Kinetic transients in the reduction of aldehydes catalysed by liver alcohol dehydrogenase.

The transient-state kinetics of enzymic reduction of acetaldehyde and benzaldehyde by NADH, catalyzed by horse liver alcohol dehydrogenase, have been examined under single-turnover conditions, obtained by carrying out reactions either with limiting amounts of enzyme in the presence of 20 mM pyrazole or with limiting amounts of substrate. Analysis of the variation with substrate, coenzyme, and enzyme concentrations of amplitudes and time constants for the exponential transients observed at 328 nm and 300 nm shows that the kinetics of enzymic aldehyde reduction are qualitatively and quantitatively consistent with the relationships derived in the preceding paper for an ordered ternary-complex mechanism involving identical and independent catalytic sites. It is concluded that there is no evidence whatsoever for the kinetic significance of a half-of-the-sites reactivity or any other kind of subunit interaction in the liver alcohol dehydrogenase system. The biphasic transients observed at 328 nm for the reduction of aromatic aldehydes such as benzaldehyde are a normal kinetic characteristic of the ordered ternary-complex mechanism, being attributable to accumulation of the ternary enzyme-NAD-product complex when product dissociation from this complex is slow in comparison to its formation by ternary-complex interconversion.     

Conversion of green note aldehydes into alcohols by yeast alcohol dehydrogenase

Green note aldehydes were successfully reduced into their corresponding alcohol by commercial yeast alcohol dehydrogenase. Among different yeasts tested for their ability to convert (Z)-3-hexenal into (Z)-3-hexenol, Pichia anomala gave the best results. Conversion yields higher than 90% were also obtained by directly conducting the reaction in the medium where (Z)-3-hexenal is produced by the action of lipoxygenase and hydroperoxide lyase on linolenic acid.     

Catalytic properties of human liver alcohol dehydrogenase isoenzymes

Human liver alcohol dehydrogenase (ADH) exists in multiple molecular forms which arise from the association of eight different types of subunits, alpha, beta 1, beta 2, beta 3, gamma 1, gamma 2, pi, and chi, into active dimeric molecules. A genetic model accounts for this multiplicity as products of five gene loci, ADH1 through ADH5. Polymorphism occurs at two loci, ADH2 and ADH3, which encode the beta and gamma 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.     

Oxidation and reduction of 4-hydroxyalkenals catalyzed by isozymes of human alcohol dehydrogenase

4-Hydroxyalkenals, natural cytotoxic products of lipid peroxidation, are substrates for human alcohol dehydrogenases (ADH). Class I and II ADHs reduce aliphatic 4-hydroxyalkenals with chain lengths of from 5 to 15 carbons at pH 7 with kcat and Km values comparable to simple aliphatic aldehydes of the same chain length. Class II is particularly effective in the reduction with kcat values as high as 3300 min-1 for 4-hydroxyundecenal. Class III ADH is essentially inactive toward all of these substrates. The class I and II isozymes also catalyze the oxidation of the 4-hydroxy group at pH 10. However, during the reaction, an NAD(+)-dependent irreversible partial inactivation of the alpha beta 1 isozyme is observed which is attributed, with the aid of computer graphics modeling, to selective modification of the alpha subunit. Both ethanol and 1,10-phenanthroline, known to compete with conventional substrates, instantaneously, reversibly, and competitively inhibit 4-hydroxyalkenal reduction and oxidation, indicating that 4-hydroxyalkenals bind at the same site as do conventional substates. The fact that the class II enzyme pi pi-ADH so far is found only in the liver and that the 4-hydroxyalkenals are the best substrates known for this isozyme suggest that it may play a significant role in cellular defenses in the conversion of the cytotoxic aldehydes to the less reactive alcohols.     

Thermodynamics of the reduction of NADP with 2-propanol catalyzed by an NADP-dependent alcohol dehydrogenase

The apparent equilibrium constant of the biochemical reaction, 2-propanol+NADP(ox) = acetone+NADP(red), was determined at I = 0.25 M over a wide range of pH (5.63 to 8.02) and temperature (5 to 40 degrees C). The reaction was catalyzed by an NADP-dependent alcohol dehydrogenase. The results were used to calculate thermodynamic quantities for the chemical (ionic) reference reaction: 2-propanol+NADP(ox)(3-) = acetone+NADP(red)(4-)+H(+). The thermodynamic quantities for this reference reaction are as follows: equilibrium constant K = (5.98+/-0.46) x 10(-10); standard molar Gibbs energy change Delta(r)G(0) = (52.65+/-0.19) kJmol(-1); standard molar enthalpy change Delta(r)H(0) = (38.9+/-0.6) kJmol(-1); and standard molar entropy change Delta(r)S(0) = -(46.1+/-2.2)J K(-1)mol(-1). All of these results pertain to 25 degrees C (298.15 K) and I = 0. The results also lead, in conjunction with tabulated thermodynamic quantities, to the standard electromotive force E(0) = -0.140 V for the reduction of NADP(ox)(3-) to NADP(red)(4-).     

