Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

Purification and properties of cytosolic malic enzyme from human skeletal muscle

1. An NADP+-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein. 2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column. 3. Either Mn2+ or Mg2+ was required for activity: the pH optima with Mn2+ and Mg2+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent Km values with Mn2+ and Mg2+ for L-malate and NADP+ were 0.246 mM and 5.8 microM, and 0.304 mM and 5.8 microM, respectively. The Km values with Mn2+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 microM and 22.2 mM, respectively. 4. The enzyme was also able to decarboxylate malate in the presence of NAD+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP+, with a Km value for NAD+ of 13.9 mM. 5. The following physical parameters were established: s0(20.w) = 10.48, Stokes' radius = 5.61 nm, pI = 5.72 Mr of the dissociated enzyme = 61,800. The estimates of the native apparent Mr yielded a value of 313,000 upon gel filtration, and 255,400 with f/fo = 1.33 by combining the chromatographic data with the sedimentation measurements. 6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure. 7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme.     

Regulation of coenzyme utilization by mitochondrial NAD(P)-dependent malic enzyme

1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, L-malate:NAD+ oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent Km values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, V(s-NADP)/V(NAD) or V(NADP)/V(s-NAD), are 6 and 26, respectively.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

Structure and properties of malic enzyme from Bacillus stearothermophilus

The malic enzyme (EC 1.1.1.38) gene of Bacillus stearothermophilus was cloned in Escherichia coli, and the enzyme was purified to homogeneity from the E. coli clone. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, the enzyme catalyzes the decarboxylation of oxalacetate. The enzyme is a tetramer of Mr 200,000 consisting of four identical subunits of Mr 50,000. The pH optima for malate oxidation and pyruvate reduction are 8.0 and 6.0, respectively; and the optimum temperature is 55 degrees C. The enzyme strictly requires divalent metal cations for its activity, and the activity is enhanced 5-7 times by NH4+ and K+. Kinetic study shows that the values of the dissociation constant of the enzyme-coenzyme complex are 77 microM for NAD and 1.0 mM for NADP, indicating that the enzyme has a higher affinity for NAD than for NADP. The nucleotide sequence of the gene and its flanking regions was also found. A single open reading frame of 1434 base pairs encoding 478 amino acids was concluded to be that for the malic enzyme gene because the amino acid composition of the enzyme and the sequence of 16 amino acids from the amino terminus of the enzyme agreed well with those deduced from this open reading frame.     

Malic enzymes of salmon trout heart mitochondria: separation and some physicochemical properties of NAD-preferring and NADP-specific enzymes

Mitochondria isolated from the heart of the Baltic salmon trout Salmo trutta contain two distinct malic enzymes. One of these enzymes (NAD-preferring malic enzyme) catalyses the oxidative decarboxylation of malate in the presence of either NAD or NADP. The specific activity with NAD was six times that with NADP as coenzyme. The second enzyme is specific for NADP. These two malic enzymes have been separated by: ion exchange chromatography of DEAE-Sephacel, affinity chromatography on 2',5'ADP-Sepharose 4B, gel filtration on Sephacryl S-300 and polyacrylamide gel electrophoresis. The mol. wts of the two native malic enzymes determined by gel filtration were found to be 280,000 and 190,000 for NAD-preferring and NADP-specific malic enzyme, respectively. Chromatofocusing revealed the isoelectric points of the two enzymes at pH 5.45 and 5.85 for NAD-preferring and NADP-specific malic enzyme, respectively.     

Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.

Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex.

