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. 1981 Oct;78(10):6221–6225. doi: 10.1073/pnas.78.10.6221

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

M W Makinen, M B Yim
PMCID: PMC349010  PMID: 6273859

Abstract

The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (EPR) methods with use of the active-site-specific Co2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme--NAD+ complex shows approximate axial symmetry of the metal ion site, indicating that binding of the coenzyme induces a structural alteration in the active-site region. This environment is not significantly altered further by binding of the competitive inhibitor pyrazole. To assign the coordination number of the active-site metal ion, the zero-field splitting was determined on the basis of the temperature dependence of the spin--lattice relaxation of the Co2+ ion. The zero-field splitting energies are approximately 9 cm-1 for the free Co2+-reconstituted enzyme and approximately 46 and approximately 47 cm-1 for the enzyme--NAD+ and enzyme--NAD+--pyrazole complex, respectively. On the basis of studies of structurally defined small molecule complexes, these values are compatible with a tetracoordinate metal ion in the active site of the free enzyme but a pentacoordinate metal ion in the binary enzyme--NAD+ complex and in the ternary enzyme--NAD+--inhibitor complex and, therefore, presumably also in the catalytically active ternary enzyme--NAD+--alcohol complex formed in the course of alcohol oxidation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aasa R., Hanson M., Lindskog S. Low temperature magnetic susceptibility of a human Co(II) carbonic anhydrase B sulphonamide complex. Biochim Biophys Acta. 1976 Nov 26;453(1):211–217. doi: 10.1016/0005-2795(76)90266-x. [DOI] [PubMed] [Google Scholar]
  2. Argos P., Garavito R. M., Eventoff W., Rossmann M. G., Brändén C. I. Similarities in active center geometries of zinc-containing enzymes, proteases and dehydrogenases. J Mol Biol. 1978 Dec 5;126(2):141–158. doi: 10.1016/0022-2836(78)90356-x. [DOI] [PubMed] [Google Scholar]
  3. Bignetti E., Rossi G. L., Zeppezauer E. Microspectrophotometric measurements on single crystals of coenzyme containing complexes of horse liver alcohol dehydrogenase. FEBS Lett. 1979 Apr 1;100(1):17–22. doi: 10.1016/0014-5793(79)81122-9. [DOI] [PubMed] [Google Scholar]
  4. Boiwe T., Bränden C. I. X-ray investigation of the binding of 1,10-phenanthroline and imidazole to horse-liver alcohol dehydrogenase. Eur J Biochem. 1977 Jul 1;77(1):173–179. doi: 10.1111/j.1432-1033.1977.tb11655.x. [DOI] [PubMed] [Google Scholar]
  5. Cook P. F., Cleland W. W. pH variation of isotope effects in enzyme-catalyzed reactions. 2. Isotope-dependent step not pH dependent. Kinetic mechanism of alcohol dehydrogenase. Biochemistry. 1981 Mar 31;20(7):1805–1816. doi: 10.1021/bi00510a015. [DOI] [PubMed] [Google Scholar]
  6. Desideri A., Morpurgo L., Raynor J. B., Rotilio G. An electron spin resonance study of high spin forms of cobalt(II) bovine carbonic anhydrase. Biophys Chem. 1978 Sep;8(4):267–270. doi: 10.1016/0301-4622(78)80009-x. [DOI] [PubMed] [Google Scholar]
  7. Dworschack R. T., Plapp B. V. pH, isotope, and substituent effects on the interconversion of aromatic substrates catalyzed by hydroxybutyrimidylated liver alcohol dehydrogenase. Biochemistry. 1977 Jun 14;16(12):2716–2725. doi: 10.1021/bi00631a020. [DOI] [PubMed] [Google Scholar]
  8. Eklund H., Nordström B., Zeppezauer E., Söderlund G., Ohlsson I., Boiwe T., Söderberg B. O., Tapia O., Brändén C. I., Akeson A. Three-dimensional structure of horse liver alcohol dehydrogenase at 2-4 A resolution. J Mol Biol. 1976 Mar 25;102(1):27–59. doi: 10.1016/0022-2836(76)90072-3. [DOI] [PubMed] [Google Scholar]
  9. Eklund H., Samma J. P., Wallén L., Brändén C. I., Akeson A., Jones T. A. Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 A resolution. J Mol Biol. 1981 Mar 15;146(4):561–587. doi: 10.1016/0022-2836(81)90047-4. [DOI] [PubMed] [Google Scholar]
  10. Horrocks W. D., Jr, Ishley J. N., Holmquist B., Thompson J. S. Structural and electronic mimics of the active site of cobalt(II)-substituted zinc metalloenzymes. J Inorg Biochem. 1980 Apr;12(2):131–141. doi: 10.1016/s0162-0134(00)80124-5. [DOI] [PubMed] [Google Scholar]
  11. Kvassman J., Pettersson G. Effect of pH on the process of ternary-complex interconversion in the liver-alcohol-dehydrogenase reaction. Eur J Biochem. 1978 Jun 15;87(2):417–427. doi: 10.1111/j.1432-1033.1978.tb12391.x. [DOI] [PubMed] [Google Scholar]
  12. Kvassman J., Pettersson G. Unified mechanism for proton-transfer reactions affecting the catalytic activity of liver alcohol dehydrogenase. Eur J Biochem. 1980 Feb;103(3):565–575. doi: 10.1111/j.1432-1033.1980.tb05981.x. [DOI] [PubMed] [Google Scholar]
  13. Maret W., Andersson I., Dietrich H., Schneider-Bernlöhr H., Einarsson R., Zeppezauer M. Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state. Eur J Biochem. 1979 Aug 1;98(2):501–512. doi: 10.1111/j.1432-1033.1979.tb13211.x. [DOI] [PubMed] [Google Scholar]
  14. Plapp B. V., Eklund H., Brändén C. I. Crystallography of liver alcohol dehydrogenase complexed with substrates. J Mol Biol. 1978 Jun 15;122(1):23–32. doi: 10.1016/0022-2836(78)90105-5. [DOI] [PubMed] [Google Scholar]
  15. Shore J. D., Theorell H. Substrate inhibition effects in the liver alcohol dehydrogenase reaction. Arch Biochem Biophys. 1966 Nov;117(2):375–380. doi: 10.1016/0003-9861(66)90425-5. [DOI] [PubMed] [Google Scholar]
  16. Sloan D. L., Young J. M., Mildvan A. S. Nuclear magnetic resonance studies of substrate interaction with cobalt substituted alcohol dehydrogenase from liver. Biochemistry. 1975 May 6;14(9):1998–2008. doi: 10.1021/bi00680a030. [DOI] [PubMed] [Google Scholar]
  17. Theorell H., Tatemoto K. The transient state kinetics of horse liver alcohol dehydrogenase. Acta Chem Scand. 1970;24(8):3069–3070. doi: 10.3891/acta.chem.scand.24-3069. [DOI] [PubMed] [Google Scholar]
  18. WRATTEN C. C., CLELAND W. W. PRODUCT INHIBITION STUDIES ON YEAST AND LIVER ALCOHOL DEHYDROGENASES. Biochemistry. 1963 Sep-Oct;2:935–941. doi: 10.1021/bi00905a007. [DOI] [PubMed] [Google Scholar]
  19. Zarem H. A., Gray G. F., Jr, Morehead D., Edgerton M. T. Heterotopic brain in the nasopharynx and soft palate: report of two cases. Surgery. 1967 Mar;61(3):483–486. [PubMed] [Google Scholar]

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