Catalytic properties of alcohol dehydrogenase isozymes specified by duplicate genes in the diploid plant Stephanomeria exigua

The alcohol dehydrogenase (ADH) isozymes induced in flooded roots of the diploid plant Stephanomeria exigua are specified by tightly linked genes comprising a complex locus, Adh1. Individuals homozygous for a complex with two active genes which specify electrophoretically different subunits have three ADH-I isozymes, two intragenic homodimers and an intergenic heterodimer. Individual isozymes were partially purified from plants homozygous for several different Adh1 complexes and apparent K _m values for acetaldehyde, ethanol, NAD, and NADH and responses to temperature, pH, and two different alcohols were determined. The two homodimeric enzymes specified by a particular Adh1 complex generally differed in one or more of the properties studied, and in three of four cases, intergenic heterodimers differed significantly from intermediacy, often having lower K _m values than either homodimer. None of the isozymes studied could be considered greatly divergent or defective. Constraints on evolution of duplicate genes which form intergenic heterodimers are considered.     

Catalytic and spectroscopic characterisation of a copper-substituted alcohol dehydrogenase from yeast

Yeast alcohol dehydrogenase (Y-ADH) is widely studied for its biotechnological importance and various attempts to improve its catalytic properties have been made. In this paper, a catalytically active metal-substituted Y-ADH was prepared in vitro by substituting one zinc atom with copper. EPR and Raman spectroscopy suggest that copper maintains the same co-ordination geometry as zinc in native Y-ADH. The active Cu-ADH shows lower substrate affinity and lower specific activity (SA) than native ADH, but greater than a previously obtained Co-ADH. Furthermore, Cu-ADH maintains its catalytic efficiency in a wider pH range than native enzyme.     

Catalytic properties and active-site structural features of immobilized horse liver alcohol dehydrogenase

Alcohol dehydrogenase from horse liver was immobilized by covalent attachment to CNBr-Sepharose and by adsorption to octyl-Sepharose CL-4B, a hydrophobic analog of Sepharose. In each case, rate constants for the binding and release of coenzyme and for the oxidation of substrates were measured based on the concentration of accessible active-site zinc atoms determined by titration with a paramagnetic inhibitor. All rate constants were substantially reduced upon immobilization; however, the rate constant of immobilized enzyme for ethanol oxidation was independent of the immobilization method, whereas the rate constant for cyclohexanol oxidation was lower for enzyme immobilized to octyl-Sepharose. Consequently, the substrate specificity of the two immobilized enzyme samples differed by an order of magnitude. Moreover, EPR spectroscopy studies and computer graphic analyses of spin labels occupying three defined regions of the active-site domain indicated that the active-site conformation adjacent to the catalytic zinc atom was similar in the two samples while the conformation slightly further from the zinc atom was different. This result may explain why the two immobilized enzyme preparations exhibited the same rate constant toward a small substrate (ethanol) yet different rate constants toward a larger substrate (cyclohexanol), whose rate constant is expected to be sensitive to a larger portion of the active site.     

Production of a perillyl alcohol dehydrogenase by site-directed mutagenesis of a benzyl alcohol dehydrogenase

Benzyl alcohol dehydrogenase from Acinetobacter calcoaceticus oxidises a wide range of aromatic and other cyclic alcohols and it has high specificity constants for these substrates, but it does not oxidise short- or long-chain aliphatic alcohols. Mutation of an active-site arginine to a histidine can switch the substrate specificity of the enzyme so that it has a very much greater preference for perillyl alcohol than for benzyl alcohol. © Rapid Science Ltd. 1998     

Inhibition of alcohol dehydrogenase by bismuth

Bismuth compounds have been widely used for the treatment of ulcers and Helicobacter pylori infection, and enzyme inhibition was thought to be crucial for bismuth anti-microbial activity. We have investigated the interaction of colloidal bismuth subcitrate (CBS) with alcohol dehydrogenase and our results demonstrate that bismuth can effectively inhibit the enzyme. Kinetic analysis revealed that CBS acted as a non-competitive inhibitor of yeast alcohol dehydrogenase. Both UV-vis and fluorescence data show that interaction of CBS with the enzyme exhibits biphasic processes. Bismuth can replace only half of Zn(II) from the enzyme (i.e., about one Zn(II) per monomer). Surprisingly, binding of CBS also induces the enzyme dissociation from its native form, tetramer into dimers. The inhibition of Bi(III) on the enzyme is probably due to its direct interference with the zinc sites. This study is likely to provide an insight into the mechanism of action of bismuth drugs.     

