title: Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase.
abstract: BACKGROUND
  The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme.
  
  
  RESULTS
  Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination 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 and enzyme denaturation) to be evaluated. In addition, molecular modelling studies 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 stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.
authors.affiliation: Laboratoire d' Enzymologie, Centre d' Ingénierie des Protéines, Institut de Chimie, Université de Liège, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Lejeune
authors.forename: M
authors.initials: M
authors.lastname: Vanhove
authors.forename: J
authors.initials: J
authors.lastname: Lamotte-Brasseur
authors.forename: R H
authors.initials: RH
authors.lastname: Pain
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
authors.forename: A
authors.initials: A
authors.lastname: Matagne
mesh_terms: D000215:Acylation; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D013211:Staphylococcus aureus / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*; D047090:beta-Lactams
chemical_list: D000900:Anti-Bacterial Agents; D047090:beta-Lactams; C054232:beta-lactamase PC1; D001618:beta-Lactamases