The Use of Biochemical Solid‐Phase Techniques in the Study of Alcohol Dehydrogenase

Abstract

Iodoacetate is known to inhibit horse liver alcohol dehydrogenase by selectively reacting with Cys‐46 in each of the two active sites of the dimeric enzyme. In this study, a method is described in which horse liver alcohol dehydrogenase is carboxymethylated while the enzyme is strongly bioaffinity‐bound to N 6 ‐[ N ‐(6‐aminohexyl)carbamolymethyl]‐NADH‐substituted Sepharose 2B as a ternary complex in the presence of isobutyramide. A low degree of coenzyme‐ligand substitution (about 20 nmol/ml settled gel) was used in order to restrict enzyme‐coenzyme interaction on the gel to a single subunit. After the modification reaction by iodoacetate on the solid phase, the enzyme was eluted from the NADH‐substituted gel and its properties studied in free solution. Based on the incorporation of [ 14 C]carboxymethyl groups, it is concluded that the reaction takes place at the active center of the ‘free’ (not bioaffinity‐bound) subunit; amino acid analysis showed that S ‐carboxymethylcysteine had been formed. Also, the monoalkylated enzyme preparation was half as active as native enzyme when ethanol or benzaldehyde were used as substrates. Furthermore, after alkylation on the solid phase the enzyme loses about 50% of its original NADH‐binding capacity which was determined fluorometrically by titrating with NADH in the presence of isobutyramide. From these results it is suggested that both subunits in native horse liver alcohol dehydrogenase are kinetically equivalent.