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1.
《Biocatalysis and Biotransformation》2013,31(1):34-38
AbstractThe effect of NADP+ and glucose-6-phosphate (G6P) on the biotransformation of D-xylose to xylitol by cells of Candida guilliermondii permeabilized with surfactant Triton X-100 was evaluated. The experimental runs were performed with 12 g L?1 of permeabilized cells and a reaction medium composed of Tris–HCl buffer (0.1 M pH 7), D-xylose (57 g L?1), and MgCl2.6H2O (5 mM). The levels of NADP+ (from 0.0 to 1.7 mM) and G6P (from 0.00 to 0.17 M) were varied according a 22-full factorial composed design. Under optimized conditions (NADP+ 0.5 mM and 0.05 M G6P), the xylitol volumetric productivity (QP) and yield factor (YP/S) predicted were 1.86 ± 0.03 g L?1 h? 1 and 0.64 ± 0.03 g g?1, respectively. These values were 94% and 19% higher than those obtained with unpermeabilized cells under fermentation conditions (0.97 g L?1 h?1 and 0.53 g g?1, respectively). On the basis of the results, it can be concluded that xylitol production by biotransformation with cells of C. guilliermondii permeabilized with Triton X-100 is a promising alternative to the fermentative process. 相似文献
2.
Summary A preliminary test for the enzymatic conversion of D-xylose into xylitol by the intact cells of Candida pelliculosa (xylose reductase) coupled with the intact cells of a formate-utilizing methanogen strain HU (hydrogenase and F420-NADP oxidoreductase) was conducted by using H2 as an electron donor of NADP+. In the system, NADP(H) was well regenerated via the methanogen cells and about 90% conversion of xylose to xylitol (ca. 8 g/l) could be achieved at 35°C and pH 7.5 after 24 h incubation. 相似文献
3.
《Journal of Fermentation and Bioengineering》1989,67(1):20-24
Cell-free extracts of the xylose fermenting yeast Pichia stipitis exhibited xylitol dehydrogenase activity with NAD+ and NADP+. During the purification step on DEAE-sephadex A-50 a NAD+-dependent xylitol dehydrogenase could be separated from a NADP+-dependent. The NAD+-xylitol dehydrogenase was further purified to electrophoretic homogeneity via gel and affinity chromatography. The purified enzyme was most active at pH 9 and 35°C. Its molecular weight was determined to be 63,000 dalton by Sephadex G-200 column chromatography, and that of its subunit was 32,000 dalton by sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the results of substrate specificity, the enzyme should be named l-iditol:NAD+-5-oxidoreductase (EC 1.1.1.14, sorbitol dehydrogenase). 相似文献
4.
Li Lulu Zhang Ling Wang Dongmei Gao Xiaolian Tamaki Hisanori Kumagai Hidehiko Hong Jiong 《Molecular biotechnology》2013,53(2):159-169
Xylitol dehydrogenase (XDH) (EC 1.1.1.9) is one of the key enzymes in the xylose fermentation pathway in yeast and fungi. A xylitol dehydrogenase gene (XYL2) encoding a XDH was cloned from Kluyveromyces marxianus NBRC 1777, and the in vivo function was validated by disruption and complementation analysis. The highest activity of KmXDH could be observed at pH 9.5 during 55°C. The values of k cat/K m indicate that KmXDH prefers NAD+ to NADP+ (k cat/K m NAD + 3681/min mM and k cat/K m NADP + 1361/min mM). The different coenzyme preference between KmXR and KmXDH caused an accumulation of NADH in the xylose utilization pathway. The redox imbalance may be one of the reasons to cause the poor xylose fermentation under oxygen-limited conditions in K. marxianus NBRC1777. 相似文献
5.
The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NADP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water. 相似文献
6.
Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD+- and NADP+-isocitrate dehydrogenases, NAD+- and NADP+-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP+-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP+-malate dehydrogenase activity is probably due to unspecificity of the NAD+-malate dehydrogenase. NADP+-specific isocitrate dehydrogenase had much lower Kms for NADP+ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD+-specific enzyme (101 micromolar for NAD+ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP+-specific isocitrate dehydrogenase whereas the NAD+-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP+ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP+ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP+-reducing activities of malate dehydrogenase and the NADP+-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes. 相似文献
7.
