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 i n close proximity to the active-site. Tyr-105 in beta-lactamase from B acillus licheniformis was converted into Phe by site-directed mutagene sis. This mutation caused no significant effect on the structure of th e enzyme and had only small effects on the catalytic properties. In pa rticular, 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 perturbatio ns 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 substitu tion on the catalytic efficiency for different pencillin and cephalosp orin substrates ranged from 14% to 56% compared with the wild-type act ivity. We conclude that Tyr-105 is not an essential residue for beta-l actamase catalysis, but does contribute to substrate binding.