US EPA

626109 

Journal Article 

Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase 

Sanghani, PC; Stone, CL; Ray, BD; Pindel, EV; Hurley, TD; Bosron, WF 

2000 

Yes 

Biochemistry
ISSN: 0006-2960
EISSN: 1520-4995 

39 

35 

10720-10729 

English 

10978156 

10.1021/bi9929711 

WOS:000089283600014 

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.