Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite

Biochemistry. 1995 Mar 21;34(11):3544-52. doi: 10.1021/bi00011a008.


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)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alcohol Dehydrogenase / antagonists & inhibitors*
  • Alcohol Dehydrogenase / chemistry*
  • Alcohol Dehydrogenase / metabolism
  • Cysteine / chemistry
  • Histidine / chemistry
  • Hydrogen Peroxide / pharmacology
  • Hypochlorous Acid / metabolism
  • Hypochlorous Acid / pharmacology*
  • Kinetics
  • Molsidomine / analogs & derivatives
  • Molsidomine / pharmacology
  • Nitrates / metabolism
  • Nitrates / pharmacology*
  • Sulfhydryl Compounds / chemistry
  • Taurine / analogs & derivatives
  • Taurine / pharmacology
  • Zinc / chemistry*


  • Nitrates
  • Sulfhydryl Compounds
  • Taurine
  • peroxynitric acid
  • Histidine
  • N-chlorotaurine
  • linsidomine
  • Hypochlorous Acid
  • Hydrogen Peroxide
  • Molsidomine
  • Alcohol Dehydrogenase
  • Zinc
  • Cysteine