Purification and properties of two malate dehydrogenases from Candida sp. N-16 grown on methanol

Two malate dehydrogenases (MDH-M1 and MDH-M2) were found in a methanol-using yeast, Candida sp. N-16. MDH-M2 was induced with methanol. These enzymes were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The molecular weights of MDH-M1 and MDH-M2 were estimated to be about 78,000 (homodimer) and 160,000 (homotetramer). Several kinetic properties were significantly different between the two enzymes. The value (2.07) of Vmax(oxaloacetate)/Vmax(malate) and Kcats (555 s(-1) for oxaloacetate, 481 s(-1) for NADH) of MDH-M2 were higher than the ratio (1.37) of Vmax and Kcats (241 s(-1) for oxaloacetate, 271 s(-1) for NADH) of MDH-M1, respectively. The activity of MDH-M2 was inhibited by a high concentration of NAD+ and the activity of MDH-M1 by oxaloacetate.     

Cloning, sequencing and functional expression of cytosolic malate dehydrogenase from Taenia solium: Purification and characterization of the recombinant enzyme

We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517 Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409 U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. K_cat values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962 s⁻¹, respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56 Kb genomic contig assembly is also reported.     

Biochemical differences between cytoplasmic malate dehydrogenase allozymes of Drosophila virilis

Two allozymes (MDH^f and MDH^s) of cytoplasmic malate dehydrogenase of Drosophila virilis were partially purified and their biochemical properties were compared. MDH^f has a pH optimum of 9.75 and MDH^s one of 9.25 for malate oxidation. Optimal pH for oxaloacetate reduction is 6.75 and 8.0 for MDH^f and MDH^s, respectively. The K_m value for oxaloacetate of MDH^s is approximately twice as that of MDH^f. No differences were found with respect to thermostability and K_m's for malate, NAD⁺, or NADH. These results are discussed in terms of the physiological role of cytoplasmic malate dehydrogenase of D. virilis.This work was supported in part by grants from the Ministry of Education, Japan, Nos. 134050 and 154205.     

Identification and Biochemical Characterization of a Thermostable Malate Dehydrogenase from the Mesophile Streptomyces coelicolor A3(2)

We identified and characterized a malate dehydrogenase from Streptomyces coelicolor A3(2) (ScMDH). The molecular mass of ScMDH was 73,353.5 Da with two 36,675.0 Da subunits as analyzed by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry (MALDI-TOF-MS). The detailed kinetic parameters of recombinant ScMDH are reported here. Heat inactivation studies showed that ScMDH was more thermostable than most MDHs from other organisms, except for a few extremely thermophile bacteria. Recombinant ScMDH was highly NAD⁺-specific and displayed about 400-fold (k _cat) and 1,050-fold (k _cat?K _m) preferences for oxaloacetate reduction over malate oxidation. Substrate inhibition studies showed that ScMDH activity was inhibited by excess oxaloacetate (K _i=5.8 mM) and excess L-malate (K _i=12.8 mM). Moreover, ScMDH activity was not affected by most metal ions, but was strongly inhibited by Fe²⁺ and Zn²⁺. Taken together, our findings indicate that ScMDH is significantly thermostable and presents a remarkably high catalytic efficiency for malate synthesis.     

Effect of sulphite on phosphoenolpyruvate carboxylase and malate formation in extracts of Zea mays

The effect of SO₃^2? on the activity of PEP-carboxylase and on subsequent malate formation has been studied in leaf extracts of Zea mays. PEP-carboxylase was assayed by incorporation of H¹⁴CO₃ - into oxaloacetate dinitrophenylhydrazone and by a spectrophotometric method. In contrast to ribulose diphosphate carboxylase, PEP-carboxylase was not inhibited by 10 mM SO₃^2? with respect to PEP. As was the case with ribulose diphosphate carboxylase, the activity of PEP-carboxylase was inhibited non-competitively with respect to Mg²⁺. However, the K_i value (84.5 mM) was found to be very high. With respect to HCO₃^?, like ribulose diphosphate carboxylase, PEP-carboxylase was inhibited competitively, but the K_i value (27 mM SO₃^2?) increased by about the same factor (× 9) as the K_m, (0·5 mM HCO₃^?) is decreased. This indicates that the replacement of HCO₃^? by SO₃^2?, common to both enzymes, is facilitated by decreasing the affinity of the enzyme for HCO₃^?. At substrate saturating conditions malate formation by the combined action of PEP-carboxylase and endogenous NADH-dependent malate dehydrogenase in leaf extracts was not inhibited by 10 mM SO₃^2?. Although the malate dehydrogenase is inhibited at this SO₃^2? concentration to about 85%, malate formation is unaffected, as PEP-carboxylase is the rate limiting step its turnover rate being only about 8% of NADH-dependent malate dehydrogenase.     

