.beta.2 (Oriental) human liver alcohol dehydrogenases do not exhibit subunit interaction: oxidation of cyclohexanol by homo- and heterodimers

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Abstract
The steady-state kinetics of isozymes of human liver alcohol dehydrogenase (ADH) containing the .beta.2 (Oriental) subunit were investigated in order to confirm the supposition [Fong, W. P., and Keung, W. M. (1987) Biochemistry (preceding paper in this issue)] that the subunits of such heterodimeric ADHs act independently and noncooperatively. The ADH isozymes .alpha..beta.2, .beta.2.beta.2, .beta.2.gamma.1, and .beta.2.gamma.2 as well as .gamma.1.gamma.1 were purified by chromatography on DEAE-cellulose, 4-[3-[N-(6-aminocapropyl)amino]propyl]pyrazole-Sepharose, and CM-cellulose. Their kinetics were studied at pH 9.0 with cyclohexanol since this substrate permits maximal differentiation between activities of the heterodimeric subunits. Oxidation of cyclohexanol by the homodimers .beta.2.beta.2 and .gamma.1.gamma.1 follows conventional Michaelis-Menten kinetics. The values of Km and kcat determined for .beta.2.beta.2 and .gamma.1.gamma.1 are 0.11 M and 260 min-1 and 79 .mu.M and 45 min-1, respectively, indicating that .beta.2.beta.2, like the previously studied .beta.1.beta.1, has an unusually low binding affinity for cyclohexanol compared to that of the ADH isozymes formed by the combination of .alpha., .gamma.1, and .gamma.2 chains. Cyclohexanol oxidation by the heterodimers .alpha..beta.2, .beta.2.gamma.1, and .beta.2.gamma.2 follows biphasic kinetics which can be fully accounted for by the individual subunits, one exhibiting a high and the other a low substrate-binding affinity. Eadie-Hofstee plots resolve the biphasic kinetics into two linear components, each of which yields a set of kinetic parameters. The Km values for the low-affinity components of the .alpha..beta.2 (0.082 M), .beta.2.gamma.1 (0.079 M), and .beta.2.gamma.2 (0.091 M) heterodimers are similar to that of the .beta.2.beta.2 (0.11 M) homodimer, while the Km values of the high-affinity components fall into the same concentration range (micromolar) as those determined for the .alpha., .gamma.1, and .gamma.2 chains [Wagner, F. W., Burger, A. R., and Vallee, B. L. (1983) Biochemistry 22, 1857-1863]. Furthermore, the substrate concentration vs. activity profile of .beta.2.gamma.1 fits the theoretical curve obtained by averaging the kinetic responses of the parent homodimers, .beta.2.beta.2 and .gamma.1.gamma.1. Thus, the individual subunits of .beta.2-containing heterodimeric ADHs do not exhibit subunit interaction. This conclusion is supported by studies of the effects of pH and thiourea on the activities of the high- and low-affinity components of the heterodimers.

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