Sulfolobus solfataricus carboxypeptidase, (CPSso), is a heat- and pressare-resistant zinc-metalloprotease. Thanks to its properties it is an ideal tool for investigating the role of non-covalent interactions in substrate binding. It has a broad substrate specificity as it can cleave any N-blocked amino acid (except for N-blocked proline). Its catalytic and kinetic mechanisms are well understood, and the hydrolytic reaction is easily detectable spectrophotometrically. Here, we report investigations on the pressure- and temperature-dependence of the kinetic parameters (turnover number and Michaelis constant) of CPSso using several benzoyl- and 3-(2-furyl)acryloyl-amino acids as substrates. This approach enabled us to study these parameters in terms of individual rate constants and establish that the release of the free amino acid is the rate-limiting step, making it possible to dissect the individual non-covalent interactions participating in substrate binding. In keeping with molecular docking experiments performed on the 3D model of CPSso available to date, our results show that both hydrophobic and energetic interactions (i.e., stacking and van der Waals) are mainly involved, but their contribution varies strongly, probably due to changes in the conformational state of the enzyme.

Occhipinti, E., Bec, N., Gambirasio, B., Baietta, G., Martelli, P., Casadio, R., et al. (2007). Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions. Sulfolobus solfataricus carboxypeptidase as a model enzyme. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1764(3), 563-572 [10.1016/j.bbapap.2005.12.007].

Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions. Sulfolobus solfataricus carboxypeptidase as a model enzyme.

TORTORA, PAOLO
2007

Abstract

Sulfolobus solfataricus carboxypeptidase, (CPSso), is a heat- and pressare-resistant zinc-metalloprotease. Thanks to its properties it is an ideal tool for investigating the role of non-covalent interactions in substrate binding. It has a broad substrate specificity as it can cleave any N-blocked amino acid (except for N-blocked proline). Its catalytic and kinetic mechanisms are well understood, and the hydrolytic reaction is easily detectable spectrophotometrically. Here, we report investigations on the pressure- and temperature-dependence of the kinetic parameters (turnover number and Michaelis constant) of CPSso using several benzoyl- and 3-(2-furyl)acryloyl-amino acids as substrates. This approach enabled us to study these parameters in terms of individual rate constants and establish that the release of the free amino acid is the rate-limiting step, making it possible to dissect the individual non-covalent interactions participating in substrate binding. In keeping with molecular docking experiments performed on the 3D model of CPSso available to date, our results show that both hydrophobic and energetic interactions (i.e., stacking and van der Waals) are mainly involved, but their contribution varies strongly, probably due to changes in the conformational state of the enzyme.
Articolo in rivista - Articolo scientifico
Sulfolobus solfataricus; carboxypeptidase; high pressure; enzyme catalysis; non-covalent interactions
English
563
572
Occhipinti, E., Bec, N., Gambirasio, B., Baietta, G., Martelli, P., Casadio, R., et al. (2007). Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions. Sulfolobus solfataricus carboxypeptidase as a model enzyme. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1764(3), 563-572 [10.1016/j.bbapap.2005.12.007].
Occhipinti, E; Bec, N; Gambirasio, B; Baietta, G; Martelli, P; Casadio, R; Balny, C; Lange, R; Tortora, P
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/2655
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