Fs. Guo et al., The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors, BBA-PROT ST, 1431(1), 1999, pp. 132-147
Citations number
69
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
The role of the non-conserved amino acid residue at position 104 of the cla
ss A beta-lactamases, which comprises a highly conserved sequence of amino
acids at the active sites of these enzymes, in both the hydrolysis of beta-
lactam substrates and inactivation by mechanism-based inhibitors was invest
igated. Site-directed mutagenesis was performed on the penPC gene encoding
the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corre
sponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained wi
th the purified Aspl04Ala B, cereus 569/H beta-lactamase I was compared to
that obtained from the wild-type B. cereus and S. aureus enzymes. Replaceme
nt of amino acid residue 104 had little effect on the Michaelis parameters
for the hydrolysis of both S- and A-type penicillins. Relative to wild-type
enzyme, the Aspl04Ala P-lactamase I had 3-fold higher K-m values for benzy
lpenicillin and methicillin, but negligible difference in K-m for ampicilli
n and oxacillin. However, k(cat) values were also slightly increased result
ing in little change in catalytic efficiency, k(cat)/K-m. In contrast, the
Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its res
ponse to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluo
romethane sulfonyl)amido-penicillanic acid sulfone with respect to both res
ponse to the inhibitors and subsequent enzymatic properties. Based on the k
nown three-dimensional structures of the Bacillus licheniformis 749/C. Esch
erichia coli TEM and S. aureus PCI beta-lactamases. a model for the role of
the non-conserved residue at position 104 in the process of inactivation b
y mechanism-based inhibitors is proposed. (C) 1999 Elsevier Science B.V. Al
l rights reserved.