Site Of Horse Liver Alcohol Dehydrogenase Research Articles

Zinc(II) N2S2 Schiff-base complexes incorporating pyrazole: syntheses, characterization, tautomeric equilibria and racemization kinetics

O. P. Anderson, A. la Cour, M. Findeisen, L. Hennig, O. Simonsen, L. F. Taylor and H. Toftlund, J. Chem. Soc., Dalton Trans., 1997, 111 DOI: 10.1039/A603158G

Metal ion substitution at the catalytic site of horse-liver alcohol dehydrogenase: results from solvent magnetic relaxation studies. 2. Binding of manganese(2+) and competition with zinc(2+) and cadmium(2+) ions

The interaction of Mn2+ aquo ions with native horse-liver alcohol dehydrogenase demetalized specifically at the catalytic sites has been investigated by studying the magnetic field dependence and time dependence of the magnetic spin-lattice relaxation rate of solvent water protons. We find no detectable binding of Mn2+ ions to the catalytic sites in times on the order of hours; however, we do find that these ions bind to the enzyme at two previously unreported types of sites: one, characterized by a low dissociation constant (0.01 mM at pH 7.7, 5 degrees C), low relaxivity, and a stoichiometry of one per two catalytic sites, and a second, with a high dissociation constant (1.5 mM at pH 7.7, 5 degrees C) and high relaxivity. The stoichiometry of the second type of site could not be determined because of the relatively weak bindng of Mn2+ ions to these sites. Both Zn2+ and Cd2+ ions bind to the newly found tight-binding sites, displacing Mn2+ ions and thereby altering the relaxation rates of solvent protons. By monitoring the return to equilibrium of these altered rates, we find that Zn2+ ions enter the catalytic sites from the new tight-binding sites with an on-rate of approximately 0.1 M-1 s-1. It is not clear whether binding to these new sites is an obligatory intermediate for reintroduction of Zn2+ ions into the catalytic sites, but a small excess of Zn2+ ions beyond one per monomer causes the protein to precipitate. Cd2+ ions, by contrast, enter the catalytic sites at least 1 order of magnitude more rapidly than do Zn2+ ions, a rate too rapid to observe by our techniques. However, once the catalytic sites are filled, Cd2+ ions displace Mn2+ ions at the new sites as do Zn2+ ions.

Metal ion substitution at the catalytic site of horse-liver alcohol dehydrogenase: results from solvent magnetic relaxation studies. 1. Copper(II) and cobalt(II) ions.

Metal ion substitution at the catalytic site of horse-liver alcohol dehydrogenase: results from solvent magnetic relaxation studies. 1. Copper(II) and cobalt(II) ions.