Kinetics of the interaction of chymotrypsin with eglin c

• Published in: Biochemical journal 1991-11, Vol.280 (1), p.27-32
• Main Authors: FALLER, B, BIETH, J. G
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biological and medical sciences; Cattle; Chymotrypsin - antagonists & inhibitors; Chymotrypsin - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Interactions. Associations; Intermolecular phenomena; Kinetics; Leukocyte Elastase; Mathematics; Models, Theoretical; Molecular biophysics; Molecular Sequence Data; Pancreas - enzymology; Pancreatic Elastase - antagonists & inhibitors; Pancreatic Elastase - metabolism; Protein Binding; Proteins; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Serine Proteinase Inhibitors - metabolism; Serine Proteinase Inhibitors - pharmacology; Serpins; Substrate Specificity; Thermodynamics
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2800027

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.