S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme

Abstract

An enzyme isolated from rat liver cytosol (native molecular mass 78. 3 kDa; polypeptide molecular mass 42.5 kDa) is capable of catalysing the NADH/NADPH-dependent degradation of S-nitrosoglutathione (GSNO). The activity utilizes 1 mol of coenzyme per mol of GSNO processed. The isolated enzyme has, as well, several characteristics that are unique to alcohol dehydrogenase (ADH) class III isoenzyme: it is capable of catalysing the NAD+-dependent oxidations of octanol (insensitive to inhibition by 4-methylpyrazole), methylcrotyl alcohol (stimulated by added pentanoate) and 12-hydroxydodecanoic acid, and also the NADH/NADPH-dependent reduction of octanal. Methanol and ethanol oxidation activity is minimal. The enzyme has formaldehyde dehydrogenase activity in that it is capable of catalysing the NAD+/NADP+-dependent oxidation of S-hydroxymethylglutathione. Treatment with the arginine-specific reagent phenylglyoxal prevents the pentanoate stimulation of methylcrotyl alcohol oxidation and markedly diminishes the enzymic activity towards octanol, 12-hydroxydodecanoic acid and S-hydroxymethylglutathione; the capacity to catalyse GSNO degradation is also checked. Additionally, limited peptide sequencing indicates 100% correspondence with known ADH class III isoenzyme sequences. Kinetic studies demonstrate that GSNO is an exceptionally active substrate for this enzyme. S-Nitroso-N-acetylpenicillamine and S-nitrosated human serum albumin are not substrates; the activity towards S-nitrosated glutathione mono- and di-ethyl esters is minimal. Product analysis suggests that glutathione sulphinamide is the major stable product of enzymic GSNO processing, with minor yields of GSSG and NH3; GSH, hydroxylamine, nitrite, nitrate and nitric oxide accumulations are minimal. Inclusion of GSH in the reaction mix decreases the yield of the supposed glutathione sulphinamide in favor of GSSG and hydroxylamine.

Nitric Oxide and Cancer Development

“…However it has a Km for GSNO of ~20 µM and a Kcat/Km of 94,300 mM -1 min -1 , which is much higher than the other ADHIII substrates 108,109 . Based on this and all the other evidence 109 it is considered that ADHIII is the primary enzyme responsible for metabolizing GSNO 109,110 , and that the two enzymes, ADHIII and GDFDH, are the same 108 .…”

“…Based on this and all the other evidence 109 it is considered that ADHIII is the primary enzyme responsible for metabolizing GSNO 109,110 , and that the two enzymes, ADHIII and GDFDH, are the same 108 . In addition to ADHIII, the Cu/Zn superoxide dismutase enzyme (Cu/Zn SOD) has been shown to effectively degrade GSNO in the presence of GSH with a Km of 5.6 µM 111 .…”

“…With regard to nitrosoglutathione (GSNO) there are several pathways whereby it has been shown to be metabolized. GSNO is degraded in a NADH/ NADPH-dependent process by a rat liver cytosolic enzyme that has been identified as alcohol dehydrogenase (ADH) class III enzyme 109 . This enzyme has GSH-dependent formaldehyde dehydrogenase (GDFDH) activity also.…”

Inflammatory Modulation of Hepatocyte Apoptosis by Nitric Oxide: In Vivo, In Vitro, and In Silico Studies

“…to proceed via reduction of NO • by SOD, [42] ferrocytochrome c, [43] and ubiquinol, [44] and/or reduction of GSNO by GSH and the alcohol dehydrogenase system. [45][46][47] Several groups have reported that HNO, per se a strong reductant, [48] can trigger reactions of oxidation.…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…GSNHOH can either react with GSH to produce glutathione disulfide (GSSG) and hydroxylamine (NH 2 OH) (eq. 3-6), which is rapidly reduced by thiolate anions to ammonia [50], or rearranges and then spontaneously hydrolyses to produce GSO 2 H and ammonia [51]. GSSG is then reduced back to GSH by GR using NADPH as a cofactor.…”