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A B s m A c r : Yeast alcohol dehydrogenase has previously been shown to contain two distinct active-site sulfhydryl groups, one which reacts specifically with iodoacetate (and by inference also with iodoacetamide) and one which reacts with butyl isocyanate. The characteristics of the reactions of these sulfhydryl groups with butyl isocyanate and with iodoacetamide and some properties of the inactive derivatives have been studied further. The inactivation of dehydrogenase by the two reagents was compared in parallel experiments and found to be affected very similarly by protective agents such as NAD+, NADH, NAD+ + pyrazole, NADH + acetamide.The two sulfhydryl groups per active site cannot both be modified; when one has reacted, the other becomes unreactive towards either reagent, and it is concluded that the two sulfhydryl groups are closely associated in the active site. The reaction with iodoacetamide at p H 6.5 was found to involve both sulfhydryl groups, thus providing further evidence for Y east alcohol dehydrogenase is a tetramer made up of four identical or very similar subunits (Pfleiderer and Auricchio, 1964; Harris, 1964a). Based on coenzyme binding (Hayes and Velick, 1954), on Zn binding (Kagi and Vallee, 1960), and on the specific inactivation of the enzyme by the reaction of 4 mol of sulfhydryl groups/mol of enzyme (Whitehead and Rabin, 1964), it has been concluded that the tetramer contains four active sites. Dickinson (1970), however, showed that the ternary NADH-acetamide-enzyme complex only formed with three of the active sites, and consequently suggested that negative interaction between sites may make the fourth site essentially inoperative in the normal action of the enzyme. We recently reported (Twu and Wold, 1973) that butyl isocyanate acts as an active-site-specific reagent for yeast alcohol dehydrogenase and in support of Dickinson's hypothesis found that the enzyme was completely inactivated after the incorporation of only 3 mol of reagent/@ of enzyme. The butylcarbamoylation was specific for sulfhydryl groups, but rather surprisingly the isolated butylcarbamoylated peptide wqs found to be different from that isolated from dehydrogenase inactivated with iodoacetate (Harris, 1964b). Thus, it was proposed that each of the active sites of yeast alcohol dehydrogenase contains two reactive sulfhydryl groups, one which reacts preferentially with butyl isocyanate and appears to reflect the proposed anticooperative interaction (for clarity of discussion this group will be given the designation X) and one which reacts preferentially with iodoacetate and which appears not to be affected by subunit interaction (designated Y ) . the existence of two distinct "essential" sulfhydryl groups, and also establishing that the specificity of a given reagent for a given sulfhydryl group is not absolute. Some spectral properties of binary and ternary coenzyme and analog complexes of inactive butylcarbamoyl-dehydrogenase and carboxamidomethyl-dehydrogenase were determined and compared to those of the active enz...

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