Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase

Background: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this ph enomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzy me unfolding was followed by monitoring the progressive decrease of the rat e of substrate utilization by the Staphylococcus aureus PCI P-lactamase, at temperatures above the melting point of the enzyme. Results: Enzyme inactivation was directly followed by spectrophotometric me asurements. In the presence of substrate concentrations above the K,, value s, significant stabilization was observed with all tested compounds. A comb ination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis a nd enzyme denaturation) to be evaluated. In addition, molecular modelling s tudies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. Conclusions: Our analysis indicates that the enzyme is substantially stabil ized towards heat-induced denaturation, independently of the relative propo rtions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediate s are expected to be significantly populated, both species (ES and ES*) app ear to be similarly stabilized. This analysis contributes a new quantitativ e approach to the problem. (C) 2001 Elsevier Science Ltd. All rights reserv ed.