Selmihan Sahin 《Preparative biochemistry & biotechnology》2013,43(6):590-596
AbstractIn this study, 6-phosphogluconate dehydrogenase was covalently immobilized onto the N-2-aminoethyl-3-aminopropyltriethoxysilane (APTES) modified core-shell Fe3O4@SiO2 magnetic nanoparticles (ASMNPs) using glutaraldehyde (GA). Immobilization of 6PGDH on ASMNPs was confirmed using fourier transform-infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) analysis. The NADP+ conversion ratio, the reusability, thermal, and storage stability of the immobilized 6PGDH were determined and compared with those of the free enzyme. The maximum retention of enzyme activity reached to 96% when the enzyme was immobilized on ASMNPs activated with monomer form of GA. Although the thermal stability of free and immobilized enzymes was similar, at 30?°C, the immobilized 6PGDH showed the improved thermal stability at 40?°C and 50?°C compared with free 6PGDH. While the free 6PGDH only converted 33% of NADP+ in reaction medium upon 480?s, the immobilized 6PGDH performed 56% conversion of NADP+ at same time. The immobilized 6PGDH retained 62% of its initial activity up to the fifth cycle and 35% of its initial activity after 22?days of storage at 4?°C. 相似文献
8.
Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors
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The steady state kinetics of glyceraldehyde 3-phosphate:NADP+ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting Km values of 2 to 5 micromolar for NADP+ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP+ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP+ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP+ (Ki 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP+ and G3P (Ki 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP+ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (Ki 7 micromolar) and Pi (Ki 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP+) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts. 相似文献
9.
A sonicate of Achromobacter parvulus IFO-13182 produced NADPH from NADP+by an NADP+-linked malic enzyme [l-malate: NAD(P)+oxidoreductase, EC 1.1.1.39–40] reaction in the presence of l-malic acid and divalent metal ions. Malic enzyme of A. parvulus was stabilized by 5% l-malic acid, and activity was maintained at 60°C for 1 h. Contaminating phosphatase (orthophosphoricmonoester phosphohydrolase, EC 3.1.3.1–2) was completely inactivated by this treatment. Among the conditions tested, the optimum NADPH production was done using 36 μmol NADP+, 67 μmol l-malic acid, 63 μmol MgCl2 and 1 unit of the malic enzyme in 3 ml of 55 mm phosphate buffer (pH 7.8). Conversion ratio of NADPH from NADP+ reached 100% after 4 h incubation at 30°C and the amount of NADPH accumulated was ~12 μmol ml?1of the reaction mixture. No dephosphorylation of NADP+to NAD+or of NADPH to NADH was found by high performance liquid chromatography. The NADPH produced by such enzymatic reduction was purified by ethanol precipitation and dried in vacuo in powdered form with 97% purity, judged from the ratio of the absorbances at 340 and 260 nm. The purity of the NADPH produced was determined to be 95% from its coenzyme activity with NAD(P)+-linked glutathione reductase [NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2]. 相似文献
10.
Matsushika A Watanabe S Kodaki T Makino K Inoue H Murakami K Takimura O Sawayama S 《Applied microbiology and biotechnology》2008,81(2):243-255
A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor
imbalance between NADPH-preferring XR and NAD+-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity
toward NADP+. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP+-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain
named MA-N5, constructed by chromosomal integration of the gene for NADP+-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and
decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose
but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g
of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose
and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain. 相似文献
11.
Isoenzymes of glucose 6-phosphate dehydrogenase from the plant fraction of soybean nodules 总被引:2,自引:1,他引:1
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Two isoenzymes of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been separated from the plant fraction of soybean (Glycine max L. Merr. cv Williams) nodules by a procedure involving (NH4)2SO4 gradient fractionation, gel chromatography, chromatofocusing, and affinity chromatography. The isoenzymes, which have been termed glucose 6-phosphate dehydrogenases I and II, were specific for NADP+ and glucose 6-phosphate and had optimum activity at pH 8.5 and pH 8.1, respectively. Both isoenzymes were labile in the absence of NADP+. The apparent molecular weight of glucose 6-phosphate dehydrogenases I and II at pH 8.3 was estimated by gel chromatography to be approximately 110,000 in the absence of NADP+ and double this size in the presence of NADP+. The apparent molecular weight did not increase when glucose 6-phosphate was added with NADP+ at pH 8.3. Both isoenzymes had very similar kinetic properties, displaying positive cooperativity in their interaction with NADP+ and negative cooperativity with glucose 6-phosphate. The isoenzymes had half-maximal activity at approximately 10 micromolar NADP+ and 70 to 100 micromolar glucose 6-phosphate. NADPH was a potent inhibitor of both of the soybean nodule glucose 6-phosphate dehydrogenases. 相似文献
12.
