Complexes of liver alcohol dehydrogenase. Further studies on the rate of inactivation

• Published in: European journal of biochemistry Vol. 14; no. 1; pp. 14 - 26
• Main Authors: Reynolds, C H, Morris, D L, McKinley-McKee, J S
• Format: Journal Article
• Language: English
• Published: England 01.05.1970
• Subjects: Adenine Nucleotides; Alcohol Oxidoreductases - antagonists & inhibitors; Animals; Binding Sites; Chelating Agents; Coenzymes; Halogens; Horses; Hydrocarbons; Hydrogen-Ion Concentration; Imidazoles; Iodoacetates; Kinetics; Liver - enzymology; Mathematics; NAD; Osmolar Concentration; Phenanthrolines; Protein Binding; Ribose; Zinc
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1970.tb00255.x

The Michaelis‐Menten‐type kinetics of inactivation of horse‐liver alcohol dehydrogenase with iodoacetate have been used to study the binding of a number of known ligands. Adenine nucleotides protect competitively with iodoacetate as do halides and aromatic acids. Uncharged chelating‐agents (orthophenanthroline, and 2,2′‐bipyridyl) protect non‐competitively. Uncharged monodentate ligands ( e. g. imidazole) stimulate inactivation. The effect of imidazole on the binding of various ligands at pH 7.4 and at pH 10 was studied. Multiple inhibition analysis was used to examine the mutual interaction of pairs of substances which protected the enzyme from iodoacetate. ADP‐ribose and decanoate were mutually competitive, while AMP and orthophenanthroline were non‐competitive. Increasing the ionic strength was found to affect differently the mutual interactions of pairs of anions, as well as to weaken their binding to the enzyme. In some cases, kinetic studies of reduction of NAD + were used to complement the inactivation studies. It is concluded that, in the coenzyme, the phosphate group nearer to adenosine is important for binding. Most, but not all, of the ligand‐ligand interactions can be explained by steric or electrostatic effects; it is tentatively suggested that conformational changes may also be involved. Precise interpretation, however, is complicated: there are two different types of zine in the enzyme, and anions might bind to either, or neither.