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.     

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.     

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.     

Effects of ferulic acid on L-malate oxidation in isolated soybean mitochondria

The effects of ferulic acid on L-malate oxidation in mitochondria isolated from soybean (Glycine max L.) seedlings were investigated. Oxygen uptake and the products of L-malate oxidation were measured under two conditions: pH 6.8 and 7.8. At acidic pH, the activity of the NAD+-linked malic enzyme (L-malate:NAD+oxidoreductase [decarboxylating] EC 1.1.1.39) was favoured, whereas at alkaline pH a predominance of the L-malate dehydrogenase activity (L-malate:NAD+oxidoreductase EC 1.1.1.37) was apparent. Ferulic acid inhibited basal and coupled respiration during L-malate oxidation either at acidic or alkaline pH, reducing also the amounts of pyruvate or oxaloacetate produced. The results suggest that the site of ferulic acid action is situated at some step that precedes the respiratory chain. An interference with the L-malate entry into the mitochondria could be an explanation for the effects of ferulic acid, but the possibility of a direct inhibition of both enzymes involved in L-malate oxidation cannot be ruled out. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Studies on regulatory functions of malic enzymes. VI. Purification and molecular properties of NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme [EC 1.1.1.40] was highly purified from Escherichia coli W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471,000, 438,000, and 495,000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6 M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76,000 and 82,000, respectively. The sedimentation coefficient (S(0)20, W) was 13.8S, and the molecular activity was 44,700 min-1 at 30 degrees C. The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g. The Km value for L-malate was 2.3 mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and alpha-keto acid reductase activities. Based on the above results, the molecular properties of the enzymatic reaction are discussed.     

Tartrate dehydrogenase catalyzes the stepwise oxidative decarboxylation of D-malate with both NAD and thio-NAD

Tartrate dehydrogenase catalyzes the divalent metal ion- and NAD-dependent oxidative decarboxylation of D-malate to yield CO(2), pyruvate, and NADH. The enzyme also catalyzes the metal ion-dependent oxidation of (+)-tartrate to yield oxaloglycolate and NADH. pH-rate profiles and isotope effects were measured to probe the mechanism of this unique enzyme. Data suggest a general base mechanism with likely general acid catalysis in the oxidative decarboxylation of D-malate. Of interest, the mechanism of oxidative decarboxylation of D-malate is stepwise with NAD(+) or the more oxidizing thio-NAD(+). The mechanism does not become concerted with the latter as observed for the malic enzyme, which catalyzes the oxidative decarboxylation of L-malate [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103]. It appears the change in mechanism observed with malic enzyme is specific to its transition state structure and not a generalized trait of metal ion- and NAD(P)-dependent beta-hydroxy acid oxidative decarboxylases. The V/K(malate) pH-rate profile decreases at low and high pH and exhibits pK(a) values of about 6.3 and 8.3, while that for V/K(tartrate) (measured from pH 7.5 to pH 9) exhibits a pK(a) of 8.6 on the basic side. A single pK(a) of 6.3 is observed on the acid side of the V(max) pH profile, but the pK(a) seen on the basic side of the V/K pH profiles is not observed in the V(max) pH profiles. Data suggest the requirement for a general base that accepts a proton from the 2-hydroxyl group of either substrate to facilitate hydride transfer. A second enzymatic group is also required protonated for optimum binding of substrates and may also function as a general acid to donate a proton to the enolpyruvate intermediate to form pyruvate. The (13)C isotope effect, measured on the decarboxylation of D-malate using NAD(+) as the dinucleotide substrate, decreases from a value of 1.0096 +/- 0.0006 with D-malate to 1.00787 +/- 0.00006 with D-malate-2-d, suggesting a stepwise mechanism for the oxidative decarboxylation of D-malate. Using thio-NAD(+) as the dinucleotide substrate the (13)C isotope effects are 1.0034 +/- 0.0007 and 1.0027 +/- 0.0002 with D-malate and D-malate-2-d, respectively.     

Staining of argininosuccinate lyase activity in polyacrylamide gel.

A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.     

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.     

Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism

Malic enzymes catalyze the oxidative decarboxylation of L-malate to pyruvate and CO(2) with the reduction of the NAD(P)(+) cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P)(+)-dependent malic enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD(+) or NADH, the divalent cation Mn(2+), and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.     

Active oxidative decarboxylation of malate by mitochondria isolated from L-1210 ascites tumor cells

Mitochondria from L-1210 mouse ascites tumor show a very high rate of oxidation of L-malate in comparison with mitochondria from normal tissues. They were found to contain large amounts of malic enzyme (E.C.1.1.1.39) catalyzing oxidative decarboxylation of L-malate to pyruvate. Malic enzyme in extracts of tumor mitochondria requires Mn²⁺ or Mg²⁺, utilizes either NAD⁺ or NADP⁺ as electron acceptor, and shows positive cooperativity in binding of L-malate. These observations suggest that L-1210 tumor mitochondria actively convert excess tricarboxylate cycle intermediates and their precursors into pyruvate for further oxidation.     

