Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase.

Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues.     

Studies on methylated yeast alcohol dehydrogenase

The incubation of yeast alcohol dehydrogenase with formaldehyde in the presence of NaBH₄ methylates lysine residues to form ?N,?N-dimethyl lysine with a concurrent decrease in enzymic activity which is not alleviated by the presence of coenzymes. The modification causes structural change(s) in yeast alcohol dehydrogenase as evidenced by a hyperchromic shift in the uv spectrum, the sensivitity to heat inactivation, the reactivity to sulfhydryl reagents, and a change in Stokes' radius. Kinetic studies indicate that the reduced activity of the methylated enzyme to oxidize alcohols is associated with decreased maximum velocities by retarding the interconversion of the ternary complexes. The catalytic efficiency of the control enzyme to oxidize primary alcohols is affected by the steric interaction which is absent in the methylated enzyme.     

2-Mercaptoethanol as a substrate for liver alcohol dehydrogenase.

An activity was identified in a phosphate buffer extract of calf liver acetone powder which utilized 2-mercaptoethanol and NAD⁺ as substrates and formed NADH as one product. The activity responsible for catalyzing this reaction is associated with calf liver alcohol dehydrogenase based on copurification, similarity in pH optima, and similarity in response to chelating agents and other inactivating agents. Crystalline horse liver alcohol dehydrogenase also catalyzes the formation of NADH from NAD⁺ using 2-mercaptoethanol as the substrate. Although the K_m for mercaptoethanol is much lower than that for ethanol, 30 μm as compared to 0.625 mm, the maximum velocity with mercaptoethanol as the substrate is only 7% of that when ethanol is the substrate. Because of this difference in maximum velocity, 2-mercaptoethanol is an apparent competitive inhibitor with respect to ethanol with crystalline horse liver alcohol dehydrogenase, consistent with ethanol and 2-mercaptoethanol binding at the same site. The apparent K_i for 2-mercaptoethanol is 14 μm. 2-Butanethiol is a competitive inhibitor with respect to both 2-mercaptoethanol and ethanol with horse and beef liver alcohol dehydrogenases.     

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study.

The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic.Addition of two [¹⁴C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD⁺-pyrazole complex. However, the ability to bind NAD⁺-pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD⁺-pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD⁺-pyrazole binding capacity remains; under these reaction conditions one [¹⁴C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD⁺-pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD⁺ during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD⁺-pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD⁺-pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase.Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.     

The reaction of horse-liver alcohol dehydrogenase with glyoxal.

Horse liver alcohol dehydrogenase was reacted with glyoxal at different pH values ranging from 6.0 to 9.0. At pH 9.0 the enzyme undergoes a rapid activation over the first minutes of reaction, followed by a decline of activity, which reaches 10% of that of the native enzyme. Chemical analysis of the inactivated enzyme after sodium borohydride reduction shows that 11 argi-ine and 11 lysine residues per mole are modified. At pH 7.7 the enzyme activity increases during the first hour of the reaction with glyoxal and then decreases slowly. Chemical analysis shows that 4 arginine and 3 lysine residues per mole are modified in the enzyme at the maximum of activation. At pH 7.0 the enzyme undergoes a 4-fold activation. Chemical analysis shows that in this activated enzyme 3 lysine and no arginine residues per mole have been modified. Steady-state kinetic analysis suggests that the activated enzyme is not subjected to substrate inhibition and that its Michaelis constant for ethanol is three times larger than that of the native enzyme. The possible role of arginine and lysine residues in the catalytic function of liver alcohol dehydrogenase is discussed.     

Human liver cytosolic malate dehydrogenase: Purification,kinetic properties,and role in ethanol metabolism

