Evidence of dimerisation among class D beta-lactamases: kinetics of OXA-14 beta-lactamase.
OXA-14 enzyme, a class D beta-lactamase, gave biphasic kinetics with all penicillin and cephalosporin substrates tested, such that the catalytic rate declined more swiftly than was explicable by substrate depletion. This biphasic behaviour was independent of temperature or extraneous protein but was lost if the enzyme was diluted to occupy almost the total assay volume before addition of a small amount of concentrated substrate. The presence of substrate could partially protect the enzyme against conversion to the less active form, with protection greatest at substrate concentration above the K(m). These observations are compatible with the hypothesis that the biphasic kinetics depended on the enzyme existing as a highly active dimer at high concentration and as a less active monomer at low concentration. Direct evidence supporting this hypothesis came from the observation that gel exclusion chromatography indicated a higher molecular weight for concentrated enzyme than for dilute. Biphasic kinetics are not so universal for different substrates amongst beta-lactamases (OXA-10, -11, -13, -16 and -17) that differ from OXA-14 by only one to two amino acid substitutions. It may be that the monomer:dimer equilibrium is more rapidly achieved with these enzymes than with OXA-14, or that the kinetic properties of the dimers and monomers of these enzymes are similar, masking any biphasic trait.
Department of Medical Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Turner Street, London E1 2AD, UK. franck.danel@roche.com
F
F
Danel
J M
JM
Frère
D M
DM
Livermore
D002511:Cephalosporins / Q000378:metabolism; D019281:Dimerization; D007700:Kinetics; D008956:Models, Chemical; D010406:Penicillins / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D002511:Cephalosporins; D010406:Penicillins; C096781:beta-lactamase OXA-14; D001618:beta-Lactamases