Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase

P. C. Sanghani, C. L. Stone, B. D. Ray, E. V. Pindel, T. D. Hurley, W. F. Bosron

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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 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.

Original languageEnglish (US)
Pages (from-to)10720-10729
Number of pages10
JournalBiochemistry
Volume39
Issue number35
DOIs
StatePublished - Sep 5 2000

ASJC Scopus subject areas

  • Biochemistry

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    Sanghani, P. C., Stone, C. L., Ray, B. D., Pindel, E. V., Hurley, T. D., & Bosron, W. F. (2000). Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase. Biochemistry, 39(35), 10720-10729. https://doi.org/10.1021/bi9929711