Cytosolic malate dehydrogenase from human liver was isolated and its physical and kinetic properties were determined. The enzyme had a molecular weight of 72,000 ± 2000 and an amino acid composition similar to those of malate dehydrogenases from other species. The kinetic behaviour of the enzyme was consistent with an Ordered Bi Bi mechanism. The following values (μm) of the kinetic parameters were obtained at pH 7.4 and 37 °C: K_a, 17; K_ia, 3.6; K_b, 51; K_ib, 68; K_p, 770; K_ip, 10,700; K_q, 42; K_iq, 500, where a, b, p, and q refer to NADH, oxalacetate, malate, and NAD⁺, respectively. The maximum velocity of the enzyme in human liver homogenates was 102 μmol/min/g wet wt of liver for oxalacetate reduction and 11.2 μmol/min/g liver for malate oxidation at pH 7.4 and 37 °C. Calculations using these parameters showed that, under conditions in vivo, the rate of NADH oxidation by the enzyme would be much less than the maximum velocity and could be comparable to the rate of NADH production during ethanol oxidation in human liver. The rate of NADH oxidation would be sensitive to the concentrations of NADH and oxalacetate; this sensitivity can explain the change in cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ redox state during ethanol metabolism in human liver.     

Deuterium isotope effects in the oxidation of alcohols in vitro and in vivo

The oxidation of ethanol and isopropanol by liver alcohol dehydrogenase was studied in vitro and in vivo. Oxidation of ethanol by horse liver alcohol dehydrogenase was carried out in the presence of lactaldehyde and other aldehydes which reoxidized enzyme-bound NADH. Under these conditions the oxidation of ethanol was accelerated 7 to 22-fold, depending on the nature of the aldehyde. (An acceleration of ethanol oxidation by lactaldehyde was previously reported by Gupta and Robinson [(1966) Biochim, Biophys. Acta118, 431]. In the presence of lactaldehyde ping-pong kinetics were observed and a deuterium isotope effect on V of 4.2 was seen. In the absence of acceptor aldehyde no, or small, isotope effects (Baker, R. H. (1962) Biochemistry1, 41) are observed. Therefore, when dissociation of NADH is no longer rate limiting the hydrogen transfer step becomes largely rate determining. Oxidation of isopropanol shows an isotope effect on V of 2.5 in the absence of acceptor aldehyde. With mouse liver alcohol dehydrogenase results similar to those obtained with the horse liver enzyme were obtained.When ethanol metabolism was examined in vivo, in mice by measuring blood alcohol levels, no isotope effect was observed with ethanol-1-d₂. On the other hand, an isotope effect of 2.0 was observed when the metabolism of isopropanol and isopropanol-2-d₁ were compared. This isotope effect is very close to that observed in vitro with the mouse liver enzyme. The relative rate of metabolism of ethanol and isopropanol in vivo was similar to that observed in vitro with the mouse liver enzyme (ethanol:isopropanol, 2.1 in vivo:2.2 in vitro). It was concluded that in the metabolism of ethanol and isopropanol, alcohol dehydrogenase is partially rate determining. Administration to mice of lactaldehyde, as well as other aldehydes, ketones, or fructose, simultaneously with ethanol produced no increase in the rate of ethanol metabolism.     

Redox potential dependence of photophosphorylation and electron transfer in continuous illumination of Rhodopseudomonas sphaeroides chromatophores

The rate of ethanol elimination in fed and fasted rats can be predicted based on the liver content of alcohol dehydrogenase (EC 1.1.1.1), the steady-state rate equation, and the concentrations of substrates and products in liver during ethanol metabolism. The specific activity, kinetic constants, and multiplicity of enzyme forms are similar in fed and fasted rats, although the liver content of alcohol dehydrogenase falls 40% with fasting. The two major forms of the enzyme were separated and found to have very similar kinetic properties. The rat alcohol dehydrogenase is subject to substrate inhibition by ethanol at concentrations above 10 mM and follows a Theorell-Chance mechanism. The steady-state rate equation for this mechanism predicts that the in vivo activity of the enzyme is limited by NADH product inhibition at low ethanol concentrations and by both NADH inhibition and substrate inhibition at high ethanol concentrations. When the steady-state rate equation and the measured concentrations of substrates and products in freeze-clamped liver of fed and fasted rats metabolizing alcohol are employed to calculate alcohol oxidation rates, the values agree very well with the actual rates of ethanol elimination determined in vivo.     

Activation and inactivation of horse liver alcohol dehydrogenase with pyridoxal compounds.

