The association behaviour of beta-lactamases. Sedimentation equilibrium studies in ammonium sulphate solutions.

摘要

The beta-lactamases (EC 3.5.2.6) from TEM plasmid RP4, Bacillus licheniformis 749/C and Enterobacter cloacae P99 were studied in solution over a wide concentration range by equilibrium sedimentation. Though crystal symmetries indicate that all three enzymes are potentially dimeric in their crystal forms, in 50 mM-sodium cacodylate at pH 6.5 the enzymes show only a small tendency to associate, indicated by a weight-average Mr (Mw) at 3% (w/v) concentration about 9% greater than that of the monomer. Although the mode of association could not be determined, this extent of association corresponded to a dimerization constant of about 2 X 10(2) M-1. In 2.1 M-(NH4)2SO4 the B. licheniformis enzyme shows some association at concentrations over 1%, displaying an Mw value at 7% concentration about 60% more than the monomer. Under the same conditions Mw for the Entero. P99 enzyme is about 60% greater than the monomer near the solubility limit of about 2%. However, the Mw for the TEM enzyme is over twice that of the monomer at its solubility limit (3%) in 1.7 M-(NH4)2SO4. Fitting the sedimentation data of the TEM enzyme in 1.7 M-(NH4)2SO4 with a dimerization model and an indefinite-isodesmic-association model yielded equilibrium constants of 1.5 X 10(4) and 3.3 X 10(2) M-1 respectively, with the indefinite-isodesmic model giving the better fit. Fitting the data for the other two enzymes yielded values of 1.4 X 10(3) and 1.7 X 10(2) M-1 respectively for the Entero. P99 enzyme and 4.5 X 10(2) and 45 M-1 respectively for the B. licheniformis enzyme. It could not be determined which model was the better fit for these two enzymes. Since none of the beta-lactamases studied here showed strong evidence of the terminal aggregate being a dimer, we conclude that crystalline dimers, if they exist, will not be tightly associated or physiologically significant.