Influence of L-Leucine on Glutamate Dehydrogenase Activity in Isolated Rat Diaphragm

Glutamate dehydrogenase (NAD) activity was measured in liver and diaphragm mitochondria from 48 h fasted rats. Kinetic studies were performed with diaphragm enzyme and the effects of L-leu, ADP and L-ala on the K¹m and V¹max for NH₄+, and α-ketoglutarate were evaluated. L-leucine increases by 2-8 fold the V¹max for all three substrates with no significant changes in the K¹m. ADP increased by 3-7 fold the V¹max for all three substrates and the K¹m for NADH and α-ketoglutarate by 1.5-7.0 fold. L-alanine had no effect on either the V¹max or the K¹m of any substrate. The results suggest that muscle has the capacity to form glutamate through the glutamate dehydrogenase reaction and that L-leucine may stimulate this reaction in muscle.     

GLYOXYLATE CYCLE ENZYME ACTIVITIES IN THE CYANOBACTERIUM ANACYSTIS NIDULANS1

The enzymes of the glyoxylate cycle, isocitrate lyase (EC.4.1.3.1) and malate synthase (EC.4.1.3.2), were measured in cell-free extracts from the cyanobacterium Anacystis nidulans Drouet during photoautotrophic growth in medium aerated with ordinary air (0.03% CO₂). Isocitrate lyase had an average specific activity of 112 nmoles·min^?1·mg protein^?1 whereas malate synthase had an average specific activity of 12.5 nmoles·min^?1·mg protein^?1. Unpurified isocitrate lyase showed classical Michaelis kinetics with a K_m of 8 mM. Isocitrate lyase activity was strongly inhibited by numerous cellular metabolites at 10 mM concentration. The previously reported low specific activity for isocitrate lyase may be due to metabolite inhibition caused by growth in high CO₂ concentrations. The activities reported for isocitrate lyase and malate synthase suggest the operation of the glyoxylate cycle in Anacystis nidulans under CO₂-limiting growth conditions.     

Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> MA-4680

A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42°C and displays a half-life (t _1/2) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k _cat value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k _cat/K _m ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over l-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP⁺ were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess l-malate. Enzymatic activity is enhanced by the addition of Na⁺, NH₄ ⁺, Ca²⁺, Cu²⁺ and Mg²⁺ and inhibited by addition of Hg²⁺ and Zn²⁺. MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

Kinetic characterization and thermostability of C. elegans cytoplasmic and mitochondrial malate dehydrogenases

Malate dehydrogenase (MDH) catalyzes the conversion of NAD⁺ and malate to NADH and oxaloacetate in the citric acid cycle. Eukaryotes have one MDH isozyme that is imported into the mitochondria and one in the cytoplasm. We overexpressed and purified Caenorhabditis elegans cytoplasmic MDH-1 and mitochondrial MDH-2 in E. coli. Our goal was to compare the kinetic and structural properties of these enzymes because C. elegans can survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we measured K_M values for oxaloacetate of 54 and 52 μM and K_M values for NADH of 61 and 107 μM for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH-1 and MDH-2 from a mixed population of worms and separated them using anion exchange chromatography. Both endogenous enzymes had a K_M for oxaloacetate similar to that of the corresponding recombinant enzyme. Recombinant MDH-1 and MDH-2 had maximum activity at 40 °C and 35 °C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Protein homology modeling predicted that MDH-1 had more intersubunit salt-bridges than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer intersubunit ionic interactions compared to mammalian MDH2 enzymes. These results suggest that the increased stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.     

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria

Mitochondria isolated from spinach leaves oxidized malate by both a NAD⁺-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumulation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition.Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD⁺-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate.The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition.The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD⁺/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD⁺-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD⁺-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.     

Kinetic and functional properties of human mitochondrial phosphoenolpyruvate carboxykinase

The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn²⁺ ions for activity; Mg²⁺ ions reduce the K_m for Mn²⁺ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (k_cat=2.7 s^?1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis.     

