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Titolo:
Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase
Autore:
Lejeune, A; Vanhove, M; Lamotte-Brasseur, J; Pain, RH; Frere, JM; Matagne, A;
Indirizzi:
Univ Liege, Ctr Ingn Prot, Enzymol Lab, Inst Chim B6, B-4000 Liege, Belgium Univ Liege Liege Belgium B-4000 Lab, Inst Chim B6, B-4000 Liege, Belgium Jozef Stefan Inst, Dept Biochem & Mol Biol, Ljubljana 1000, Slovenia JozefStefan Inst Ljubljana Slovenia 1000 Biol, Ljubljana 1000, Slovenia
Titolo Testata:
CHEMISTRY & BIOLOGY
fascicolo: 8, volume: 8, anno: 2001,
pagine: 831 - 842
SICI:
1074-5521(200108)8:8<831:QAOTSB>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
TRANSITION-STATE; PROTEIN STABILITY; THERMAL-STABILITY; CRYSTAL-STRUCTURE; FOLDING KINETICS; LOW-TEMPERATURE; MECHANISM; LYSOZYME; BINDING; ENZYME;
Keywords:
enzyme kinetics; beta-lactamase; molecular modelling; protein stability; thermal unfolding;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
48
Recensione:
Indirizzi per estratti:
Indirizzo: Matagne, A Univ Liege, Ctr Ingn Prot, Enzymol Lab, Inst Chim B6, B-4000 Liege, Belgium Univ Liege Liege Belgium B-4000 him B6, B-4000 Liege, Belgium
Citazione:
A. Lejeune et al., "Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase", CHEM BIOL, 8(8), 2001, pp. 831-842

Abstract

Background: The stabilization of enzymes in the presence of substrates hasbeen 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 PCI P-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,, 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 parameterscharacteristic 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. (C) 2001 Elsevier Science Ltd. All rights reserved.

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Documento generato il 04/04/20 alle ore 02:41:25