PMID:7980393
| Citation |
van der Linden, MP, de Haan, L, Dideberg, O and Keck, W (1994) Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli. Biochem. J. 303 ( Pt 2):357-62 |
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| Abstract |
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 (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis. |
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| Keywords |
Acylation; Amino Acid Sequence; Bacterial Proteins; Carboxypeptidases/metabolism; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Catalysis; Conserved Sequence; Electrophoresis, Polyacrylamide Gel; Escherichia coli/genetics; Escherichia coli/metabolism; Hexosyltransferases; Kinetics; Molecular Sequence Data; Molecular Weight; Muramoylpentapeptide Carboxypeptidase/chemistry; Muramoylpentapeptide Carboxypeptidase/genetics; Muramoylpentapeptide Carboxypeptidase/metabolism; Mutagenesis, Site-Directed; Mutation/genetics; Penicillin-Binding Proteins; Penicillins/metabolism; Peptide Mapping; Peptidyl Transferases; Restriction Mapping; Sequence Alignment |
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