Beta 2 (Oriental) human liver alcohol dehydrogenases do not exhibit subunit interaction: oxidation of cyclohexanol by homo- and heterodimers.
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., & 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-aminocaproyl)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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Fong
W P
WP
Department of Biochemistry, Chinese University of Hong Kong, Shatin, N.T.
Keung
W M
WM
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D003511:Cyclohexanols / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D044469:Racial Groups
D003511:Cyclohexanols; D007527:Isoenzymes; D046911:Macromolecular Substances; D000426:Alcohol Dehydrogenase