Effects of site-specific mutagenesis of tyrosine 105 in a class A beta-lactamase.
Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site. Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis. This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties. In particular, in comparison to the wild-type, kcat. for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates. For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme. Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation. Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity. We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding.
Escobar
W A
WA
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
Miller
J
J
Fink
A L
AL
D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002511:Cephalosporins / Q000378:metabolism; D002863:Chromogenic Compounds; D002942:Circular Dichroism; D017124:Conserved Sequence; D017931:DNA Primers / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010400:Penicillin G / Q000378:metabolism; D010404:Penicillin V / Q000378:metabolism; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics* / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D002511:Cephalosporins; D002863:Chromogenic Compounds; D017931:DNA Primers; D007202:Indicators and Reagents; D014443:Tyrosine; C034313:pyridine-2-azo-4-dimethylaniline cephalosporin; D001618:beta-Lactamases; C021720:nitrocefin; D010400:Penicillin G; D010404:Penicillin V