Rat and calf adrenal cortex homogenates were found to contain three different malic enzymes. Two were strictly NADP+-dependent and were localized, one each, in the cytosol and the mitochondrial fractions, respectively. These two enzymes appear to be identical to those described by Simpson and Estabrook (Simpson, E. R., and Estabrook, R. W. (1969) Arch. Biochem. Biophys. 129, 384-395). The third was NAD(P)+-linked and was present in the mitochondrial fraction only. All three malic enzymes separated as distinct bands during electrophoresis on 5 percent polyacrylamide slab gels at pH 9.0. Marker enzymes and the mitochondrial malic enzymes migrated together in intact mitochondria during sucrose density gradient centrifugations despite changes in the equilibrium position of the mitochondria promoted by energy-dependent calcium phosphate accumulation. In adrenal cortex mitochondria subfractionated by the method of Sottocasa et al. (SOTTOCASA, G.L., KUYLENSTIERNA, B., ERNSTER, L., and BERGSTAND, A. (1967) J. Cell Biol. 32, 415-438), both malic enzymes were associated with the inner membrane-matrix space. Sonication solubilized the two malic enzymes along with the matrix space marker enzymes. The NAD(P)+-dependent malic enzyme was purified 100-fold from calf adrenal cortex mitochondria. The final preparation was free of malic dehydrogenase, fumarase, the strictly NADP+-linked malic enzyme and adenylate kinase. Either Mn24 orMg2+ was required for activity and 1 mol of pyruvate was formed for each mole of NAD+ and NADP+ reduced. The pH optima with NAD+ and NADP+ were 6.5 tp 7.0 and 6.0 to 6.5, respectively. Michaelis-Menten kinetics were observed on the alkaline side. Fumarate, succinate, and isocitrate were positive and ATP and ADP were negative modulators of the regulatory enzyme. The modulators did not influence the stoichiometry and they were not metabolized during the reaction. Under Vmax conditions the ratios for the rate of NAD+:NADP+ reduction were 1.76 and 1.15 at pH 7.4 and 6.0, respectively. The apparent Michaelis constants also differed depending on the pH and the coenzyme. At pH 7.4 (in the presence of 5 mM fumarate) and at pH 6.0 (no fumarate) the Km values for (-)-malate, NAD+, and Mn2+ were 1.7, 0.16, and 0.15 mM, and 0.31, 0.06, and 0.09 mM, respectively. At pH 7.4 (5MM fumarate) and pH 6.0 (no fumarate), the Km values for (-)-malate, NADP+, and Mn2+ were 6.5, 0.62, and 0.59 mM, and 0.68. 0.12, and 0.31 mM, respectively. The apparent Ki values for ATP with NAD+ and NADP+ as coenzyme were 0.42 and 0.27 mM, respectively.     

Study of properties of NADP malate dehydrogenase from corn leaves]

Kinetic properties of purified chloroplast isoenzyme of the "malic" enzyme from corn leaves were studied. The enzyme had optimum activity at pH 8.0 and 36 degrees C. Under standart conditions the Michaelis constants for the "malic" enzyme with Mn2+ as cofactor are 0.091 mM for malate and 0.04 mM for NADP. In case of Mg2+ as cofactor they are 0.66 and 0.02 mM respectively. Respective Km values for the cofactors Mn2+ and Mg2+ are 0.018 and 0.091 mM. The activity of the "malic" enzyme was inhibited by reduced NADP and NAD, ATP, ADP, fructose-1,6-diphosphate, oxaloacetic, oxalic, glyoxylic, glycolic and alpha-ketoglutaric acids, as well as by phosphate anions and pyrophosphate. The inhibitory effect of all metabolites and ions is more pronounced in case of Mn, rather than Mg, used as cofactors for the reaction. A possibility of metabolic regulation of NADP-"malic" enzyme activity in the leaves of C4-plants, is discussed.     

Coupling of "malic" enzyme and NADPH:NAD transhydrogenase in the energetics of Hymenolepis diminuta (Cestoda)

Acetylpyridine NADP replaced NADP in promoting the Mn2+ ion-requiring mitochondrial "malic" enzyme of Hymenolepis diminuta. Disrupted mitochondria displayed low levels of an apparent oxaloacetate-forming malate dehydrogenase activity when NAD or acetylpyridine NAD served as the coenzyme. Significant malate-dependent reduction of acetylpyridine NAD by H. diminuta mitochondria required Mn2+ ion and NADP, thereby indicating the tandem operation of "malic" enzyme and NADPH:NAD transhydrogenase. Incubation of mitochondrial preparations with oxaloacetate resulted in a non-enzymatic decarboxylation reaction. Coupling of malate oxidation with electron transport via the "malic" enzyme and transhydrogenase was demonstrated by polarographic assessment of mitochondrial reduced pyridine nucleotide oxidase activity.     

