Advertisement

Kinetic and Mechanistic Studies of Oxidation of Vitamin a Alcohol to Vitamin a Aldehyde by Horse Liver Alcohol Dehydrogenase. The Inhibition by Ethanol and Pyrazole

  • Y. Pocker
  • K. W. Raymond

Abstract

The present investigation shows that all-trans retinol (Vitamin A alcohol), an alcohol of great physiological importance, is efficiently oxidized to all-trans retinaldehyde by the enzyme horse liver alcohol dehydrogenase. We observe a Km retinol value of 145μM and a turnover number of 0.45s−1 for the oxidation of all-trans retinol in the presence of Triton X-100, a surfactant used as a solubilizer. Over the concentration range of surfactant used (up to 0.1% Triton X-100) our studies on the oxidation of ethanol and all-trans retinol show that turnover numbers for both reactions remain constant as does the value for Km ethanol. On increasing the concentration of Triton X-100 from 0.025% to 0.10%, however, the Km retinol value increases by a factor of two. This behavior for retinol oxidation can be attributed to the partitioning of retinol between enzyme and surfactant. Pyrazole, a known inhibitor of alcohol oxidation by horse liver alcohol dehydrogenase, is a competitive inhibitor of both all-trans retinol and ethanol, with observed Ki values of 3.3×10−7 M and 3.9×10−7 M, respectively. We also find that ethanol inhibits all-trans retinol oxidation in a complex fashion, an observation which may have important consequences in view of the physiological role of retinol and its oxidation products. Our present studies indicate that all-trans retinol binds in the same region of the enzyme as does ethanol and is oxidized with an efficiency approaching that of ethanol itself.

