Substrate specificity of human class I alcohol dehydrogenase homo- and heterodimers containing the .beta.2 (Oriental) subunit

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Abstract
In order to gain a better understanding of the metabolism of ethanol in Orientals, the kinetic properties of human alcohol dehydrogenase (ADH) isozymes containing the .beta.2 (Oriental) subunit, i.e., .alpha..beta.2, .beta.2.gamma.1, .beta.2.beta.2, .beta.2.gamma.2, as well as .gamma.1.gamma.1, were examined by using primary and secondary alcohol substrates of various chain lengths and compared with those of the corresponding .beta.1 (Caucasian) subunit containing isozymes already on record [Wagner, F. W., Burger, A. R., and Vallee, B. L. (1983) Biochemistry 22, 1857-1863]. With primary alcohols, these isozymes follow typical Michaelis-Menten kinetics with a preference for long-chain alcohols, as indicated by Km and kcat/Km values. The kcat values obtained with primary alcohols, except methanol, do not vary greatly, i.e., less than 3-fold, whereas the corresponding Km values span a 3600-fold range, i.e., from 26 .mu.M to 94 mM, indicating that the specificity of these isozymes manifests principally in substrate binding. As a consequence, ethanol.sbd.which might be thought to be the principal in vivo substrate for ADH.sbd.is oxidized rather poorly, i.e., from 50- to 90-fold less effectively than octanol. Secondary alcohol oxidation by the homodimers .beta.2.beta.2 and .gamma.1.gamma.1 also follows normal Michaelis-Menten kinetics. Again, values of Km and kcat/Km reveal that both isozymes prefer long carbon chains. For all secondary alcohols studied, the Km and kcat values for .beta.2.beta.2 are much higher than those for .gamma.1.gamma.1, i.e., 25- to 360-fold and 6- to 16-fold, respectively. However, for each isozyme the values of kcat for different alcohols are lessentially invariant. For a given chain length the isozymes favor primary over secondary alcohols. Secondary alcohol oxdiation by the heterodimers .alpha..beta.2 and .beta.2.gamma.1 follows non Michaelis-Menten kinetics, which can be interpreted in terms of a high-affinity and a low-affinity enzyme component. Values of Km and kcat estimated from the Eadie-Hofstee plot curves for both of these isozymes indicate that the high-affinity activity of these heterodimers is contributed by the .alpha. and .gamma.1 subunits, respectively, while the low-affinity activity is largely due to the .beta.2 subunit. These results suggest that the subunits of .beta.2-containing heterodimeric ADH act independently and noncooperatively. The observed difference between the .beta.2 chain and the .alpha. and .gamma.1 chains in their binding affinities toward secondary alcohols can be related to structural differences in the substrate-binding sites of these isozymes.