Human glucose 6-phosphate dehydrogenase (G6PD) has both the “catalytic” NADP+ site and a “structural” NADP+ site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PDWisconsin (R393G) and G6PDNashville (R393H), and G6PDFukaya (G488S) and G6PDCampinas (G488V), in which the mutations are in the vicinity of the “structural” NADP+ site, showed elevated Kd values of the “structural” NADP+, ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of l-Arg, NADP+ and DTT at 25 °C. The refolding yields of the mutants exhibited strong NADP+-dependence and ranged from 1.5% to 59.4% with 1000 μM NADP+, in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of “structural” NADP+ can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state. 相似文献
13.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1983,742(2):391-398
The kinetics of the NADP+- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism. The Km for dl-pyrroline-5-carboxylic acid is 2.5 mM, for NADP+ is 0.05 mM and for phosphate is 0.35 mM. TheVmax is approx. 8.0 units per mg protein. The reaction is highly specific for the dl-pyrroline-5-carboxylic acid and NADP+, but a number of divalent anions can substitute for phosphate. NADPH is competitive with respect to all three substrates and an analog of γ-glutamyl phosphate, 3-(phosphonoacetylamido)-l-alanine, is competitive with respect to dl-pyrroline-5-carboxylic acid and non-competitive with respect to NADP+ and phosphate, suggesting dead-end complex formation. 相似文献
14.
Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD+-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP+, which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP+ and NAD+. One of the XDH variants utilized NADP+ about 4 times more efficiently than NAD+. This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation. 相似文献
15.
The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP+. Magnesium ions were required not only for light-induced electron transport from water to NADP+ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg2+ lost the capacity to photoreduce NADP+ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP+ by DCIPH2 was independent of Mg2+ (or Cl?) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentrations of MgCl2 produced a strong inhibition of NADP+ photoreduction with DCIPH2 without significantly affecting the rate of NADP+ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP+ with different electron donors are discussed. 相似文献
16.
The subcellular distribution of NADP+ and NAD+-dependent glucose-6-phosphate and galactose-6-phosphate dehydrogenases were studied in rat liver, heart, brain, and chick brain. Only liver particulate fractions oxidized glucose-6-phosphate and galactose-6-phosphate with either NADP+ or NAD+ as cofactor. While all of the tissues examined had NADP+-dependent glucose-6-phosphate dehydrogenase activity, only rat liver and rat brain soluble fractions had NADP+-dependent galactose-6-phosphate dehydrogenase activity. Rat liver microsomal and rat brain soluble galactose-6-phosphate dehydrogenase activities were kinetically different (Km's 0.5 mm and 10 mm, respectively, for galactose-6-phosphate), although their reaction products were both 6-phosphogalactonate. Rat brain subcellular fractions did not oxidize 6-phosphogalactonate with either NADP+ or NAD+ cofactors but phosphatase activities hydrolyzing 6-phosphogalactonate, galactose-6-phosphate and galactose-1-phosphate were found in crude brain homogenates. In addition, galactose-6-phosphate and 6-phosphogalactonate were tested as inhibitors of various enzymes, with largely negative results, except that 6-phosphogalactonate was a competitive inhibitor (Ki = 0.5 mM) of rat brain 6-phosphogluconate dehydrogenase. 相似文献
17.
The activity of NADP+-specific isocitrate dehydrogenase (NADP+-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP+-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP+-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP+-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K m values of the enzymes from green and ripe pericarp for NADP+, isocitrate and Mg2+ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP+-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C6 organic acids and in glutamate accumulation in ripe tissues. 相似文献
18.
Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves 总被引:1,自引:1,他引:0
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Randall DD 《Plant physiology》1981,68(1):70-73
The subcellular location of NADP+-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP+-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP+-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP+-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent Km values of 11 micromolar (NADP+), 35 micromolar (isocitrate), 78 micromolar (Mn2+), 0.3 millimolar (Mg2+) and showed optimum activity at pH 8 to 8.5 with Mn2+ and 8.8 to 9.2 with Mg2+. The NADP+-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP+-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action. 相似文献
19.
Ulrike Demmer Eberhard Warkentin Ankita Srivastava Daniel Kockelkorn Markus P?tter Achim Marx Georg Fuchs Ulrich Ermler 《The Journal of biological chemistry》2013,288(9):6363-6370
Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP+ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP+ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP+, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP+. Evolution from an NADP+ to a bispecific NADP+ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP+ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis. 相似文献
20.
NADP+-isocitrate dehydrogenase (threo-DS-isocitrate: NADP+ oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg2+ or Mn2+, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP+, dl-isocitrate, Mn2+, and Mg2+. Their Km values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP+. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP+ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP+ and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP+-IDH in situ is likely to be controlled by intracellular NADPH to NADP+ ratio as well as by the concentration of various substrates and products. 相似文献