title: Kinetics of the interaction of chymotrypsin with eglin c.
abstract: The kinetics of binding of recombinant eglin c to bovine pancreatic chymotrypsin was studied by conventional and stopped-flow techniques. With nanomolar enzyme and inhibitor concentrations, the inhibition was fast and pseudo-irreversible (k(assoc.) = 4 x 10(6) m-1.s-1 at 7.4 and 25 degrees C). Reaction of the enzyme-inhibitor complex with alpha 1-proteinase inhibitor, an irreversible chymotrypsin ligand, resulted in a slow release of free eglin c, which was monitored by electrophoresis (k(dissoc.) approximately 1.6 x 10(-6) s-1, t1/2 approximately 5 days). The proflavin displacement method and a stopped-flow apparatus were used to monitor the association of chymotrypsin with eglin c under a wide range of inhibitor concentration and under pseudo-first-order conditions. At pH 7.4 and 25 degrees C or 5 degrees C, or at pH 5.0 and 25 degrees C, the pseudo-first-order rate constant of proflavin displacement increased linearly with eglin c up to the highest concentration tested, suggesting a one-step bimolecular association reaction: E + I in equilibrium with EI. However, kassoc. is much lower than the rate constant for a bimolecular reaction and its activation energy (66 kJ.mol-1 at pH 7.4 and 78 kJ.mol-1 at pH 5.0) is far too high for a diffusion-controlled step. The enzyme-inhibitor association may therefore occur via a loose pre-equilibrium complex EI* (Ki* much greater than 5 x 10(-4) M) that rapidly isomerizes (k2 much greater than 2 x 10(3) s-1) into an extremely stable final complex (Ki approximately 4 x 10(-13) M). Unlike other proteinase-inhibitor systems, the chymotrypsin-eglin association is virtually pH-independent.
authors.affiliation: INSERM U 237, Université Louis Pasteur de Strasbourg, Illkirch, France.
authors.forename: B
authors.initials: B
authors.lastname: Faller
authors.forename: J G
authors.initials: JG
authors.lastname: Bieth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D015843:Serpins; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C028656:eglin proteinase inhibitors; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase