Mpg. Vanderlinden et al., SITE-DIRECTED MUTAGENESIS OF PROPOSED ACTIVE-SITE RESIDUES OF PENICILLIN-BINDING PROTEIN-5 FROM ESCHERICHIA-COLI, Biochemical journal, 303, 1994, pp. 357-362
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.