Km Values For NADP Research Articles

Effects of ethanol and other organic solvents on the kinetic behaviour of purified human placental oestradiol-17β-dehydrogenase

The effects of acetone, ethanol, dimethylsulphoxide, glycerol, and propanediol on the formation of reduced coenzyme in the presence of purified human placental oestradiol dehydrogenase were measured fluorometrically at 10 microM oestradiol-17 beta and 500 microM NAD. Addition of increasing amounts of each solvent except glycerol resulted in an initial increase followed by a decline in rate of the enzyme catalyzed reaction. Glycerol caused a linear decline in rate. A range (0.1-25%, v/v) of ethanol concentrations was tested for its effect upon the Km and Vmax values for oestradiol, NAD, and NADP. Over this range the Km value for NADP remained nearly constant (0.6-0.8 microM). The Km value for NAD increased rapidly with an upward convex shape of the curve (6.6-25 microM), and the Km value for oestradiol with NAD or NADP as cofactor increased slowly with an upward concave shape (6.7-25 microM). With increasing ethanol concentration, the maximum velocity increased, reaching its highest values at 10 and 20% ethanol for NAD and NADP, respectively, and then declined. At the maximum Vmax values for NAD and NADP were, respectively, 165 and 200% the values obtained with 0.1% ethanol.

Malic enzyme of Chromatium vinosum

Malic enzyme of the phototropic bacterium Chromatium vinosum strain D that lacks malate dehydrogenase was partially purified yielding a specific activity of 55 units/mg protein. The constitutive enzyme with a molecular weight of 110,000 and a pH optimum of 8.0 was absolutely dependent on the presence of a monovalent cation (NH4+, K+, Cs+, or Rb+) as well as a divalent cation (Mn2+, or Mg2+). The enzyme was inhibited by oxaloacetate, glyoxyate, and NADPH. The K0.5 value for L-malate and the inhibition constants for oxaloacetate and glyoxylate are dependent on the concentration of the monovalent cation, whereas the Km value for NADP (18 microM) and the KI value for NADPH (42 microM) are independent. Throughout all kinetic measurements hyperbolic saturation curves and linear double reciprocal plots were obtained.

Purification and properties of NADP-dependent maltose dehydrogenase produced by alkalophilic Corynebacterium sp. No. 93-1.

NADP-dependent maltose dehydrogenase (NADP-MalDH) was completely purified from the cell free extract of alkalophilic Corynebacterium sp. No. 93-1. The molecular weight of the enzyme was estimated as 45, 000_??_48, 000. The enzyme did not have a subunit structure. The isoelectric point of the enzyme was estimated as pH 4.48. The pH optimum of the enzyme activity was pH 10.2, and it was stable at pH 6 to 8. The temperature optimum was 40°C, and the enzyme was slightly protected from heat inactivation by 1mM NADP. The enzyme oxidized D-xylose, maltose and maltotriose, and the Km values for these substrates were 150mM, 250mM and 270mM, respectively. Maltotetraose and maltopentaose were suitable substrates. The Km value for NADP was 1.5mM with 100mM maltose as substrate. The primary product of this reaction from maltose was estimated as maltono-b-lactone, and it was hydrolyzed non-enzymatically to maltobionic acid. The enzyme was inhibited completely by PCMB, Ag+ and Hg2+.

Properties of NADP-dependent isocitrate dehydrogenase from the mussel Mytilus edulis L.

1.1. NADP-dependent isocitrate dehydrogenase (ICDH) activity has been measured in the digestive gland, mantle, gill, adductor muscle and foot of Mytilus edulis. The highest specific activities were found in the digestive gland and the lowest activities in the adductor muscle and the mantle tissue.2.2. ICDH was partially purified from the digestive gland and mantle tissue. The enzyme from the digestive gland was mainly cytoplasmic. The molecular weight by gel filtration was 71,000 (±7000), for both the digestive gland and mantle enzymes.3.3. ICDH was inactive in the absence of metal ion and in vitro both Mn2+ and Mg2+ were shown to be activators.4.4. The Km values for d-isocitrate, measured over a range of pH from 6.5–8.0 were found to be constant and identical in both tissues (approx 3 μm). The Km value for NADP was approx 3 μM.5.5. The Km values for d-isocitrate and maximal reaction rates were measured over a range of temperatures (5–25°C). The Km values increased with increasing temperature. Q10 values (Vmax) were approx 3 (5–15°C) and 2 (15–25°C) for both tissues.6.6. ICDH from the digestive gland of M. edulis was not inhibited by ATP or palmitoyl-CoA. It was inhibited slightly at 1 mM levels of either glyoxalate or oxaloacetate and was inhibited strongly when the two effectors were added together.