Pyridoxal compounds can either activate or inactivate horse liver alcohol dehydrogenase in differential labeling experiments. Amino groups outside of the active sites were modified with ethyl acetimidate, while the amino groups in the active sites were protected by the formation of the complex with NAD-plus and pyrazole. After removal of the NAD-plus and pyranzole, the partially acetimidylated enzyme was reductively alkylated with pyridoxal and NaBH4, with the incorporation of one pyridoxal group per subunit of the enzyme. The turnover numbers for the reaction of NAD-plus and ethanol increased by 15-fold, and for NADH and acetaldehyde by 32-fold. The Michaelis and inhibition constants increased 80-fold or more. Pyridoxal phosphate and NaBH4 also modified one group per subunit, but the turnover numbers decreased by 10-fold and the kinetic constants were intermediate between those obtained for pyridoxyl alcohol dehydrogenase and the partially acetimidylated enzyme. With native enzyme, the rates of dissociation of the enzyme-coenzyme complexes are rate-limiting in the catalytic reactions. The pyridoxyl enzyme is activated because the rates of dissociation of the enzyme-coenzyme complexes are increased. The rates of binding of coenzyme to phosphopyridoxyl enzyme have decreased due to the introduction of the negatively charged phosphate. The size of the group is not responsible for this decrease since these rates are not greatly decreased by the incorporation of pyridoxal. For both pyrodoxal and phosphopyridoxyl alcohol dehydrogenases, the interconversion of the ternary complex is at least partially rate-limiting. Chymotryptic-tryptic digestion of pryidoxyl enzyme produced a major peptide corresponding to residues 219 to 229, in which Lys 228 had reacted with pyridoxal. The same lysine residue reacted with pyridoxal phosphate.     

Carboxymethyl horse-liver alcohol dehydrogenase. Ligand-binding and kinetic properties of the cysteine-46-modified enzyme.

Horse-liver alcohol dehydrogenase was carboxymethylated with iodoacetate, which is known to selectively alkylate cysteine-46 in the polypeptide sequence. Carboxymethyl and native enzyme had the same electrophoretic mobility on starch or polyacrylamide gel, but some separation was achieved when isobutyramide and a low concentration of NADH were present (under these conditions NADH was bound by native enzyme but not by Carboxymethyl enzyme).The Carboxymethyl enzyme formed ternary complexes with NAD⁺ and pyrazole or decanoate. The fluorescence emission of NADH was enhanced 7- to 8-fold (at 410 nm), and a dissociation-constant of 1.7 μM was calculated at pH 7.4; but, in contrast to native enzyme, neither the affinity nor fluorescence were increased by amides (acetamide or isobutyramide).Carboxymethyl alcohol dehydrogenase possesses catalytic activity. Higher alcohols gave maximum velocities up to 7-fold higher than ethanol (reaching nearly 20% of the activity of native enzyme) while [²H]ethanol showed an isotope-rate effect of 3.3. Although the affinity for aldehydes was considerably increased, the maximum velocity of aldehyde-reduction was always at least 20% of that shown by native enzyme, and at pH 9.9 it was almost 2-fold greater than with native enzyme. The rate-limiting step in alcohol-oxidation is likely to be the interconversion of ternary complexes (possibly the hydride-transfer step), while in aldehyde-reduction it could still be the dissociation of the enzyme/NAD⁺ complex. This is also indicated by inhibition experiments with decanoate, pyrazole, and isobutyramide.These results suggest that a major effect of carboxymethylation is upon ternary complexes of enzyme and NADH, which become much more reluctant to form, either by combination of NADH and ligand with the modified enzyme, or by catalytic conversion of the enzyme/NAD ⁺/alcohol complex.     

Horse liver alcohol dehydrogenase. A study of the essential lysine residue.

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.     

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.     

Characterization of a nonhepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach.

