title: Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
authors.forename: P C
authors.initials: PC
authors.lastname: Sanghani
authors.forename: C L
authors.initials: CL
authors.lastname: Stone
authors.forename: B D
authors.initials: BD
authors.lastname: Ray
authors.forename: E V
authors.initials: EV
authors.lastname: Pindel
authors.forename: T D
authors.initials: TD
authors.lastname: Hurley
authors.forename: W F
authors.initials: WF
authors.lastname: Bosron
mesh_terms: 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
chemical_list: 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