title: Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G.
abstract: We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.
mesh_terms: D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000097:blood; D004347:Drug Interactions; D005069:Evaluation Studies as Topic; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000097:blood; D011506:Proteins / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology*; D012697:Serine Endopeptidases; D015843:Serpins; D013696:Temperature
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; C028656:eglin proteinase inhibitors; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase