| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 87 === Retinoic acid plays a critical role in cellular differentiation and embryogenesis in the vertebrates. Oxidation of retinol appears to be rate-limiting in the biosynthesis of retinoic acid. Native human class IV mm alcohol dehydrogenase (ADH) exhibits 10-110 fold greater catalytic efficiency for retinol oxidation as compared with other ADH family members. To investigate kinetic mechanism of oxidation of retinol, human class IV ADH expressed in Escherichia coli transformed with vectors harboring the cDNA of mm, was isolated to apparent homogeneity via DEAE-cellulose, AMP-Sepharose and Mono-Q FPLC chromatographic procedures. The subunit molecular weight of mm was determined to be 40 kD. Mechanism of the oxidation of retinol was studied by steady-state kinetic approaches. Intersecting patterns from initial velocity experiments suggested the reaction follows Sequential Bi mechanism. Product inhibition studies indicated that NADH and retinal are competitive inhibitors against the NAD+ and retinol, respectively. Dead-end inhibition studies showed that trifluoroethanol acts as a competitive inhibitor against retinol and a noncompetitive inhibitor against NAD+. The results of the above inhibition studies as well as 10-fold difference in the turnover numbers for retinol and ethanol support that for oxidation of retinol, human class IV ADH obeys the Rapid Equilibrium Random Bi mechanism and that hydride transfer can be the rate-limiting step in catalysis.
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