Ethanol oxidation by the soluble fraction of a rat hepatoma was compared to that of the liver. Ethanol oxidation by the hepatoma was NAD⁺-dependent and sensitive to pyrazole, suggesting the presence of alcohol dehydrogenase. At low concentrations of ethanol (10.8 mm) the alcohol dehydrogenase activities of hepatoma and liver supernatant fractions were comparable. When the concentration of ethanol was raised to 108 mm, the activity of the liver enzyme decreased, whereas the activity in hepatoma supernatant fractions was strikingly elevated. m-Nitrobenzaldehyde-reducing activity was also conspicuously higher in hepatoma supernatant fractions. By contrast the ability to metabolize steroids and cyclohexanone was less than that in supernatant fractions of the liver.Electrophoresis of the liver supernatant fractions on ionagar at pH 7.0 revealed only one component that oxidized ethanol. On the other hand, hepatoma supernatant fractions contained two components with alcohol dehydrogenase activity; one with the same electrophoretic mobility as the liver enzyme, the other showing a slower rate of migration. The latter component, which is absent in the liver, is referred to as hepatoma alcohol dehydrogenase. By electrophoresis on starch gels at pH 8.5, it could be demonstrated that the liver and hepatoma enzymes moved in opposite directions.The liver and hepatoma enzymes differ in electrophoretic mobility, susceptibility to heat treatment, pH activity optimum and some catalytic properties. The substrate specificity of the hepatoma enzyme is narrower than that of liver alcohol dehydrogenase; cyclohexanone or 3β-hydroxysteroids of A/B cis configuration and the corresponding 3-ketones are not substrates for the hepatoma enzyme. The overall substrate specificity characteristics are, however, similar to those of the liver enzyme in that the effectiveness of substrates increases with an increase in chain length and introduction of unsaturation or an aromatic group. Both liver and hepatoma alcohol dehydrogenase cross-react with antibody to horse liver alcohol dehydrogenase EE. The Michaelis constant for ethanol with the hepatoma enzyme is 223 mm, compared to 0.3 mm for liver alcohol dehydrogenase; at 1.0 m ethanol the hepatoma enzyme is not fully saturated with substrate. The Michaelis constant for 2-hexene-1-ol is 0.3 mm, indicating that the hepatoma enzyme is better suited for dehydrogenation of longer chain alcohols. Stomach alcohol dehydrogenase has kinetic properties comparable to those of the hepatoma enzyme, as well as similar electrophoretic mobility. The hepatoma enzyme can be detected in the serum of rats bearing hepatomas.     

Horse liver alcohol dehydrogenase SS: purification and characterization of the homogenous isoenzyme.

Alcohol dehydrogenase SS was prepared from horse liver by salt fractionation, ion-exchange chromatography, and affinity chromatography. The purified isoenzyme is free from extraneous protein and other alcohol dehydrogenase isoenzyme contaminants and contains four Zinc atoms per molecule. The substrate specificity with saturated aliphatic alcohols and aldehydes of two to six carbon chain lengths has been investigated. The K_m values and turnover numbers at maximal velocity (k_cat) are presented. Values of k_cat are constant within a substrate category and independent of the substrate chain length, while the K_m values decrease with the increase of the substrate chain length. The K_m values for two- and three-carbon substrates are large, that for ethanol (40 mm) is two orders of magnitude larger than that reported for classical preparations of horse liver alcohol dehydrogenase. At pH 7, the k_cat values for alcohol oxidation are almost 30 times smaller than for aldehyde reduction. The enzyme has been characterized with regard to specific activity with several nonsteroidal substrates and with two steroids: 3-oxo-5β-androstan-17β-ol and 5β-pregnan-21-ol-3,20-dione hemisuccinate. NAD(H) is the preferred coenzyme. Values of K_m for NADH with constant steroidal substrates are an order of magnitude smaller than the corresponding K_m values with nonsteroidal substrates. A possible explanation for this observation is presented.     

Tomato alcohol dehydrogenase: Purification and substrate specificity

Alcohol dehydrogenase of tomato (Lycopersicon esculentum) has been purified to homogeneity, using affinity chromatography on Cibacron F3GA-agarose. The enzyme is a dimer, M_r 90,000–100,000. The coenzyme is NAD⁺; no NADP⁺-dependent activity was detected even in crude extracts. Among saturated substrates, ethanol and acetaldehyde show the lowest apparent K_m values (2.67 and 0.174 mm, respectively) and highest V values, supporting a primary role in acetaldehyde metabolism, with action also on “flavor aldehydes”; 2-unsaturated alcohols show still lower K_m values, probably due to a more favorable K_eq. This enzyme and other plant alcohol dehydrogenases form a definite class, intermediate in specificity between liver and yeast alcohol dehydrogenases: they differ from the former in being essentially inactive on secondary and aromatic substrates, from the latter in showing only a mild decrease in V with increasing chain length of alkyl substrates, and from both in showing the lowest K_m as well as highest V on ethanol and acetaldehyde. The tomato enzyme differs from other reported plant enzymes in showing substantial activity on geraniol. Kinetic studies are in agreement with an ordered sequential mechanism. The enzyme is inhibited slowly by iodoacetamide, and reversibly by acetamide and zinc-chelating compounds.     

