Mechanism of inactivation of human leukocyte elastase by a chloromethyl ketone: kinetic and solvent isotope effect studies.
The mechanism of inactivation of human leukocyte elastase (HLE) by the chloromethyl ketone MeOSuc-Ala-Ala-Pro-Val-CH2Cl was investigated. The dependence of the first-order rate constant for inactivation on concentration of chloromethyl ketone is hyperbolic and suggests formation of a reversible "Michaelis complex" prior to covalent interaction between the enzyme and inhibitor. However, the observed Ki value is 10 microM, at least 10-fold lower than dissociation constants for complexes formed from interaction of HLE with structurally related substrates or reversible inhibitors, and suggests that Ki is a complex kinetic constant, reflecting the formation and accumulation of both the Michaelis complex and a second complex. It is proposed that this second complex is a hemiketal formed from attack of the active site serine on the carbonyl carbon of the inhibitor. The accumulation of this intermediate may be a general feature of reactions of serine proteases and chloromethyl ketones derived from specific peptides and accounts for the very low Ki values observed for these reactions. The solvent deuterium isotope effect (SIE) on the inactivation step (ki) is 1.58 +/- 0.07 and is consistent with rate-limiting, general-catalyzed attack of the active site His on the methylene carbon of the inhibitor with displacement of chloride anion. The general catalyst is thought to be the active site Asp. In contrast, the SIE on the second-order rate constant for HLE inactivation, ki/Ki, is inverse and equals 0.64 +/- 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D003903:Deuterium; D017666:Deuterium Oxide; D004121:Dimethyl Sulfoxide; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D008433:Mathematics; D008954:Models, Biological; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000097:blood*; D012997:Solvents; D014867:Water
D000590:Amino Acid Chloromethyl Ketones; D012997:Solvents; D014867:Water; C026704:methoxysuccinyl-alanyl-alanyl-prolyl-valine chloromethyl ketone; D003903:Deuterium; D010196:Pancreatic Elastase; D017666:Deuterium Oxide; D004121:Dimethyl Sulfoxide