期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1981
卷号：78
期号：10
页码：6221-6225
DOI：10.1073/pnas.78.10.6221
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