Light dependent reduction of nitrate by pea chloroplasts in the presence of nitrate reductase and C4-dicarboxylic acids

In the presence of purified nitrate reductase (NR) and 1 mM NADH, illuminated pea chloroplasts catalysed reduction of NO₃^? to NH₃ with the concomitant evolution of O₂. The rates were slightly less than those for reduction of NO₂^? to NH₃ and O₂, evolution by chloroplasts in the absence of NR and NADH (ca 6 μg atoms N/mg Chl/hr). Illuminated chloroplasts quantitatively reduced 0.2 mM oxaloacetate (OAA) to malate. In the presence of an extrachloroplast malate-oxidizing system comprised of NAD-specific malate dehydrogenase (NAD-MDH), NAD, NR and NO₃^?, illuminated chloroplasts supported OAA-dependent reduction of NO₃^? to NH₃ with the evolution of O₂. The reaction did not proceed in the absence of any of these supplements or in the dark but malate could replace OAA. The results are consistent with the reduction of NO₃^?by reducing equivalents from H₂O involving a malate/OAA shuttle. The ratios for O₂, evolved: C₄-acid supplied and N reduced: C₄-acid supplied in certain experiments imply recycling of the C₄-acids.     

A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity

The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V_max with no apparent effect on K_m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.     

Determination of NAD Malic Enzyme in Leaves of C(4) Plants : EFFECTS OF MALATE DEHYDROGENASE AND OTHER FACTORS

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn²⁺-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.     

Evaluation of kinetic data: What the numbers tell us about PRMTs

Protein arginine N-methyltransferase (PRMT) kinetic parameters have been catalogued over the past fifteen years for eight of the nine mammalian enzyme family members. Like the majority of methyltransferases, these enzymes employ the highly ubiquitous cofactor S-adenosyl-l-methionine as a co-substrate to methylate arginine residues in peptidic substrates with an approximately 4-μM median K_M. The median values for PRMT turnover number (k_cat) and catalytic efficiency (k_cat/K_M) are 0.0051 s⁻¹ and 708 M⁻¹ s⁻¹, respectively. When comparing PRMT metrics to entries found in the BRENDA database, we find that while PRMTs exhibit high substrate affinity relative to other enzyme-substrate pairs, PRMTs display largely lower k_cat and k_cat/K_M values. We observe that kinetic parameters for PRMTs and arginine demethylase activity from dual-functioning lysine demethylases are statistically similar, paralleling what the broader enzyme families in which they belong reveal, and adding to the evidence in support of arginine methylation reversibility.     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

Purification,catalytic and regulatory properties of malate dehydrogenase from the foot of Patella caerulea (L.)

• 1.1. Malate dehydrogenase has been purified from the foot muscle of Patella caerulea by ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Blue Agarose and gel filtration on Sephadex G-150.
• 2.2. The yield was 23.5% of the initial activity with a final specific activity of 257 U/mg of protein.
• 3.3. The apparent mol. wt of the native enzyme is approx. 75,000 and it consists of two subunits of mol. wts in the range of 36,000–39,000.
• 4.4. The enzyme exhibits hyperbolic kinetics with respect to oxaloacetate, NADH and l-malate. The K_m values were determined to be 0.055 mM for oxaloacetate, 0.010 mM for NADH and 0.37 mM for l-malate. The pH optima are around 8.4 for the reduction of oxaloacetate and 9.2–9.6 for the reduction of oxaloacetate and 9.2–9.6 for the l-malate oxidation. V_max and K_m values for oxaloacetate change in an opposite manner with respect to pH values.
• 5.5. Of the various compounds tested, only α-ketoglutarate, citrate and adenylate phosphates were found to inhibit the enzyme activity.
• 6.6. From the above properties it appears that the reaction of cytoplasmic malate dehydrogenase of P. caerulea foot muscle is a key reaction in the anaerobic pathway and it occurs with the production of malate.

     

Kinetic studies of killer toxin K1 binding to yeast cells indicate two receptor populations

A recently described new method for determination of killer toxin activity was used for kinetic measurenments of K1 toxin binding. The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates. The low-affinity and high-velocity binding component (K _T1=2.6x10⁹ L.U./ml, V _max1=0.19 s^-1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (K _T2=3.2x10⁷ L.U./ml, V _max2=0.03 s^-1) presumably indicates the binding of the toxin to plasma membrane receptors. Adsorption of most of the killer toxin K1 to the surface of sensitive cells occured within 1 min and was virtually complete within 5 min. The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S. cerevisiae S6.