The reactions of 1,10-phenanthroline with yeast alcohol dehydrogenase.

Freshly prepared samples of yeast alcohol dehydrogenase (EC 1.1.1.1) were inhibited by 1,10-phenanthroline at pH 7.0 and 0 degrees C in a two-stage process. The first step appeared to be slowly established, but was rendered reversible by removal of reagent or by addition of excess Zn2+ ions. The second step was irreversible and was associated with the dissociation of the tetrameric enzyme. The presence of saturating concentrations of NAD+ or NADH promoted and enhanced inhibition by the slowly established reversible process, but prevented dissociation of the enzyme. For the incubation mixtures containing NAD+, removal of the 1,10-phenanthroline resulted in virtually complete recovery of activity, whereas, for the incubation mixtures containing NADH, removal of the reagent gave only partial re-activation. The presence of NAD+ and pyrazole, or NADH and acetamide, in incubation mixtures with the enzyme gave rise to ternary complexes that gave protection against both forms of inactivation by 1,10-phenanthroline. The results support the view that at least some of the Zn2+ ions associated with yeast alcohol dehydrogenase have a catalytic, as opposed to a purely structural, role.     

The effect of thiamine and its metabolites on the activity of tissue and purified lactate dehydrogenase

It is shown that thiamine and its metabolites effect lactate dehydrogenase activity and lactate content in the tissues. Thiochrome and thiamine phosphate increase the lactate level in the liver and small intestine. The given effect correlates with the inhibition of the tissue and purified lactate dehydrogenase by thiochrome.     

The conformation of adenosine diphosphoribose and 8-bromoadenosine diphosphoribose when bound to liver alcohol dehydrogenase.

8-Bromo-adenosine diphosphoribose (br8 ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr8AD+) which are analogues of the coenzyme NAD+, were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in hydrogen transport and br8ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase. X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution. The conformations of these analogues were determined from the X-ray data. It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD+, when NAD+ is bound to lactate and malate dehydrogenase. br8ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD+, in contrast to the syn conformation found in 8-bromo-adenosine. The overcrowding at the 8-position is relieved in br8ADP-Rib by having the ribose in the 2' endo condormation instead of the usual 3' endo as in ADP-Rib and NAD+.     

Interaction of NAD(H)-dependent dehydrogenases with active dyes and their complexes with transitional metal ions

The interaction of NAD(H)-dependent dehydrogenases--yeast alcohol dehydrogenase and rabbit muscle lactate dehydrogenase--with reactive dyes produced in the USSR was studied. The essential role of metal ions in specific binding of alcohol dehydrogenase and dyes was demonstrated by differential spectroscopy, circular dichroism spectroscopy and chromatography. Lactate dehydrogenase in contrast with alcohol dehydrogenase does not require metal ions for the binding of the above-said dyes. A comparative study of eluting abilities of selected desorption agents (imidazole, adenine, 8-oxyquinoline-5-sulfonic acid, NAD, AMP, EDTA) by alcohol dehydrogenase chromatography on adsorbents with light-resistant yellow 2KT-Cu(II) and orange 5K revealed the differences in competition of the dyes for NAD-binding sites of alcohol dehydrogenase. The participation of light-resistant yellow 2KT-Cu(II) in the formation of mixed complexes with imidazole, adenine, 8-oxyquinoline-5-sulfonic acid, NAD and EDTA suggests that the specific binding of alcohol dehydrogenase to light-resistant yellow 2KT-Cu(II) is due to coordination between the Cu(II) ion and the amino acid residue in alcohol dehydrogenase.     

Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram-negative bacterium Pseudomonas putida, were purified and characterized. Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible. Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes. The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 microM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 microM. Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH-blocking agents such as N-ethylmaleimide. Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa. Both enzymes transfer hydride to the pro-R side of the prochiral C4 of the pyridine ring of NAD+.     

STUDIES ON THE PROPERTIES OF RETINAL ALCOHOL DEHYDROGENASE FROM THE RAT

An NAD-dependent alcohol dehydrogenase (alcohol:NAD oxidoreductase; EC 1.1.1.1) has been isolated and partially purified from the retinal cytosol of the rat. Its substrate specificity and sensitivity to inhibitors of hepatic alcohol dehydrogenase have been investigated. Ethanol, 1-propanol and 1-butanol served as substrates for this enzyme but the K_m values were more than 100-fold higher than those reported for hepatic alcohol dehydrogenase. Methanol and retinol were unreactive with this alcohol dehydrogenase. Inhibition by pyrazole was observed but the K_t was about 100-fold higher than the value observed for hepatic alcohol dehydrogenase. n-Butyraldoxime inhibited retinal alcohol dehydrogenase with a K_t of 2 μM, a value which approximates its K_t for hepatic alcohol dehydrogenase. 1, 10-Phenanthroline was ineffective as an inhibitor. Oxidation of retinol was observed in retinal homogenates in the presence of NADP but no inhibition was observed with ethanol, methanol or pyrazole. We conclude that oxidation of retinol is not catalysed by soluble retinal alcohol dehydrogenase.     

