title: Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry.
abstract: Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis-Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37°C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis-Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays.
authors.affiliation: Centre of New Technologies, University of Warsaw, 02-097 Warsaw, Poland. Electronic address: k.maximova@cent.uw.edu.pl.
authors.forename: Ksenia
authors.initials: K
authors.lastname: Maximova
authors.affiliation: Centre of New Technologies, University of Warsaw, 02-097 Warsaw, Poland. Electronic address: joanna@cent.uw.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Trylska
mesh_terms: D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D055162:Biocatalysis; D002151:Calorimetry / Q000379:methods*; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011487:Protein Conformation; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
chemical_list: D001550:Benzamidines; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; D010664:Phenylmethylsulfonyl Fluoride; D014357:Trypsin; C032157:benzamidine
keywords: Benzamidine; Casein; Enzyme kinetics; Isothermal titration calorimetry; Trypsin; α1-Antitrypsin