Article (Scientific journals)
Quantitative Analysis of the Stabilization by Substrate of Staphylococcus Aureus Pc1 Beta-Lactamase
Lejeune, Annabelle; Vanhove, Marc; Lamotte-Brasseur, Josette et al.
2001In Chemistry and Biology, 8 (8), p. 831-42
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Keywords :
enzyme kinetics; bêta-lactamases; molecular modelling; protein stability; thermal unfolding
Abstract :
[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Lejeune, Annabelle ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie, Centre d'Ingénierie des Protéines
Vanhove, Marc;  Université de Liège - ULiège > Sciences de la Vie > Enzymologie, Centre d'Ingénierie des Protéines
Lamotte-Brasseur, Josette ;  Université de Liège - ULiège > Centre d'Ingénierie des protéines
Pain, R. H.;  Jožef Stefan Institute > Department of Biochemistry & Molecular Biology
Frère, Jean-Marie ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie, Centre d'Ingénierie des protéines
Matagne, André  ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie, Centre d'Ingénierie des protéines
Language :
English
Title :
Quantitative Analysis of the Stabilization by Substrate of Staphylococcus Aureus Pc1 Beta-Lactamase
Publication date :
August 2001
Journal title :
Chemistry and Biology
ISSN :
1074-5521
eISSN :
1879-1301
Publisher :
Cell Press, Cambridge, United States - Massachusetts
Volume :
8
Issue :
8
Pages :
831-42
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 23 November 2009

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