The Site of Auramine O Binding to Horse Liver Alcohol Dehydrogenase

In agreement with an earlier report (Conrad, R. H., Heitz, J. R., and Brand, L., Biochemistry, 9, 1540 (1970)), auramine O was found to bind to horse liver alcohol dehydrogenase at two equivalent noninteracting sites per enzyme molecule, with an intrinsic dissociation constant Kr = 1.65 x 10-5 m in 0.05 m phosphate buffer at pH 7.0 at 25°. The dye was displaced competitively from the enzyme by substrates (ethanol and 1-butanol), substrate inhibitors (decanoic acid, imidazole, and pyrazole), and by inhibitors binding to the zinc ion presumed to be at the active site (1,10-phenanthroline and 2,2-bipyridine). The dye forms ternary complexes, with comparable affinity, with the binary complexes of the enzyme with NAD+, NADH, and adenosine diphosphoribose. In all three cases, the dissociation constant of auramine O from the ternary complex is significantly lower than the dissociation constant for the enzyme-auramine O binary complex. On the basis of the noncompetitive inhibition of liver alcohol dehydrogenase by auramine O in initial rate studies, Conrad et al. suggested that the dye binding site is distinct from the coenzyme and substrate binding sites. This interpretation is incompatible with the data summarized above. The apparent noncompetitive inhibition kinetic data are shown here to be the consequence of the unique ability of the dye to form ternary complexes with the enzyme-NAD+ and enzyme-NADH binary complexes. Thus, the auramine O-alcohol dehydrogenase interaction offers an additional case to the general correlation that most dyes which bind tightly to enzymes interact at regions overlapping binding sites for the normal ligands.