Evidence for the role of malic enzyme in the rapid oxidation of malate by cod heart mitochondria

Mitochondria isolated from the heart of cod (Gadus morrhua callarias) oxidized malate as the only exogenous substrate very rapidly. Pyruvate only slightly increased malate oxidation by these mitochondria. This is in contrast with the mitochondria isolated from rat and rabbit heart which oxidized malate very slowly unless pyruvate was added. Arsenite and hydroxymalonate (an inhibitor of malic enzyme) inhibited the respiration rate of mitochondria isolated from cod heart, when malate was the only exogenous substrate. Inhibition caused by hydroxymalonate was reversed by the addition of pyruvate. In the presence of arsenite, malate was converted to pyruvate by cod heart mitochondria. Cod heart mitochondria incubated in the medium containing Triton X-100 catalyzed the reduction of NADP+ in the presence of L-malate and Mn2+ at relatively high rate (about 160 nmoles NADPH formed/min/mg mitochondrial protein). The oxidative decarboxylation of malate was also taking place when NADP+ was replaced by NAD+ (about 25 nmol NADH formed per min per mg mitochondrial protein). These results suggest that the mitochondria contain both NAD+- and NADP+-linked malic enzymes. These two activities were eluted from DEAE-Sephacel as two independent peaks. It is concluded that malic enzyme activity (presumably both NAD+- and NADP+-linked) is responsible for the rapid oxidation of malate (as the only external substrate) by cod heart mitochondria.     

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Two malic enzymes in Pseudomonas aeruginosa

Cell-free extract supernatant fluids of Pseudomonas aeruginosa were shown to lack malic dehydrogenase but possess a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent enzymatic activity, with properties suggesting a malic enzyme (malate + NAD (NADP) --> pyruvate + reduced NAD (NADH) (reduced NADP [NADPH] + CO(2)), in agreement with earlier findings. This was confirmed by determining the nature and stoichiometry of the reaction products. Differences in heat stability and partial purification of these activities demonstrated the existence of two malic enzymes, one specific for NAD and the other for NADP. Both enzymes require bivalent metal cations for activity, Mn(2+) being more effective than Mg(2+). The NADP-dependent enzyme is activated by K(+) and low concentrations of NH(4) (+). Both reactions are reversible, as shown by incubation with pyruvate, CO(2), NADH, or NADPH and Mn(2+). The molecular weights of the enzymes were estimated by gel filtration (270,000 for the NAD enzyme and 68,000 for the NADP enzyme) and by sucrose density gradient centrifugation (about 200,000 and 90,000, respectively).     

Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 mumol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn2+ and Co2+ were by far more effective activators than Mg2+ and Ni2+, whereas the reaction did not proceed in the presence of Ca2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for L-malate and NAD were 2.82 and 0.33 mM; and in the presence of fumarate 1.18 and 0.22 mM, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.     

Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of glutamine 362

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The DeltaG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.     

Determination of the mechanism of human malic enzyme with natural and alternate dinucleotides by isotope effects.

Human malic enzyme was studied by steady state kinetics, deuterium isotope effects, and 13C isotope effects with both the physiological dinucleotide cofactor and several alternate cofactors. The log V vs pH profile with NAD revealed two pK(a) values too close to be separately determined, but with an average value of 7.33. The log V/K vs pH profile with NAD revealed two pK(a) values at 7.4 and 5.6. Deuterium and 13C isotope effects indicate that the mechanism of human malic enzyme is stepwise with both NAD and epsilonNAD, but that hyperconjugation in the transition state for hydride transfer is detectable only with the former. With thioNAD and APAD, the isotope effects do not clearly indicate whether the mechanism is stepwise or concerted. The intrinsic 13C isotope effect for decarboxylation was calculated to be 1.0485 by measurement of the partition ratio of oxaloacetate in the presence of NADH and human malic enzyme (decarboxylation to pyruvate/reduction to malate = 2.33). The isotope effect and partitioning data suggest that the energy barrier for decarboxylation of oxaloacetate is not as high relative to the barrier for reduction of oxaloacetate as with the chicken liver enzyme.     

Structural studies of the pigeon cytosolic NADP(+)-dependent malic enzyme.

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.