Active-site sulfhydryl groups of yeast alcohol dehydrogenase

Abstract

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...

Studies of NADP+-preferred secondary alcohol dehydrogenase from Thermoanaerobium brockii

“…Most sulfhydryl ADHs are readily inactivated by carboxymethylation (Li and Vallee 1965;Twu et al 1973;Biellmann et al 1979;Tsai et al 1987). The Thermoanaerobium enzyme was relatively resistant to alkylation by either iodoacetamide or iodoacetate (Fig.…”

“…Two amino acid ligands of the catalytic zinc in horse liver ADH, Cys-46 and His-67 (BrandCn et al 1975), are preserved in TADH (Peretz and Burstein 1989). Although, the catalytic cysteine of ADH is readily deactivated by sulfhydryl reagents (Li and Vallee 1965;Dickinson 1972;Twu et al 1973;Biellmann et al 1979), the active site cysteine of thermophilic ADH from Bacillus stearothermophilus is totally insensitive toward sulfhydryl modifications (Bridgen et al 1973). Our study on chemical modifications of TADH also indicates the presence of the catalytically important histidine and cysteine residues.…”

Covalent binding of acetaldehyde selectively inhibits the catalytic activity of lysine-dependent enzymes

“…Acetaldehyde can also react with sul&ydryl groups, but the complete absence of cysteine residues in glucose-6-phosphate dehydrogenase from L. mesenteroides further implicates Schiff base formation with essential lysyl residues in the inhibitory process (22). Moreover, yeast alcohol dehydrogenase, which depends on sulfhydryl groups for activity (14) was not affected by acetaldehyde adduct formation. These factors suggest that, under the conditions employed here, modification of lysyl residues was the primary mechanism of acetaldehyde-related inhibition.…”

Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol

“…Yeast ADH has cysteine residues (ligated to the zinc in the active site) that are required for enzymatic activity and react especially readily with alkylating agents to destroy activity [35, 36]. Either or both of the two cysteine residues react differentially with chemical agents [37]. Since yeast growing aerobically on YPD do not require ADH, it does not appear that the ADH is inactivated substantially by acrolein in vivo , because if it were, toxicity would be prevented.…”