Kinetic effects of a single-amino acid mutation in a highly variable loop (residues 114–120) of class IV ADH

Class IV alcohol dehydrogenase shows a deletion at position 117 with respect to class I enzymes, which typically have a Gly residue. In class I structures, Gly117 is part of a loop (residues 114–120) that is highly variable within the alcohol dehydrogenase family. A mutant human class IV enzyme was engineered in which a Gly residue was inserted at position 117 (G117ins). Its kinetic properties, regarding ethanol and primary aliphatic alcohols, secondary alcohols and pH profiles, were determined and compared with the results obtained in previous studies in which the size of the 114–120 loop was modified. For the enzymes considered, a smaller loop was associated with a lower catalytic efficiency towards short-chain alcohols (ethanol and propanol) and secondary alcohols, as well as with a higher K_m for ethanol at pH 7.5 than at pH 10.0. The effect can be rationalized in terms of a more open, solvent-accessible active site in class IV alcohol dehydrogenase, which disfavors productive binding of ethanol and short-chain alcohols, specially at physiological pH.     

Comparison of the Kinetic Behavior toward Pyridine Nucleotides of NAD-Linked Dehydrogenases from Plant Mitochondria

In this article we compare the kinetic behavior toward pyridine nucleotides (NAD⁺, NADH) of NAD⁺-malic enzyme, pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine decarboxylase extracted from pea (Pisum sativum) leaf and potato (Solanum tuberosum) tuber mitochondria. NADH competitively inhibited all the studied dehydrogenases when NAD⁺ was the varied substrate. However, the NAD⁺-linked malic enzyme exhibited the weakest affinity for NAD⁺ and the lowest sensitivity for NADH. It is suggested that NAD⁺-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I.     

Rat liver mitochondrial malate dehydrogenase: purification, kinetic properties, and role in ethanol metabolism

Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 37 degrees C, yielding the following values (microM): Ka, 72; Kia, 11; Kb, 110; Kp, 1600; Kip, 7100; Kq, 170; Kiq, 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib was estimated to be about 100 microM. The maximum velocities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37 degrees C, were 380 +/- 40 mumol/min per gram of liver, wet weight, for oxalacetate reduction and 39 +/- 3 mumol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in vivo were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 mumol min-1 per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 microM.     

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD⁺ ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD⁺ ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD⁺-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD⁺ ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD⁺ ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD⁺ redox couple and the effects of ethanol on methionine metabolism in the liver.     

Regulation of ethanol metabolism in the rat

The purpose of these experiments was to examine the factors which regulate ethanol metabolism in vivo. Since the major pathway for ethanol removal requires flux through hepatic alcohol dehydrogenase, the activity of this enzyme was measured and found to be 2.9 mumol/(min X g liver). Ethanol disappearance was linear for over 120 min in vivo and the blood ethanol fell 0.1 mM/min; this is equivalent to removing 20 mumol ethanol/min and would require that flux through alcohol dehydrogenase be about 60% of its measured maximum velocity. To test whether ethanol metabolism was limited by the rate of removal of one of the end products (NADH) of alcohol dehydrogenase, fluoropyruvate was infused to reoxidize hepatic NADH and to prevent NADH generation via flux through pyruvate dehydrogenase. There was no change in the rate of ethanol clearance when fluoropyruvate was metabolized. Furthermore, enhancing endogenous hepatic NADH oxidation by increasing the rate of urea synthesis (converting ammonium bicarbonate to urea) did not augment the steady-state rate of ethanol oxidation. Hence, transport of cytoplasmic reducing power from NADH into the mitochondria was not rate limiting for ethanol oxidation. In contrast, ethanol oxidation at the earliest time periods could be augmented by increasing hepatic urea synthesis.