SITE-DIRECTED MUTAGENESIS OF PROPOSED ACTIVE-SITE RESIDUES OF PENICILLIN-BINDING PROTEIN-5 FROM ESCHERICHIA-COLI

Alignment of the amino acid sequence of penicillin-binding protein 5 ( PBP5) with the sequences of other members of the family of active-site -serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser(44)), Lys(47), Ser(110)-Gly-Asn, Asp(175) and Lys(213)-Thr-Gly we re identified as the residues making up the conserved boxes of this pr otein family. To determine the role of these residues, they were repla ced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity . The Ser(44)Cys and the Ser(44)Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys(47)Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduce d rate. Mutants in the Ser(110)-Gly-Asn fingerprint were affected in b oth acylation and deacylation upon reaction with penicillin and lost t heir DD-carboxypeptidase activity with the exception of Asn(112)Ser an d Asn(112)Thr. The Asp(175)Asn mutant showed wild-type penicillin-bind ing but a complete loss of DD-carboxypeptidase activity. Mutants of Ly s(213) lost both penicillin-binding and DD-carboxypeptidase activity e xcept for Lys(213)His, which still bound penicillin with a k(+2)/K' of 0.2% of the wild-type value. Mutation of His(216) and Thr(217) also h ad a strong effect on DD-carboxypeptidase activity. Thr(217)Ser and Th r(217)Ala showed augmented hydrolysis rates for the penicillin acyl-en zyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicilli n-binding, and confirms them to play crucial roles in catalysis.