Interactions of substrates, inhibitors, and coenzymes at the active site of horse liver alcohol dehydrogenase.

2,2-Bipyridine chelates 2 zinc ions of horse liver alcohol dehydrogenase with a dissociation constant of 4.0 X low4 M. The complex shows an absorption maximum at 308 rnp with a difference extinction coefficient of 1 .l X lo4 M+ cm-r per zinc ion. Because bipyridine binds the enzyme less tightly than o-phenanthroline, and because the principal absorption maximum of the difference spectrum appears at longer wave lengths, this reagent is preferred as a tool in the determination of the binding constants of inhibitors, substrates, and coenzymes. The dissociation constants for binary complexes of enzyme with alcohols, aldehydes, mercaptans, carboxylic acids, and amides, as well as with imidazole and pyrazole, have been determined by measuring the diminution that they cause in the intensity of the enzyme-bipyridine spectrum. The competitive inhibitors ADP-ribose, AMP, ADP, and 4-biphenylcarboxylic acid do not interfere with the enzymebipyridine spectrum, whereas the coenzymes NAD+ and NADH diminish this spectrum. These results confirm the conclusion, previously advanced by Yonetani, that the nicotinamide moiety of the coenzyme lies near the zinc ion of the active site, and that this zinc ion is part of the substrate-binding site. Ternary complexes of enzyme, coenzyme, and inhibitor have been detected from the changes in enzyme-bipyridine spectrum; these complexes therefore require the enzymic zinc ion at the active site, or form near it, or both.