Hydrophobic anion activation of human liver .chi..chi. alcohol dehydrogenase

Abstract

Class III alcohol dehydrogenase (chi chi-ADH) from human liver binds both ethanol and acetaldehyde so poorly that their Km values cannot be determined, even at ethanol concentrations up to 3 M. However, long-chain carboxylates, e.g., pentanoate, octanoate, deoxycholate, and other anions, substantially enhance the binding of ethanol and other substrates and hence the activity of class III ADH up to 30-fold. Thus, in the presence of 1 mM octanoate, ethanol displays Michaelis-Menten kinetics. The degree of activation depends on the size both of the substrate and of the activator; generally, longer, negatively charged activators result in greater activation. At pH 10, the activator binds to the E-NAD+ form of the enzyme to potentiate substrate binding. Pentanoate activates methylcrotyl alcohol oxidation and methylcrotyl aldehyde reduction 14- and 30-fold, respectively. Such enhancements of both oxidation and reduction are specific for class III ADH; neither class I nor class II shows this effect. The implications as to the nature of the physiological substrate(s) of class III ADH are discussed in light of the recent finding that this ADH and glutathione-dependent formaldehyde dehydrogenase are identical. A new rapid purification procedure for chi chi-ADH is presented.

Arabidopsis Formaldehyde Dehydrogenase

“…Amino acid sequences were compared using the program Clustal W (Thompson et al, 1994 viously used for class I11 . Class P was inactive with S-hydroxymethylglutathione, while pentanoate (5-50 mM) did not activate the oxidation of methyl crotyl alcohol, although class 111 shows a 12-fold activation under identical conditions (Moulis et al, 1991). We can therefore conclude that Argl15 and other active site features common to classes P and I11 are insufficient to develop class 111 properties in class P. This confirms that additional residues typical of the class 111 active site, such as Tyr93 and Asp57, are essential for class transitions and for the class I11 specificity (Estonius et al, 1994).…”

Molecular Basis of Formaldehyde Detoxification

“…Formaldehyde dehydrogenases (class III alcohol dehydrogenases) were extensively characterized both structurally and biochemically (17)(18)(19)(20), whereas FGHs have received far less attention. Only three eukaryotic FGHs (from the human liver, Saccharomyces cerevisiae, and Arabidopsis thaliana) (21)(22)(23) have been purified and partially characterized, but the homologous enzyme from bacteria has not been yet purified.…”

Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

“…Moreover, its Km for ethanol is >3 M compared to 1 mM for class I isozymes; yet, it is nearly as effective as other ADHs toward long-chain primary alcohols, particularly w-hydroxy fatty acids such as 12-hydroxydodecanoate (12-HDA) (5). It is totally insensitive to inhibition by 4-methylpyrazole, a potent inhibitor of the class I and II enzymes.…”

“…An additional feature unique to yr-ADH is its activation by hydrophobic anions, particularly fatty acids, which can stimulate both alcohol oxidation and aldehyde reduction up to 30-fold (5). The nonessential activator mechanism proposed for this process indicates that the alcohol substrate and the activator bind simultaneously, with consequent enhancement of substrate binding.…”

“…This complementarity likely reflects the length of the substrate binding pocket of the ADHs (6,7). Appropriate combinations of activator and alcohol side chains apparently fit into this pocket (5) and result in maximal activity. The observations that all activators require an anionic group and that the best substrates for XX-ADH, 12-HDA and S-hydroxymethylglutathione (HMGSH), have an co-carboxylate group suggested that a corresponding positively charged residue would be located close to the active site.…”