title: Substrate specificities in class A beta-lactamases: preference for penams vs. cephems. The role of residue 237.
abstract: Site saturation mutagenesis has been carried out at Ala-237 in RTEM-1 beta-lactamase to assess the role of this site in modulating differences in specificity of beta-lactamases for penams vs. cephems as substrates. (An Ala-237 Thr mutation had previously been shown to increase activity on cephems by about 30-80%.) Screening of all 19 possible mutants on penams and cephems revealed the even more active Ala-237 Asn mutant. Detailed kinetic analysis shows that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild-type enzyme. Both mutations reduce the activity toward penams to about 10% that of RTEM-1 beta-lactamase and lower by about 5 degrees C the temperature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most interesting aspect of these results is that two quite disparate amino acids, threonine and asparagine, when introduced for Ala-237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to such a significant degree.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
authors.forename: W J
authors.initials: WJ
authors.lastname: Healey
authors.forename: M R
authors.initials: MR
authors.lastname: Labgold
authors.forename: J H
authors.initials: JH
authors.lastname: Richards
mesh_terms: D001435:Bacteriophages / Q000187:drug effects / Q000235:genetics; D015153:Blotting, Western; D002511:Cephalosporins / Q000378:metabolism; D004279:DNA, Viral / Q000302:isolation & purification; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009841:Oligonucleotides / Q000494:pharmacology; D010406:Penicillins / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000145:classification* / Q000235:genetics
chemical_list: D002511:Cephalosporins; D004279:DNA, Viral; D009841:Oligonucleotides; D010406:Penicillins; D001618:beta-Lactamases