Epoxide Inhibition of Alcohol Dehydrogenases. Identification of Modified Cysteines in Yeast Alcohol Dehydrogenase and Demonstration of Reversible and Irreversible Inhibition of Liver Alcohol Dehydrogenase by Styrene oxide

Judith P. Klinman, Katherine M. Welsh, Ruth Hogue-Angeletti

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Abstract

The inactivation of yeast alcohol dehydrogenase by styrene oxide leads to the simultaneous alkylation of two cysteines per subunit (Klinman, J. P. (1975), Biochemistry 14, 2568). The amino acid composition of one of two major radioactive peptides, obtained by tryptic digestion of enzyme alkylated with tritiated styrene oxide (StyO), indicates that cysteine-43 has been modified. Subsequent digestion of the second labeled tryptic peptide with pepsin leads to an octapeptide (StyO-Cys-Ala-Gly-Ile-Thr-Val-Tyr-Lys) which corresponds to positions 152-160 in the protein sequence. These results establish that styrene oxide alkylates cysteine-43 and -152, two of the three residues implicated as ligands for an active-site zinc. Horse liver alcohol dehydrogenase is found to be slowly inactivated by styrene oxide, t1/2 = 7.8 h at pH 8.0, 25 °C, 10 mM styrene oxide. In analogy with the yeast enzyme, inactivation is accompanied by the incorporation of 2 mol of styrene oxide per subunit, and prior alkylation with iodoacetate reduces this stoichiometry to 1 mol per subunit. The observation of saturation kinetics for inactivation in the presence of 1 mM NADH (Ki = 9.6 mM) is attributed to “nonproductive” binding by styrene oxide, since second-order rate constants for inactivation are essentially the same both in the presence and absence of NADH, k = 2.0-2.4 × 10-3 M-1 s-1. The horse liver alcohol dehydrogenase catalyzed reduction of acetaldehyde by NADH is reversibly inhibited by styrene oxide in a noncompetitive manner. The binding of styrene oxide to enzyme-NADH (obtained from slope replots) is observed to be biphasic, indicating inhibitor constants of Ki′(slope) = 1.2 mM and Ki′(slope) = 4.6 mM at pH 8.6; the similarity between Ki′(slope) and the Ki derived from enzyme inactivation suggests that the lower affinity epoxide binding site competitive with acetaldehyde is responsible for the observation of saturation kinetics in enzyme inactivation. Ki(slope) is essentially unchanged at 1.0-1.1 mM in the pH range 6.0-9.1, whereas the binding of styrene oxide to enzyme-NAD+ (obtained from intercept replots) increases from 1.2 mM at pH 6.0 to 4.8 mM at pH 9.1. These differences in the magnitude and pH dependence of Ki(intercept) vs. Ki(slope) are attributed to a stabilization of bound epoxide by hydrogen bonding to the conjugate acid of an active-site functional group of pK ≃ 7.8 in the enzyme-NAD+ complex vs. pK > 9 in the enzyme-NADH complex.

Original languageEnglish (US)
Pages (from-to)5521-5527
Number of pages7
JournalBiochemistry
Volume16
Issue number25
DOIs
StatePublished - Dec 1 1977

ASJC Scopus subject areas

  • Biochemistry

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