Keywords

Ethanol Concentration Surfactant Concentration Alcohol Dehydrogenase Turnover Number Ethanol Oxidation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Becci, P.J., H.J. Thompsom, C.J. Grubbs, R.A. Squire, C.C. Brown, M.B. Sporn, and M.C. Moon, 1978, Inhibitory effects of 13-cis retinoic acid on urinary bladder carcinogenesis induced in c57BL/6 mice by n-butyl-N-(4-hydroxybutyl)-nitrosamine, Cancer Research, 38: 4463.PubMedGoogle Scholar
  2. Bernhard, S.A., M.F. Dunn, P.L. Luisi, and P. Schack, 1970, Mechanistic studies on equine liver alcohol dehydrogenase. I. The stoichiometry relationship of the coenzyme binding sites to the catalytic sites active in the reduction of aromatic aldehydes in the transient state, Biochemistry, 9: 185.Google Scholar
  3. Bliss, A.F., 1960, Reversible enzymic reduction of retinene to vitamin A, Biol. Bull., 97: 221.Google Scholar
  4. Clarke, S., 1975, The size and detergent binding of membrane proteins, J. Biol. Chem., 250: 5459.Google Scholar
  5. Cleland, W.W., 1964, Dithiothreitol, a new protective reagent for SH groups, Biochemistry, 3: 480.PubMedCrossRefGoogle Scholar
  6. Cleland, W.W., 1963, The kinetics of enzyme-catalyzed reactions with two or more substrates or products, B.B.A., 67: 173.Google Scholar
  7. Dahlblom, R., B.R. Tolf, A. Akeson, G. Lundquist, and H. Theorell, 1974, On the inhibitory power of some further pyrazole derivatives of horse liver alcohol dehydrogenase, B.B.R.C., 57: 549.Google Scholar
  8. Dalziel, K., and F.M. Dickinson, 1966, The kinetics and mechanism of liver alcohol dehydrogenase with primary and secondary alcohols as substrates, Biochem. J., 100: 34.Google Scholar
  9. Danielsson, H., 1960, On the oxidation of 3a, 7a, 12a-trihydroxy-coprostane by mouse and rat liver homogenates, Acta Chem. Scand., 14: 348.Google Scholar
  10. Dowd, J.E., and D.S. Riggs, 1965, A comparison of estimates of Michealis-Menten kinetic constants for various linear transformations, J. Biol. Chem., 240: 863.Google Scholar
  11. Hanes, C.S., 1932, Studies on plant amylases. The effect of starch concentration upon the velocity of hydrolysis by amylase of germinated barley, Biochem. J., 1406.Google Scholar
  12. Hawkins, R.D., and H. Kalant, 1972, The metabolism of ethanol and its metabolic effects, Pharmacol. Rev., 24: 67.Google Scholar
  13. Helenius, A., and K. Simons, 1972, The binding of detergents to lipophilic and hydrophilic proteins, J. Biol. Chem., 247: 3656.Google Scholar
  14. Henderson, P.J.F., 1972, A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors, Biochem. J., 127: 321.Google Scholar
  15. Jornvall, H., and R. Pietruszko, 1972, Structural studies of alcohol dehydrogenase from human liver, Eur. J. Biochem., 25: 283.Google Scholar
  16. Khalifah, R.G., and W.M. Sutherland, 1979, Carbon-13 nuclear magnetic resonance studies on liver alcohol dehydrogenase specifically alkylated with bromo-[1–13C]acetate, Biochemistry, 18: 391.PubMedCrossRefGoogle Scholar
  17. Kim, S.K., and P.M. Horowitz, 1975, A study of dithiothreitol inactivation of the enzyme rhodanese, B.B.R.C., 67: 433.Google Scholar
  18. Koen, A.L., and C.R. Shaw, 1966, Retinol and alcohol dehydrogenase in retina and liver, B.B.A., 128: 48.Google Scholar
  19. Kutsky, R.J., 1973, “Handbook of Vitamins and Hormones,” Van Nostrand Reinhold Co., New York.Google Scholar
  20. Mezey, E., and P.R. Holt, 1971, The inhibitory effect of ethanol on retinol oxidation by human liver and cattle retina, Exper. and Molec. Pathol., 15: 148.Google Scholar
  21. Pietruszko, R., H. Theorell, and C.D. Zalenski, 1972, Heterogeneity of alcohol dehydrogenase from human liver, A.B.B., 153: 279.Google Scholar
  22. Plowman, K.M., 1972, “Enzyme Kinetics,” McGraw-Hill, New York. Pocker, Y., and R.R. Miksch, 1979, Spinach carbonic anhydrase:Google Scholar
  23. Mechanism and regulation of enzyme action, Proc. 11th Inter.Congress of Biochem., 04–7-S60.Google Scholar
  24. Reynier, M., 1969, Pyrazole inhibition and kinetic studies of ethanol and retinol oxidation catalyzed by rat liver alcohol dehydrogenase, Acta Chem. Scand., 23: 1119.Google Scholar
  25. Robbins, K.C., 1961, Enzymatic omega oxidation of fatty acids, Fed. Proc. Fed. Amer. Exp. Biol., 20: 272.Google Scholar
  26. Rubin, E., and C.S. Lieber, 1968, Malnutrition and liver disease-an overemphasized relationship, Amer. J. Med., 45: 1.Google Scholar
  27. Segel, I.H., 1975, “Enzyme Kinetics,” John Wiley and Sons, New YorkGoogle Scholar
  28. Shore, J.D., and M.J. Gilleland, 1970, Binding and kinetic studies of liver alcohol dehydrogenase-coenzyme-pyrazole complexes, J. Biol. Chem., 245: 3422.Google Scholar
  29. Sporn, M.B., 1977, Vitamin A and its analogs (retinoids) in cancer prevention, Curr. Concepts Nutr., 6: 119.Google Scholar
  30. Sund, H., and H. Theorell, 1963, Alcohol Dehydrogenases, in: “The Enzymes,” P.D. Boyer, H. Lardy, K. Myrback, eds., Academic Press, New York.Google Scholar
  31. Theorell, H., A.P. Nygaard, and R. Bonnichsen, 1955, Studies on liver alcohol dehydrogenase, Acta Chem. Scand., 9: 1148.Google Scholar
  32. The orell, H., and T. Yonetani, 1963, Liver alcohol dehydrogenaseDPN-pyrazole complex: A model of a ternary intermediate in the enzyme reaction, Biochem. Z., 338: 537.Google Scholar
  33. Wald, G., 1960, The visual function of the vitamins A, Vit. Hormones, 18: 417.Google Scholar
  34. Wald, G., and R. Hubbard, 1949, The reduction of retinene to vitamin Al’ in vitro, J. Gen. Physiol., 32: 367.Google Scholar
  35. Zachman, R.D., and J.A. Olson, 1961, A comparison of retinine reductase and alcohol dehydrogenase of rat liver, J. Biol. Chem., 236: 2309.Google Scholar

Copyright information

© Springer Science+Business Media New York 1980

Authors and Affiliations

  • Y. Pocker
    • 1
  • K. W. Raymond
    • 1
  1. 1.Department of ChemistryUniversity of WashingtonSeattleUSA

Personalised recommendations