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

Abstract

The inhibition of the yeast and liver alcohol dehydrogenases' by chelating agents is accompanied by progressive changes in optical rotation.2' 3 Such alterations of the optical rotatory properties of yeast alcohol dehydrogenase are due to the binding of chelating' agents to the asymmetric zinc site of the enyme resulting in a characteristic Cotton effect. The rotatory power of this Cotton effect, when superimposed upon the background dispersion curve of the native protein, accounts for the rotational changes which appear during the course of enzymatic inhibition, and these changes can be differentiated from those due to alterations in protein conformation.4 Such observations suggested that changes in optical rotatory dispersion might be a frequent concomitant of the binding of small molecules to proteins and might, thereby, be used to identify the interactions with enzymes of optically active, chromophoric substrates, coenzymes, inhibitors, and other site specific reagents.4 To this end we have investigated the alcohol dehydrogenase of horse liver. This zinc containing enzyme5' 6 interacts stoichiometrically with two moles of coenzyme, DPN(H), to form the enzymatically holoenzyme7 and is inhibited by a number of chelating agents.' Zinc is not removed during chelation nor does the enzyme dissociate into subunits by electrophoresis or ultracentrifugation. 1,10phenanthroline, OP, reversibly inhibits the enzyme by chelation of the zinc atom forming a mixed complex: LADH-Zn OP.9 The coenzyme binds in a specific though as yet unknown manner at or near the zinc site of the enzyme. A variety of approaches indicate that zinc is a component of the active center of liver alcohol dehydrogenase.'I The present report demonstrates that the binding of reduced diphosphopyridine nucleotide to liver alcohol dehydrogenase is accompanied by the appearance of a pronounced Cotton effect at 327 m,. The magnitude of this Cotton effect is a direct function of the molar ratio of the interactants, thereby permitting a quantitative assessment of coenzyme binding. Moreover, characteristic changes in the optical rotation of the enzyme-coenzyme complex identify its interaction with substrate and inhibitors. Similar effects are observed when the analogues of DPNH bind to liver alcohol dehydrogenase. The considerations pertinent to the choice of metalloenzymes for the exploration of the molecular basis of enzyme action have been discussed3 and preliminary accounts of the present studies have been reported.2' 13 Methods and Materials.-Crystalline alcohol dehydrogenase of horse liver was obtained from C. F. Boehringer and Soehne, Mannheim, W. Germany. Prior to use, the enzyme was dialyzed for 5 days against 0.1M phosphate buffer, pH 7.5 at 4? to remove low molecular weight impurities which absorb radiation at 280