The effects of methanol on the glutamate dehydrogenase reaction at 0 degrees C

The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values.

Substrate Specificity and Kinetic Properties of NADP+—Dependent Alcohol Dehydrogenase ofPhycomyces Blakesleeanus

NADP+-dependent alcohol dehydrogenase (alcohol: NADP+: oxidoreductase, EC 1.1.1.2) was isolated from cell-free extracts of Phycomyces blakesleeanus, and the substrate specificity and kinetic properties of the enzyme were studied. Only primary alcohols were oxidized and of the aldehydes tested, only methanal (formaldehyde) was utilized as a substrate. The enzyme was inactive with NAD+ and was inhibited by pyrazole and by p-chloromercuribenzoate. The apparent Km values at pH 8.3 decreased with increasing chain length of the alcohol substrate. Ethanol had the highest value (1.98x 10M) while the value for 1-heptanol was 2.46 X 10-6M. The maximum velocities (Vmax) decreased as the chain length of the alcohols was increased. The Km value for NADP+ was 1.3 X 10-AIM.

Dehydrogenation of Indanol by Rabbit Liver 3-Hydroxyhexobarbital Dehydrogenase

1. Among the several enzyme activities in rabbit liver cytosol able to dehydrogenate 1-indanol, only the main activity was not separable from 3-hydroxyhexobarbital dehydrogenase during purification including polyacrylamide gel disc electrophoresis. 2. Results of mixed substrate method indicated that the same enzyme catalyses the dehydrogenation of 1-indanol and 3-hydroxyhexobarbital. The ratio between the two dehydrogenation activities was almost constant as the enzyme underwent thermal inactivation. The Ki values of p-chloromercuribenzoate, the Km values for NAD+, and the Km values for NADP+ were very similar for the two dehydrogenations. These results lead to the conclusion that the same enzyme catalyses the dehydrogenation of 3-hydroxyhexobarbital and 1-indanol. 3. 1-Tetralol, 1-acenaphthenol, 9-fluorenol, thiochroman-4-ol and 4-chromanol also served as substrate of the enzyme, but 2-indanol, 2-tetralol, and trans- and cis-indan-1,2-diol were not oxidized. 4. Reversibility of the reaction was also confirmed using 1-indanone as substrate.

Some observations on the NADP+-linked oxidation of methylglyoxal catalysed by 2-Oxoaldehyde dehydrogenase.

In the oxidation of methylglyoxal by 2-oxoaldehyde dehydrogenase, the apparent Km value for NADP+ was about 2.5 times lower than the corresponding Km for NAD+; the apparent Km values for methylglyoxal and for the amine activator L-2-aminopropan-1-ol, with NADP+ as cofactor, were also different from those obtained with NAD+. In the presence of NADP+, the enzyme was not activated by P1, in contrast with the activation of the enzyme when NAD+ was used. The significance of the results is discussed.

Characterization and Regulatory Properties of Glucose‐6‐Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

AbstractGlucose‐6‐phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The Km value for glucose‐6‐phosphate is 1.6 × 10−4 and 6.3 × 10−4M at low (1.0–6.0 × 10−4M) and high (6.0–30.0 × 10−4M) concentrations of the substrate, respectively. The Km value for NADP+ is 1.4 × 10−5M.The enzyme is inhibited by NADPH, 5‐phosphoribosyl‐1‐pyrophosphate, and ATP, and it is activated by Mg2+, and Mn2+. In the presence of NADPH, the plot of activity vs. NADP+ concentration gave a sigmoidal curve. Inhibition of 5‐phosphoribosyl‐1‐pyrophosphate and ATP is reversed by Mg2+ or a high pH.It is suggested that black gram glucose‐6‐phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.

