SENSITIVITY OF THE ESSENTIAL ZINC-THIOLATE MOIETY OF YEAST ALCOHOL-DEHYDROGENASE TO HYPOCHLORITE AND PEROXYNITRITE

Disruption of the zinc-thiolate center at the active site of yeast alc ohol dehydrogenase results in inactivation and zinc release. Measureme nts of activity, zinc release, and thiol/thiolate oxidation were used to assess the effects of biologically relevant oxidants on alcohol deh ydrogenase. Alcohol dehydrogenase was inactivated by 1 mM hydrogen per oxide at a rate of 1.3 M(-1) s(-1). Peroxynitrite, the near diffusion- limited reaction product of nitric oxide and superoxide, inactivated a lcohol dehydrogenase with an IC50 = 0.95 mu M when catalytic concentra tions of alcohol dehydrogenase subunit (0.074 mu M) were present. Slow , continuous production of peroxynitrite from decomposition of SIN-1 i nactivated alcohol dehydrogenase as effectively as bolus addition. The rate constants for reaction of peroxynitrite with alcohol dehydrogena se at 23 degrees C as determined by two different competition assays w ere 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 dehydr ogenase at a rate of 4 x 10(3) M(-1) s(-1). The rate constant for inac tivation by taurine choramine, the reaction product of taurine and hyp ochlorite, was only slightly slower at 2.7 x 10(3) M(-1) s(-1). Zinc r elease and thiol/thiolate oxidation were correlated with inactivation by either peroxynitrite or hypochlorite. At the concentrations of pero xynitrite or hypochlorite producing total inactivation, 0.85 zinc atom was released per subunit and 3 thiol/thiolates per subunit were oxidi zed. The structural similarity between the zinc-thiolate moiety of yea st alcohol dehydrogenase (Zn(1)Cys(2)His(1)) and that found in zinc fi nger proteins (Zn(1)Cys(2)His(2)) suggest that the widely distributed ubiquitous zinc finger moiety may be a major target for oxidant-induce d injury.