Kinetic mechanism of Human Alcohol Dehydrogenase Family Implication for Functional Classification and Ethanol/Retinol Metabolism

• Main Authors: Han Chih-Li, 韓繼利
• Other Authors: Yin Shih-Jiun
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/44055791758826399587

博士 === 國防醫學院 === 生命科學研究所 === 86 === Human ADH, principal enzyme responsible for ethanol metabolism, constitutes a complex family. The purpose of this dissertation was to investigate (1) kineti c mechanisms of the ADH family members and the implications for functional cla ssification as well as simulation of in vivo ethanol-oxidizing activity; (2) i nhibition by ethanol saturation and inhibition by ethanol saturation and inhi bition of retinol oxidizing by ethanol to assess the functional significance o f ADH family in the first-pass metabolism of alcohol as well as in the pathoge nesis of fetal alcohol syndrome.Class I aa, b1b1, b2b2, and g1g1, class II pp, amd class III cc were isolated from the livers of heart transplant dorners as well as class IV mm from the pooled surgical stomach mucosae via column chrom atographic steps. The apparent homogeneity of the purified ADH forms was judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing with the s ubunit molecular weight of 40,000.Initial velocity, product and dead-end inhib ition studies indicated that sequential mechanism, i.e. binding NAD first and releasing NADH last, with the exception of the class I g1g1 wich exhibits nega tive cooperativity for ethanol oxidation. Evidence based on the calculated rat e constants and the obtained kinetic isotope effects of deuterated ethanol sup ports that the dissociationof NADH is the rate-limiting step in catalysis. Cla ss II and class IV enzymes appear to be more similar in the aspects of kinetic constants, catalytic efficiency, equilibrium and rate constants as well as in hibition constants.Kinetic simulations of the steady-state ethanol-oxidizing a ctivity at 10 mM ethanol and the assumed in vivo concentrations of coenzymes a nd acetaldehyde indicate the decreasing order, b2b2>mm>aa>pp>b1b1.ADH is invol ved in the synthesis of retinoic acid, a potent regulator in differentiation o f the epithelial cells and embryogenesis. The catalytic efficiency for oxidizi ng all-trans-retinol was found in the following order, mm>pp>aa>b2b2>g1g1>b1b1 . Ethanol acted as a competitive inhibitor againstretinol with the inhibition constants over 37 uM-11 mM, suggesting that the retinoic acid by ethanol in pr egnant women during heavy drinking.Class I aa, b1b1, b2b2, and g1g1 as well as class II pp exhibited substrate inhibition while activity of the class IV mm was not inhibited by high concentrations of ethanol. Ethanol saturation profil es indicate that pp and uu can effectively contribute to the first-pass metabo lism of alcohol.In summary, class I b2b2 and class IV uu are the most efficien t enzymes responsible for ethanol oxidation in the liver and stomach, respecti vely in the ADH family. Class II pp is the major form which contributes to the majority pathways by ethanol may be involved in the etiology of fetal alcoho l syndrome, alcohol-related gastrointestinal tract cancers, skin disease and a spermatogenesis.