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of intersubunit interaction in horse liver alcohol dehydrogenase on the kinetics of ethanol oxidation]

The kinetics of enzymatic oxidation of ethanol in the presence of alcohol dehydrogenase within a wide range of ethanol and NAD concentrations (pH 6.0--11.5) were studied. It was shown that high concentrations of ethanol (greater than 0.7--5 mM, depending on pH) and NAD (greater than 0.4--0.8 mM) activate alcohol dehydrogenase from horse liver within the pH range of 6.0--7.9. A mechanism of activation based on negative cooperativity of ADH subunits for binding of ethanol and NAD was proposed. The catalytic and Michaelis constants for alcohol dehydrogenase were calculated from ethanol and NAD at all pH values studied. The changes resulting from the subunit cooperativity were revealed. The nature of ionogenic groups of alcohol dehydrogenase, which affect the formation of complexes between the enzyme and NAD and ethanol, and the rate constants for catalytic oxidation of ethanol was assumed. The biological significance of the enzyme capacity for activation by high concentrations of ethanol within the physiological range of pH in the blood under excessive use of alcohol is discussed.     

A two-step synthesis of new water-soluble polymers of NAD+ and ADP. The biological properties of these polymers

Alkylation at the N-1 position of the adenine moiety of NAD+, ADP or ATP with 2,3-epoxypropyl acrylate, followed by polymerization with or without acrylamide at pH 8, gave water-soluble polymers of NAD+ and ADP where the alkyl chain was located at the exocyclic adenine C-6 amino group. Cofactor incorporations were good to high: 145-447 mumol NAD+/g polymer and 667 mumol ADP/g polymer. About 30% of the bound NAD+ could be reduced with rabbit muscle lactae dehydrogenase, yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase; 84% of the bound ADP was phosphorylated with rabbit muscle creatine kinase. High cofactor activities were obtained with polymerized NAD+ with alcohol dehydrogenase as enzyme: the initial rate of NAD+ polymer reduction was 35-81% that of free NAD+. These values remained substantially high with agarose-immobilized alcohol dehydrogenase (15-36%) and should eventually allow their use in continuous enzymatic reactors. Enzymatic phosphorylation of ADP polymer by creatine kinase gave an ATP polymer with high biological activity: 480 mumol ATP/g polymer were transformed with yeast hexokinase.     

A simple steady state kinetic model for alcohol dehydrogenase. Use of reaction progress curves for parameters estimation at pH 7.4

A simple rate equation for alcohol dehydrogenase was obtained by assuming independent binding sites for ethanol and NAD+ and fully competitive inhibition by the products of the reaction, acetaldehyde and NADH. A random binding order was also assumed. The rate equation is described by six parameters: four association constants (two for the substrates and two for the products of the reaction), Vf for the forward direction, and the equilibrium constant of the reaction. The six parameters were determined at pH 7.4 by numerical analysis of progress curves of reactions started with different concentrations of ethanol and NAD+. The parameters for alcohol dehydrogenase partially purified from rat liver were: Km for ethanol = 0.746 mM, Km for NAD+ = 0.0563 mM, Km for acetaldehyde = 7.07 microM, Km for NADH = 4.77 microM and Keq = 2.36 X 10(-4). The computed values allowed a very good simulation of the experimental progress curves and little variation was observed in the kinetic parameters when the reactions were started in the presence of either NADH or acetaldehyde.     

