CATALYTIC MECHANISM OF ACTIVE-SITE SERINE BETA-LACTAMASES - ROLE OF THE CONSERVED HYDROXY GROUP OF THE LYS-THR(SER)-GLY TRIAD

The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S) G] triad has been studied for a class A and a class C beta-lactamase b y site-directed mutagenesis. Surprisingly, the disappearance of this f unctional group had little impact on the penicillinase activity of bot h enzymes. The cephalosporinase activity was much more affected for th e class A S235A (Ser(235)--> Ala) and the class C T316V (Thr(315)--> V al) mutants, but the class C T316A mutant was less impaired. Studies w ere extended to beta-lactams, where the carboxy group on C-3 of penici llins or C-4 of cephalosporins had been modified. The effects of the m utations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those o btained with a similar mutant (T299V) of the Streptomyces R61 DD-pepti dase. With this enzyme the mutation also affected the interactions wit h penicillins and severely decreased the peptidase activity. The stric t conservation of the hydroxy group on the second residue of the KT(S) G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially t hose of class C.