title: Reversible inhibition of neutrophil elastase by thiol-modified alpha-1 protease inhibitor.
abstract: We have modified the single cysteine residue of alpha 1-protease inhibitor (alpha 1-PI) with HgCl2, methylmethane thiosulfonate, oxidized glutathione (GSSG), and N-(1-anilinonaphthyl-4)maleimide (ANM). Whereas native alpha 1-PI combines rapidly and quasi-irreversibly with neutrophil elastase, the thiol-modified alpha 1-PI derivatives are dissociable reversible competitive inhibitors of the enzyme, with values of Ki in the range of 6-7 nM. Removal of the thiol modifications restores the rapid irreversible mode of inhibition. Once native alpha 1-PI has combined with neutrophil elastase, the enzyme-inhibitor complex retains a reactive thiol group, but the two proteins can no longer be dissociated by subsequent reaction with ANM, even after exposure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From kinetic measurements of fluorescence, ANM-modified alpha 1-PI combines with neutrophil elastase via an apparent biomolecular process with a second order rate constant on the order of 10(5) M-1 S-1. We estimate a dissociation rate constant on the order of 10(-3) S-1. The emission of ANM-modified alpha 1-PI is increased in intensity and blue shifted from the maximum in ANM-modified cysteine, consistent with a predominantly nonpolar environment. Association with neutrophil elastase results in an additional blue shift with further increase in intensity, consistent with a further decrease in polarity of the environment of the cysteine. Modification with methylmethane thiosulfonate or GSSG results in a small decrease in quantum yield and a red shift in the tryptophan emission spectrum of the modified inhibitor, suggestive of increased polarity of the environment of at least 1 of the 2 tryptophan residues in alpha 1-PI. These changes are reversed by dithiothreitol and are consistent with a conformational change which transforms the inhibitory activity from a rapid, irreversible mode in native alpha 1-PI to a dissociable competitive mode in the mixed disulfide derivatives.
authors.affiliation: Department of Biochemistry and Cell Biology, State University of New York, Stony Brook 11794.
authors.forename: S C
authors.initials: SC
authors.lastname: Tyagi
mesh_terms: D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D000515:alpha 1-Antitrypsin / Q000494:pharmacology*
chemical_list: D013438:Sulfhydryl Compounds; D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase