Kinetic Mechanism of Human Glutathione-Dependent Formaldehyde Dehydrogenase

• Published in: Biochemistry (Easton) 2000-09, Vol.39 (35), p.10720-10729
• Main Authors: Sanghani, Paresh C, Stone, Carol L, Ray, Bruce D, Pindel, Evgenia V, Hurley, Thomas D, Bosron, William F
• Format: Article
• Language: English
• Subjects: Aldehyde Oxidoreductases - antagonists & inhibitors; Aldehyde Oxidoreductases - chemistry; Binding, Competitive; Carbon Isotopes; Fatty Acids, Unsaturated - chemistry; Glutathione - analogs & derivatives; Glutathione - chemistry; Humans; Kinetics; Lauric Acids - chemistry; NAD - chemistry; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Protein Binding; Spectrophotometry, Ultraviolet; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9929711

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 χ-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.