Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase.
Formaldehyde, a major industrial chemical, is classified as a carcinogen because of its high reactivity with DNA. It is inactivated by oxidative metabolism to formate in humans by glutathione-dependent formaldehyde dehydrogenase. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent alcohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-ADH. The first step in the reaction involves the nonenzymatic formation of the S-(hydroxymethyl)glutathione adduct from formaldehyde and glutathione. When formaldehyde concentrations exceed that of glutathione, nonoxidizable adducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are reported to be 50 times that of formaldehyde in humans. Initial velocity, product inhibition, dead-end inhibition, and equilibrium binding studies indicate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathione and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Formation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxododecanoic acid (12-ODDA) exhibited substrate inhibition, which is consistent with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanism is consistent with the published three-dimensional structure of the formaldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coenzyme-catalytic domain conformation where substrates can bind or dissociate in any order.
Sanghani
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Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
Stone
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Ray
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Pindel
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Hurley
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Bosron
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D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001667:Binding, Competitive; D002247:Carbon Isotopes; D005231:Fatty Acids, Unsaturated / Q000737:chemistry; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D007850:Lauric Acids / Q000737:chemistry; D009243:NAD / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
C117640:12-(1',3'-hexadienyloxy)-9,11-dodecadienoic acid; D002247:Carbon Isotopes; D005231:Fatty Acids, Unsaturated; D007850:Lauric Acids; D009243:NAD; C029469:S-hydroxymethylglutathione; C031263:12-hydroxydodecanoic acid; C046049:12-aminododecanoic acid; C020145:formaldehyde dehydrogenase (glutathione); D000445:Aldehyde Oxidoreductases; D005978:Glutathione