Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase

Formaldehyde, a major industrial chemical, is classified as a carcinogen be cause of its high reactivity with DNA. it is inactivated by oxidative metab olism to formate in humans by glutathion-dependent formaldehyde dehydrogena se. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent al cohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-AD H. The first step in the reaction involves the nonenzymatic formation of th e S-(hydroxymethyl)glututhione adduct from formaldehyde and glutathione. Wh en formaldehyde concentrations exceed that of glutathione, nonoxidizable ad ducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are re ported to be 50 times that of formaldehyde in humans. Initial velocity, pro duct inhibition, dead-end inhibition, and equilibrium binding studies indic ate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathi one and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Fo rmation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxod odecanoic acid (I2-ODDA) exhibited substrate inhibition, which is consisten t with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanis m is consistent with the published three-dimensional structure of the forma ldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coe nzyme-catalytic domain conformation where substrates can bind or dissociate in any order.