Alternative substrates for malic enzyme: oxidative decarboxylation of L-aspartate

The NAD-malic enzyme from Ascaris suum will utilize L-aspartate, (2S,3R)-tartrate, and meso-tartrate as substrates with V/K values 10(-4)-10(-5) with respect to malate. There is a strict requirement for the 2S stereochemistry for all of these reactants. Since aspartate is unique as an amino acid reactant for malic enzyme, it was informative to determine the details of its mechanism of oxidative decarboxylation. The initial rate of NADH appearance is directly proportional to the concentration of aspartate, and saturation is difficult to achieve. The pH dependence of V/K(aspartate)E(t) shows a decrease at low pH, giving a pK of 5.7. The pH-independent value of V/K(aspartate)E(t) is 3 M(-1) s(-1), 12500-fold lower than that obtained with L-malate. The dissociation constant for aspartate as a competitive inhibitor of malate is 60 mM at neutral pH, allowing an estimate of about 0.18 s(-1) for V/E(t) with L-aspartate compared to a value of 39 s(-1) obtained with L-malate. The deuterium isotope effect on V/K(aspartate) is pH independent over the range 5.1-6.9 with an average value of 3.3. Data suggest that the monoanion of L-aspartate binds to enzyme and that the same general base, general acid mechanism that is responsible for the oxidative decarboxylation of malate to pyruvate applies to the oxidative decarboxylation of aspartate to iminopyruvate. In addition, the oxidation step appears to be largely rate determining with aspartate as the substrate.     

Oxidized NADP as a potential active-site-directed reagent of pigeon liver malic enzyme.

Pigeon liver malic enzyme (malate dehydrogenase (decarboxylating), EC 1.1.1.40) was reversibly inactivated by periodate-oxidized NADP in a biphasic manner. The reversibility could be made irreversible by treating the modified enzyme with sodium borohydride. The inactivation showed saturation kinetics and could be prevented by nucleotide (NADP or NADPH). Fully protection was afforded by the combination of NADP, Mn²⁺ and L-malate. Oxidized NADP was also found to be a coenzyme and noncompetitive inhibitor of L-malate in the oxidative decarboxylase reaction catalyzed by malic enzyme.     

Kinetic mechanism of the cytosolic malic enzyme from human breast cancer cell line.

The kinetic mechanism of the cytosolic NADP(+)-dependent malic enzyme from cultured human breast cancer cell line was studied by steady-state kinetics. In the direction of oxidative decarboxylation, the initial-velocity and product-inhibition studies indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism with NADP+ as the leading substrate followed by L-malate. The products are released in the order of CO2, pyruvate, and NADPH. The enzyme is unstable at high salt concentration and elevated temperature. However, it is stable for at least 20 min under the assay conditions. Tartronate (2-hydroxymalonate) was found to be a noncompetitive inhibitor for the enzyme with respect to L-malate. The kinetic mechanism of the cytosolic tumor malic enzyme is similar to that for the pigeon liver cytosolic malic enzyme but different from those for the mitochondrial enzyme from various sources.     

The pH dependence of the reductive carboxylation of pyruvate by malic enzyme

The maximum velocity of the malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) reductive carboxylation of pyruvate and V/KCO2 are pH-independent from pH 5.5 to pH 8.5. V/K for pyruvate exhibits pK values values of 6.50 +/- 0.25 and 7.25 +/- 0.25. These data suggest that the binding of pyruvate locks the protonation state of enzyme. In addition, the pK values are within experimental error identical for the pH dependence of V/Kmalate and V/Kpyruvate. Thus, the catalytic groups appear to have reverse protonation states in the two reaction directions. The ratio of (V/Kmalate)/(V/Kpyruvate) is 100, suggesting that the protonation state of enzyme is optimum in the malate oxidative decarboxylation direction. Thus, the group with a pK of about 6 is unprotonated and the group with a pK of 7.5 is protonated for malate decarboxylation, and the opposite is true for pyruvate reductive carboxylation.     

The pigeon heart 5''-nucleotidase responsible for ischaemia-induced adenosine formation.

The pH-induced reversible dissociation of pigeon liver malic enzyme (EC 1.1.1.40) was studied by combined use of chemical cross-linking and SDS/polyacrylamide-gel electrophoresis. The tetrameric enzyme showed a pH-dependent dissociation in an acidic environment. At pH values above 8.0 most molecules existed as tetramers. The enzyme was gradually dissociated at lower pH. When the pH was below 5.0 most of the enzyme was present as the monomeric forms. Reassociation of the subunits was accomplished by adjusting the pH to neutrality. The dissociation and reassociation were almost instantaneous. No trimer was detected. The pigeon liver malic enzyme was thus shown to have a double-dimer quaternary structure with D2 symmetry. In the presence of substrates, the monomer-dimer-tetramer equilibrium favours the direction of dissociation. Tartronate, an L-malate analogue, was found to be more effective than L-malate in this process. When the monomeric forms were immobilized, the enzyme subunits were found to be fully active in catalysis. A possible arrangement of the four identical subunits of the enzyme molecule is proposed to account for the results obtained in this investigation. The origin of the half-of-the-sites reactivity of pigeon liver malic enzyme is also discussed.     

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.     

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.     

Mutation of Gly-444 inactivates the S. pombe malic enzyme

A mutant malic enzyme gene, mae2⁻, was cloned from a strain of Schizosaccharomyces pombe that displayed almost no malic enzyme activity. Sequence analysis revealed only one codon-altering mutation, a guanine to adenine at nucleotide 1331, changing the glycine residue at position 444 to an aspartate residue. Gly-444 is located in Region H, previously identified as one of eight highly conserved regions in malic enzymes. We found that Gly-444 is absolutely conserved in 27 malic enzymes from various prokaryotic and eukaryotic sources, as well as in three bacterial malolactic enzymes investigated. The evolutionary conservation of Gly-444 suggests that this residue is important for enzymatic function.     

pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme.

The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme has been used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V/K for L-malate decreases when groups with pKs of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be water coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pKs displaced outward by 1.4 pH units, since the rate-limiting step is normally TPNH release, and the chemical reaction, which is pH sensitive, is 25 times faster. TPN binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of a proton to the group with pK 5.4-6, and enolpyruvate is protonated by water coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.