ANOMALOUS ROTATORY DISPERSION OF ENZYME COMPLEXES, II. THE ASYMMETRIC BINDING OF COENZYMES AND INHIBITORS TO LIVER ALCOHOL DEHYDROGENASE

Abstract

The binding of reduced diphosphopyridine nucleotide and its analogues to liver alcohol dehydrogenase is accompanied by the appearance of pronounced Cotton effects at the wavelengths of maximal absorption of the enzyme-coenzyme (analogue) complexes. The magnitudes of these effects are functions of the molar ratios of the inter-actants; this phenomenon is the basis of a new method to measure the stoichiometry of binding of small chromophoric molecules to proteins: Rotatory Dispersion Titration. A chelating inhibitor, 1,10-phenanthroline also induces a cotton effect by forming a mixed complex with the active zinc site of the enzyme: LADH [image] Zn [image] OP. The competition of this agent with DPNH can be shown spectropolarimetrically. The anomalous rotatory dispersion of these enzyme-coenzyme and enzyme-inhibitor complexes demonstrates the asymmetric nature of interactions at the active enzymatic site. Such studies provide an approach both to the three-dimensional structure of the active center and to investigation of the mechanisms of enzymatoc sterospecificity.