Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid

Chadha, V. K.; Plapp, Bryce V.

doi:10.1021/bi00297a007

Cited by 12 publications

(7 citation statements)

References 44 publications

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“…Hence, there is little effect of the substitution of the basic residues His in ß2 for Arg in ßi on iodoace tate binding at the active site of ADH. Exam ination of the specificity of inactivation of ßißi and ß2ß2 by iodoacetate indicates that a single Cys-174 is selectively and stoichiometrically alkylated [53], In a recent study, Chadha and Plapp [51] reported that Cys-174 of horse liver ADH is modified by the halo acid 3-bromopropionate. They pro posed that the carboxyl group of 3-bromopropionate interacts with the guanidino group of Arg-369 to form a MichaelisMenten complex which facilitates the alkyl ation of Cys-174.…”

Section: Inhibitors and Activators Of Human Liver Adh Isoenzymesmentioning

confidence: 99%

Catalytic Properties of Human Liver Alcohol Dehydrogenase Isoenzymes

Bosron¹,

Li²

1987

Enzyme

112

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Human liver alcohol dehydrogenase (ADH) exists in multiple molecular forms which arise from the association of eight different types of subunits, a, ß(1), ß(2), ß(3), y(1), y(2), π, and X, into active dimeric molecules. A genetic model accounts for this multiplicity as products of five gene loci, ADH(1) through ADH(5). Polymorphism occurs at two loci, ADH(2) and ADH(3), which encode the ß and y subunits. All of the known homodimeric and heterodimeric isoenzymes have been isolated and purified to homogeneity. Analysis of the steady-state kinetic properties and substrate and inhibitor specificities has shown substantial differences in the catalytic properties of the isoenzymes. For example, the Km values for NAD+ and ethanol vary as much as 1,000-fold among the isoenzymes. Some of the differences in catalytic properties can be related to specific amino acid substitutions in the ADH isoenzymes.

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Section: Inhibitors and Activators Of Human Liver Adh Isoenzymesmentioning

confidence: 99%

Catalytic Properties of Human Liver Alcohol Dehydrogenase Isoenzymes

Bosron¹,

Li²

1987

Enzyme

112

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“…A variety of cysteine-alkylating reagents have been used to investigate the structure and catalysis mechanism of HLADH. Cys 46 and Cys 174, both zinc ligands in the active site in HLADH, are selectively alkylated by iodoacetate and 3-bromopropionate, respectively, with almost complete loss of activity (Harris, 1964;Li & Vallee, 1963;Chadha & Plapp, 1984). The mechanism of inactivation of HLADH by halo acids has been established by kinetic studies (Reynolds & McKinley-McKee, 1969;Dahl & McKinley-McKee, 1981a;Chadha & Plapp, 1984) and indicates that a high-affinity, reversible enzyme inhibitor Michaelis-Menten complex is formed prior to covalent modification.…”

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confidence: 99%

Activation of Sulfolobus solfataricus Alcohol Dehydrogenase by Modification of Cysteine Residue 38 with Iodoacetic Acid

et al. 1996

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Reaction of thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus with iodoacetate at pH 9.0 and 37 degrees C significantly increases the oxidation rate of aliphatic and aromatic alcohols and decreases the reduction rate of aromatic aldehydes. The archaeal ADH is chemically modified and activated in a Michaelis-Menten-type reaction, where one molecule of the reagent binds per active site. NAD+ in micromolar concentration protects the enzyme against the inhibitor in an uncompetitive manner, while imidazole significantly increases the extent of the activation. Carboxymethylation selectively modifies one out of five cysteine residues per subunit, namely, Cys 38, located in the catalytic site, as determined by peptide and sequence analysis, and enhances by up to 25-fold the oxidation rate of benzyl alcohol. Carboxymethylated SsADH is less thermostable and shows a temperature optimum 30 degrees C lower than that of the native enzyme. The carboxymethylated enzyme exhibits a lower affinity toward the oxidized and reduced coenzyme. The dissociation constants for NAD+ and NADH determined at 25 degrees C and pH 8.8 are 60- and 200-fold higher, respectively, compared to the native enzyme. The significant isotope effect in alcohol oxidation suggests that hydride transfer partially limits the turnover rate of the reaction catalyzed by the modified enzyme, whereas the rate-limiting step for the native enzyme is NADH dissociation. Carboxymethylated enzyme probably gives higher maximum velocities of oxidation because of the faster dissociation of the modified enzyme-coenzyme complex.

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“…This finding agrees with reports on other ADHs, in which the cysteine ligand@) of the active site zinc were carboxymethylated in the native enzyme. Carboxymethylation of Cys 46 or Cys 174 of HLADH (Li & Vallee, 1963;Harris, 1964;Chadha & Plapp, 1984), Cys 43 of sorbitol dehydrogenase (Jeffery et al, 1981), and Cys 37 of TBADH (this study) abolished the catalytic activity of all three enzymes. In S. sulfaturicus, on the other hand, alkylating Cys 38 enhanced the catalytic activity of ADH (Raia et al, 1996).…”

Section: Discussionmentioning

confidence: 47%

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase

1997

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The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis( l-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[ 14C]iodoacetic acid, via S-alkylation.The respective reactions were monitored by electron paramagnetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline.This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in EWLC. The EPR data suggested a distance of 510 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 "C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.

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Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid

Cited by 12 publications

References 44 publications

Catalytic Properties of Human Liver Alcohol Dehydrogenase Isoenzymes

Catalytic Properties of Human Liver Alcohol Dehydrogenase Isoenzymes

Activation of Sulfolobus solfataricus Alcohol Dehydrogenase by Modification of Cysteine Residue 38 with Iodoacetic Acid

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase

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