title: Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions Sulfolobus solfataricus carboxypeptidase as a model enzyme.
abstract: Sulfolobus solfataricus carboxypeptidase, (CPSso), is a heat- and pressure-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.
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, I-20126 Milano, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Occhipinti
authors.forename: Nicole
authors.initials: N
authors.lastname: Bec
authors.forename: Benedetta
authors.initials: B
authors.lastname: Gambirasio
authors.forename: Gabriella
authors.initials: G
authors.lastname: Baietta
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Martelli
authors.forename: Rita
authors.initials: R
authors.lastname: Casadio
authors.forename: Claude
authors.initials: C
authors.lastname: Balny
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lange
authors.forename: Paolo
authors.initials: P
authors.lastname: Tortora
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry*; D002268:Carboxypeptidases / Q000737:chemistry*; D002384:Catalysis; D005998:Glycine / Q000737:chemistry; D006626:Hippurates / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D011312:Pressure; D011487:Protein Conformation; D055672:Static Electricity; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013696:Temperature; D013816:Thermodynamics
chemical_list: D019843:Archaeal Proteins; D006626:Hippurates; D010649:Phenylalanine; D006639:Histidine; C053764:benzoylphenylalanine; D002268:Carboxypeptidases; C030514:hippuric acid; D005998:Glycine