Spectroscopic Investigation of Binary and Ternary Coenzyme Complexes of Yeast Alcohol Dehydrogenase

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Abstract
Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes were investigated as a function of temperature. Dissociation constants were obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. The presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme .cntdot. NAD .cntdot. pyrazole complex is accompanied by a large change in the UV absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme .cntdot. NADH .cntdot. acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding were evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.