Interaction of yeast alcohol dehydrogenase with protoberberine alkaloids

Oxidation of ethanol and reduction of aldehyde catalysed by yeast alcohol dehydrogenase is inhibited by several naturally occurring as well as semi-synthetic protoberberine alkaloids. The affinity of these compounds for the enzyme depends essentially on their hydrophobicity. Corysamine and coptisine are the most potent inhibitors among the natural alkaloids of this group. The kinetics of yeast alcohol dehydrogenase inhibition with coptisine were analysed and equilibrium measurements using optical methods were carried out. The results suggest that the binding site of the enzyme for protoberberines is not identical with those for coenzyme and substrate though it should be located near the nicotinamide ring of bound NAD. The binding of protoberberines seems to be limited to rather superficially located hydrophobic groups in the vicinity of the active site of the enzyme. The inability of these alkaloids to protrude deeply into the molecule of yeast alcohol dehydrogenase at the catalytically important region is the main difference in their behaviour towards alcohol dehydrogenases from yeast and horse liver.     

Photocatalyzed oxidation of NAD dimers

A mixture of dimers of nicotinamide adenine dinucleotide, largely 4,4?-linked, obtained by electrochemical reduction of NAD⁺, can be photooxidized back to NAD⁺ in the presence of oxygen. Oxygen is consumed during the photooxidation process with the production of hydrogen peroxide. The oxidation is almost pH independent and is stimulated by light whose wavelength exceeds 300 nm. Lactate dehydrogenase and alcohol dehydrogenase added to the solutions under irradiation increased the oxygen uptake by the NAD dimers in a concentration-dependent way. These observations suggest that light induces the homolytic cleavage of NAD dimers to NAD radicals which in turn are oxidized to NAD⁺ by oxygen.     

The enzymic reduction of nicotinamide–adenine dinucleotide by 2-mercaptoethanol

1. The reduction of NAD(+), by an enzyme preparation from rat liver, in the presence of 2-mercaptoethanol is reported. 2. It is suggested that the NAD(+)-linked alcohol dehydrogenase in the extract transfers hydrogen from 2-mercaptoethanol to NAD(+). 3. Both yeast and horse-liver alcohol dehydrogenases were observed to reduce NAD(+) in the presence of 2-mercaptoethanol. 4. Some interactions of 2-mercaptoethanol, cysteine or hydroxylamine with the alcohol dehydrogenases from rat liver, horse liver and yeast are discussed.     

Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase

Formaldehyde, a major industrial chemical, is classified as a carcinogen because of its high reactivity with DNA. It is inactivated by oxidative metabolism to formate in humans by glutathione-dependent formaldehyde dehydrogenase. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent alcohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-ADH. The first step in the reaction involves the nonenzymatic formation of the S-(hydroxymethyl)glutathione adduct from formaldehyde and glutathione. When formaldehyde concentrations exceed that of glutathione, nonoxidizable adducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are reported to be 50 times that of formaldehyde in humans. Initial velocity, product inhibition, dead-end inhibition, and equilibrium binding studies indicate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathione and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Formation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxododecanoic acid (12-ODDA) exhibited substrate inhibition, which is consistent with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanism is consistent with the published three-dimensional structure of the formaldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coenzyme-catalytic domain conformation where substrates can bind or dissociate in any order.     

Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.     

The absence of quinoprotein alcohol dehydrogenase in Acinetobacter calcoaceticus

It is shown that the unusual NAD(P)+-independent quinoprotein alcohol dehydrogenase, said previously to be responsible for oxidation of ethanol during growth of Acinetobacter calcoaceticus LMD 79.39, was in fact isolated from an unidentified organism which contained cytochrome c and which has now been lost. Several genuine strains of A. calcoaceticus do not contain cytochrome c nor do they contain a quinoprotein alcohol dehydrogenase. The enzyme responsible for ethanol oxidation in these bacteria is an inducible NAD+-linked alcohol dehydrogenase.     

Enzymatic resolution of 1-phenyl-1,2-ethanediol by enantioselective oxidation: overcoming product inhibition by continuous extraction

Oxidations of alcohols by alcohol dehydrogenases often suffer from low conversions and slow reaction rates due to severe product inhibition. This can be overcome by continuous product extraction, because only the concentrations, but not the kinetic parameters, can be changed. As a consequence, it is favorable to apply a differential circulation reactor with continuous product extraction, where only a small amount of product is formed per cycle. The product is then directly extracted using a microporous hydrophobic hollow fiber membrane. This results in an increase of the relative activity of the dehydrogenase at a given conversion. The reaction investigated is the kinetic resolution of racemic 1-phenyl-1,2-ethanediol by glycerol dehydrogenase (GDH). The resulting oxidation product, 2-hydroxyacetophenone, causes a strong product inhibition. Additionally, it reacts in a chemical reaction with the cofactor lowering its active concentration. Because the GDH needs beta-nicotinamide adenine dinucleotide (NAD(+)) as a cofactor, lactate dehydrogenase is used to regenerate NAD(+) from NADH by reducing pyruvate to (L)-lactate. A conversion of 50% with respect to the racemate and an enantiomeric excess >99% of the (S)-enantiomer was reached.     

