Efficient catalysis by β-lactamase from Staphylococcus aureus PC1 accompanied by accumulation of an acyl-enzyme

The pH- and temperature-dependence of steady-state kinetic parameters for 6-β-(2-furyl)-acryloylamido-penicillanic acid showed it to be a good substrate of staphylococcal PC1 β-lactamase, and the viscosity-dependence of Km/kcat indicated that steps up to the formation of the acyl-enzyme were partially diffusion-limited. In the pH range 4–9, a pre-steady-state transient blue shift in the UV absorption spectrum of the bound furyl-acryloylamido chromophore was of constant amplitude and decayed to the spectrum of the product with a first-order rate constant equal to kcat. The spectrum of the isolated denatured acyl-enzyme was similar to that of the methyl ester of furyl-acryloylpenicilloic acid, pointing to non-covalent interactions with the folded protein, possibly associated with the charge on Glu-166, as the source of the blue-shifted spectrum. Taken together, these results point to a rapid acylation and slower deacylation at Ser-70 and imply that ionization of groups affecting enzyme activity at alkaline pH, for which likely candidates are Lys-73 and Lys-234, affect the rate of deacylation.