Catalysis of thiol/disulfide exchange: single-turnover reduction of protein disulfide-isomerase by glutathione and catalysis of peptide disulfide reduction

Protein disulfide-isomerase, a protein localized to the lumen of the endoplasmic reticulum of eukaryotic cells, catalyzes the posttranslational formation and rearrangement of protein disulfide bonds. As isolated from bovine liver, the enzyme contains 0.8 free sulfhydryl group per mole of protein monomer and 3.1 disulfide bonds. Single-turnover experiments in which the disulfide bonds of the native enzyme are reduced by glutathione reveal three distinct reduction steps corresponding to the sequential reduction of the three disulfide bonds. The fastest disulfide to be reduced undergoes a change in the rate-determining step with increasing GSH concentration from a step which is second-order with respect to GSH concentration to a step which is first-order in GSH concentration. The disulfide which is reduced at an intermediate rate displays kinetics that are first-order in GSH concentration, and the slowest disulfide to be reduced exhibits kinetics which are second-order in GSH concentration. The enzyme catalyzes the steady-state reduction of a disulfide-containing hexapeptide (CYIQNC) by GSH. Initial velocity kinetic experiments are consistent with a sequential addition of the substrates to the enzyme. Saturation behavior is not observed at high levels of both substrates (Km for GSH much greater than 14 mM, Km for CYIQNC much greater than 1 mM). Only one of the three disulfides appears to be kinetically competent in the steady-state reduction of CYIQNC by GSH. The second-order thiol/disulfide exchange reactions catalyzed by the enzyme are 400-6000-fold faster than the corresponding uncatalyzed reactions.     

Histochemical localization of alcohol dehydrogenase during the aging of seeds]

Alcohol dehydrogenase activity has been demonstrated in section of fresh and aged pea seeds, both air-dried and during germination. The activity in the various tissues has been observed using nitro-blue tetrazolium. From the comparison a general decrease appear in aged pea seeds and a tendency to a shift in the peaks of alcohol deydrogenase activity in the considered tissues (epidermis upper and lower, hypodermis, storage parenchyma and vascular system). The procambial zone forms the tracheary elements during germination, but in aged seeds this occur only rarely. The experiments here reported confirm the hypothesis that seed ageing includes changes in enzyme activity and in morphogenetic pathways.     

Medium-chain fatty acids and glutathione derivatives as inhibitors of S-nitrosoglutathione reduction mediated by alcohol dehydrogenase 3

Alcohol dehydrogenase 3 (ADH3) has emerged as an important regulator of protein S-nitrosation in its function as S-nitrosoglutathione (GSNO) reductase. GSNO depletion is associated with various disease conditions, emphasizing the potential value of a specific ADH3 inhibitor. The present study investigated inhibition of ADH3-mediated GSNO reduction by various substrate analogues, including medium-chain fatty acids and glutathione derivatives. The observed inhibition type was non-competitive. Similar to the Michaelis constants for the corresponding ω-hydroxy fatty acids, the inhibition constants for fatty acids were in the micromolar range and showed a clear dependency on chain length with optimal inhibitory capacity for eleven and twelve carbons. The most efficient inhibitors found were undecanoic acid, dodecanoic acid and dodecanedioic acid, with no significant difference in inhibition constant. All glutathione-derived inhibitors displayed inhibition constants in the millimolar range, at least three orders of magnitudes higher than the Michaelis constants of the high-affinity substrates GSNO and S-hydroxymethylglutathione. The experimental results as well as docking simulations with GSNO and S-methylglutathione suggest that for ADH3 ligands with a glutathione scaffold, in contrast to fatty acids, a zinc-binding moiety is imperative for correct orientation and stabilization of the hydrophilic glutathione scaffold within a predominantly hydrophobic active site.     

Catalytic and molecular properties of the quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase from Ralstonia eutropha strain Bo

The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent k(cat)/K(m) and K(i) values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k(cat)/K(m) value of 788 x 10(4) M(-1) s(-1). The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.     

Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase

A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD⁺-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg^–1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg^–1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg^–1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 mol g^–1 (cell dry weight) min^–1. In the presence of formate, the intracellular [NADH]/[NAD⁺] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.