-1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV-2A. These results indicate that the Ser-238-Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV-2A relative to SHV-1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser-238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV-2A. We propose that the Gly-238 to Ser-238 replacement in SHV-2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.
authors.affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis 95616.
authors.forename: K Y
authors.initials: KY
authors.lastname: Lee
authors.forename: J D
authors.initials: JD
authors.lastname: Hopkins
authors.forename: T F
authors.initials: TF
authors.lastname: O'Brien
authors.forename: M
authors.initials: M
authors.lastname: Syvanen
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002511:Cephalosporins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010405:Penicillinase / Q000737:chemistry* / Q000302:isolation & purification; D010406:Penicillins / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D002511:Cephalosporins; D010406:Penicillins; D010405:Penicillinase