Affinity chromatography of enzyme cofactors: the separation of NAD on immobilised dehydrogenase colums.

1. Alcohol dehydrogenase (EC 1.1.1.1.) has been immobilised to aminoethyl-cellulose by glutaraldehyde, to DEAE-cellulose by an s-triazine derivative and to agarose using CNBr. Lactate dehydrogenase has been immobilised to the latter two supports. 2. Their use for affinity chromatography of NAD was compared and alcohol dehydrogenase immobilised to CNBr-activated agarose chosen for detailed study due to the efficient coupling of applied enzyme and the specific nature of binding. 3. The efficiency of coupling of alcohol dehydrogenase dropped from 94.5 to 72.2% when the applied load was increased from 18 to 54 mg/g activated agarose. Activity relative to free enzyme fell from 21 to 11%. The binding of NAD was maximal between pH 5.5 and 6. With the lowest loading of enzyme, NAD binding fell from 450 to 320 mug/g support when the linear flow rate was increased from 0.84 to 3.95 cm/min. 4. NAD was completely separated from a mixture with ATP, ADP and AMP. Separation from NMN and hydrolysed RNA and DNA was evidently possible. Immobilised alcohol dehydrogenase used for 34 binding experiments over a period of weeks maintained 60% of its original enzyme activity. 5. The method was applied to yeast NAD following mechanical disruption of yeast, clarification and either ultrafiltration or hollow-fibre dialysis to permit separate purification of macromolecules and nucleotides.     

Interaction of ethanolamine with alcohol dehydrogenase from the horse liver

The pattern of kinetic behaviour of ethanolamine (EA), an ethanol structural analog, in the alcohol dehydrogenase reaction has been studied. EA has been shown to manifest a mixed type inhibition versus ethanol and a noncompetitive behaviour towards the second substrate, NAD. A graphical analysis of the experimental results as well as the construction of secondary graphs provide evidence in favour of a mechanism, according to which the interaction between EA and the enzyme results in a dead-end complex formation (ESI). A direct conversion into reaction products can be achieved only after EA separation from the complex. The Ki value for the E-EA complex is 1.3 mM; that for EA release from the E-EA is 1.8 mM. An analysis of competitive interactions with NAD showed these constants to be equal in values (2 mM). Taking account of real concentrations of tissue EA and of experimental values of Ki, a conclusion is drawn on possible participation of EA in the alcohol dehydrogenase reaction control.     

Effect of pH on the process of ternary-complex interconversion in the liver-alcohol-dehydrogenase reaction.

1. Kinetic relationships referring to multiple-turnover conditions have been derived for the slowest exponential transient appearing in two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism. The validity of these and previously derived theoretical relationships for this mechanism has been tested by application to the liver alcohol dehydrogenase reaction. 2. All essential features of the transient-state kinetics of alcohol oxidation by NAD+ in the liver alcohol dehydrogenase system can be qualitatively and quantitatively explained in view of the compulsory-order mechanism in the proposed scheme. There is no kinetic evidence for any half-of-the-sites reactivity of the enzyme. A consistent set of rate constants is reported for the enzymic oxidation of benzyl alcohol at pH 8.75. 3. Transient-state rate parameters for benzyl alcohol/benzaldehyde catalysis by liver alcohol dehydrogenase have been determined at different pH. The interpretation of such rate parameters is critically discussed with reference to their informative value for the purpose of determination of rate constants (k and k') for the process of ternary-complex interconversion in the proposed scheme. It is concluded that the apparent rate constant (k') for hydride transfer from benzyl alcohol to NAD+ is dependent on a proton dissociation step with a pKa of 6.4, whereas the rate constant (k) for hydride transfer from NADH to benzaldehyde exhibits no corresponding dependence on proton association. 4. The asymmetric pH dependence of the forward and reverse rate of ternary-complex interconversion during liver alcohol dehydrogenase catalysis appears to reflect an obligatory step of alcohol/alcoholate ion equilibration occurring at the ternary-complex level. It is suggested that the observed pKa 6.4 dependence of the transient rate of alcohol oxidation can be attributed to a coupled acid-base system involving minimally the enzyme-bound alcohol and the protein residues Ser-48 and His-51.