title: Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite.
abstract: Disruption of the zinc-thiolate center at the active site of yeast alcohol dehydrogenase results in inactivation and zinc release. Measurements of activity, zinc release, and thiol/thiolate oxidation were used to assess the effects of biologically relevant oxidants on alcohol dehydrogenase. Alcohol dehydrogenase was inactivated by 1 mM hydrogen peroxide at a rate of 1.3 M-1 s-1. Peroxynitrite, the near diffusion-limited reaction product of nitric oxide and superoxide, inactivated alcohol dehydrogenase with an IC50 = 0.95 microM when catalytic concentrations of alcohol dehydrogenase subunit (0.074 microM) were present. Slow, continuous production of peroxynitrite from decomposition of SIN-1 inactivated alcohol dehydrogenase as effectively as bolus addition. The rate constants for reaction of peroxynitrite with alcohol dehydrogenase at 23 degrees C as determined by two different competition assays were 2.6 x 10(5) M-1 s-1 and 5.2 x 10(5) M-1 s-1. The reaction with alcohol dehydrogenase represents one of the fastest reactions yet determined for peroxynitrite. Hypochlorite inactivated alcohol dehydrogenase at a rate of 4 x 10(3) M-1 s-1. The rate constant for inactivation by taurine choramine, the reaction product of taurine and hypochlorite, was only slightly slower at 2.7 x 10(3) M-1 s-1. Zinc release and thiol/thiolate oxidation were correlated with inactivation by either peroxynitrite or hypochlorite. At the concentrations of peroxynitrite or hypochlorite producing total inactivation, 0.85 zinc atom was released per subunit and 3 thiol/thiolates per subunit were oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Anesthesiology, University of Alabama at Birmingham 35233.
authors.forename: J P
authors.initials: JP
authors.lastname: Crow
authors.forename: J S
authors.initials: JS
authors.lastname: Beckman
authors.forename: J M
authors.initials: JM
authors.lastname: McCord
mesh_terms: D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009566:Nitrates / Q000378:metabolism / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013654:Taurine / Q000031:analogs & derivatives / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry*
chemical_list: D009566:Nitrates; D013438:Sulfhydryl Compounds; D013654:Taurine; C039642:peroxynitric acid; D006639:Histidine; C043410:N-chlorotaurine; C002385:linsidomine; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D008981:Molsidomine; D000426:Alcohol Dehydrogenase; D015032:Zinc; D003545:Cysteine