Effects of site-specific mutagenesis of tyrosine 105 in a Class A β-lactamase

Abstract

Tyr-105 is a conserved residue in the Class A β-lactamases and is in close proximity to the active-site. Tyr-105 in β-lactamase from Bacillus licheniformis was converted into Phe by sitedirected 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, k(cat) for benzylpenicillin was increased-slightly, whereas it was decreased slightly for several other substrates. For each substrate examined, K(m) 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 k(cat)/K(m) pH profiles due to the mutation. Overall effects of the Y105F substitution on the catalytic efficiency for different pencillin 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 β-lactamase catalysis, but does contribute to substrate binding.