INHIBITION OF THE CATALYTIC ACTIVITY OF ALCOHOL-DEHYDROGENASE BY NITRIC-OXIDE IS ASSOCIATED WITH S-NITROSYLATION AND THE RELEASE OF ZINC

Nitric oxide (NO) reacts with the sulfhydryl groups of proteins to for m nitroso thiols. Alcohol dehydrogenase (ADH) plays an important role in the metabolism of ethanol. Chronic alcohol administration stimulate s NO formation in the liver, and production of NO is increased in alco hol liver injury. The effect of exogenous and endogenous NO on rat or horse ADH activity was evaluated. incubation of intact rat hepatocytes or cytosol isolated from hepatocytes with S-nitroso-N-acetylpenicilla mine (SNAP), a nitric oxide donor, resulted in a decrease in ADH activ ity. Endogenous NO synthesis was induced in rat hepatocytes by incubat ion with a mixture of cytokines and endotoxin in the presence of L-arg inine. As NO production in hepatocytes increased over a 24 h time peri od, a significant decrease in ADH activity was observed. This effect w as blocked by the competitive inhibitor of NO synthesis, N-omega-nitro -L-arginine methyl ester, indicating that ADH was also inactivated by endogenously generated NO. The decreased activity of ADH was not relat ed to lowering of the ADH content as shown by Western blot analysis. T o evaluate the mechanism of inhibition, purified ADH from equine liver was incubated with gaseous NO or NO released from NO donors such as t he diethylamine/nitric oxide complex (DEA/NO) and SNAP. NO donors inac tivated ADH in a dose- and time-dependent manner. Trapping of NO with hemoglobin resulted in protection of ADH against inactivation by NO. T here was no effect by analogues of the NO donors which do not release NO. NAD afforded some protection against the NO inactivation of ADH, M easurements of thiol oxidation, S nitrosylation, and zinc release were used to assess the effect of NO on ADH activity. Thiol oxidation, S-n itroso thiol formation, and zinc release correlated with inactivation of ADH by NO, indicating that disruption of the zinc/thiolate active c enter due to S nitrosylation of ADH results in zinc release, followed by inactivation of the enzyme. Recovery experiments were performed by incubating the NO-treated enzyme with dithiothreitol (DTT) and/or Zn2. The inhibitory effect by NO was reversible since, after the nitrosyl ated enzyme was reduced with DTT followed by incubation with ZnCl2 to allow reincorporation of Zn2+, ADH activity was increased from 20% of control values to 70%. These results suggest that cysteine residues co ntained within the zinc/thiolate active center may be primary sites of NO interaction with ADH. NO may modulate the metabolism of ethanol an d influence metabolic actions of ethanol via interaction with ADH.