Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions Sulfolobus solfataricus carboxypeptidase as a model enzyme.
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.
Occhipinti
Emanuela
E
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, I-20126 Milano, Italy.
Bec
Nicole
N
Gambirasio
Benedetta
B
Baietta
Gabriella
G
Martelli
Pier Luigi
PL
Casadio
Rita
R
Balny
Claude
C
Lange
Reinhard
R
Tortora
Paolo
P
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
D019843:Archaeal Proteins; D006626:Hippurates; D010649:Phenylalanine; D006639:Histidine; C053764:benzoylphenylalanine; D002268:Carboxypeptidases; C030514:hippuric acid; D005998:Glycine