Human liver alcohol dehydrogenase—inhibition of methanol activity by pyrazole, 4-methylpyrazole, 4-hydroxymethylpyrazole and 4-carboxypyrazole

Abstract

With human liver alcohol dehydrogenase of high purity at pH 7.0 and 500 μM NAD the K m for methanol is 7.0 mM (ten times greater than the K m for ethanol) and the turnover number 1.4/active site/min (about one-tenth of the turnover with ethanol in the same conditions). From secondary kinetic plots it can be calculated that at saturating concentrations of both substrates, namely methanol and NAD, these constants do not change appreciably: the K m for methanol is somewhat lower (5.2 mM) and the turnover number slightly higher (1.7/active site/min). The difference in turnover numbers with methanol and ethanol as substrates suggests that the kinetic mechanism for methanol is different from that for ethanol dehydrogenation. The dissociation constant between human alcohol dehydrogenase and NAD, determined kinetically with methanol as substrate, is 127 μM. The K i values for pyrazole, 4-methylpyrazole and 4-hydroxymethylpyrazole are 0.54, 0.09 and 6.6 μM respectively; 4-carboxypyrazole (100 μM) at 3mM methanol does not inhibit human ADH. The inhibitory effect of 4-methylpyrazole is therefore not likely to be enhanced by a possible metabolic conversion to 4-hydroxymethylpyrazole and 4-carboxypyrazole.