Purification and properties of an NADP-specific 6-phosphogluconate dehydrogenase from Streptococcus faecalis.

A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.

Studies of Glucose Metabolism in <italic>Bacillus subtilis</italic><subtitle>I. Purification of Glucose-6-phosphate Dehydrogenase from the Vegetative Cell and Its Properties in Comparison with the Spore Enzyme<xref ref-type="fn" rid="fn1"><sup>1</sup></xref></subtitle>

1. Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate : NADP-+ 1-oxidoreductase, EC 1.1.1.49] from vegetative cells of Bacillus subtilis was purified about 2,400-fold. The purified enzyme was shown to be homogeneous as judged by chromatographic profile, polyacrylamide gel electrophoresis and ultracentrifugation. The correspondent enzyme from spores was also partially purified. 2. The molecular weight of the vegetative cell enzyme was estimated to be 350,000 by Sepharose 6B chromatography and sedimentation equilibrium studies, and that of its subunit was 58,000 as determined by SDS gel electrophoresis, indicating that the enzyme was found to be 240,000. 3. The vegetative cell enzyme and spore enzyme have the same Km values and pH optima. The optimum pH was about 9.2 for both enzymes and the Km values for NADP-+ and glucose-6-phosphate were determined to be 6.7 X 10-minus 6 and 7.5 X 10-minus 5M, respectively. 4. The amino acid composition of the vegetative cell enzyme was examined and was compared with those of enzymes from other sources.

A new enzyme: the 2'-nucleotidase from latex of Hevea brasiliensis

The cytoplasmic serum of Hevea brasiliensis latex contains a 2′‐nucleotidase. The monoesterase has been isolated and purified 2100 times. Its molecular weight is estimated to be about 115000–120000. Among thenucleotides, of which it hydrolyses the 2′‐phosphate group, NADP+ is utilized mainly and the nucleotides, NADPH, adenosine 2′,5′‐diphosphate, guanosine 2′,5′‐diphosphate, adenosine 2′‐phosphate, in decreasing order. In a few cases (coenzyme A, adenosine 3′,5′‐diphosphate, guanosine 3′,5′‐diphosphate), it reacts very slowly with the 3′‐phosphate group. The enzyme does not hydrolyse adenosine 3′‐phosphate, 5′‐nucleotides and others phosphorylated substrates. The 5′‐phosphate group of the nucleotidic ribose and the type of puric base seem to play an important rôle in the formation of the enzyme‐substrate complex. The optimum pH of the 2′‐nucleotidase is around 5.5 and its Km value for NADP+ is between 0.7 to 1.0mM at pH 5.5 and between 1.4 to 1.8 at pH 7.0. The ions Na+, K+, Mn2+, Mg2+, Ca2+, Co2+, Ni2+ have no influence on the enzymatic reaction; inorganic pyrophosphate, orthophosphate, adenosine, ADP and ATP are competitive inhibitors; NaF, Zn2+, Mo4+ and chiefly Cu2+ ions are strong non‐competitive inhibitors. The Ki of these effectors has been measured. It appears that these molecules, taking into account the concentration and the Ki‐value of ATP and Cu2+ in the latex, exert a physiologic effect on the 2′‐nucleotidase reaction. This enzyme of Hevea brasiliensis latex is probably an important factor in the mechanism of regulation of polysioprene synthesis.

Methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A and its repressibility by serine.

The methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A(k) was purified 100-fold. Because it is extremely labile, this enzyme required protection by 1 mm nicotinamide adenine dinucleotide phosphate (NADP(+)) during purification; 0.01 mm EADP(+) with 0.1% bovine plasma albumin stabilized the purified enzyme during storage at -20 C. Although the enzyme has properties of sulfhydryl enzymes, thiol compounds were not stabilizers. Oxidation of methylenetetrahydrofolate, catalyzed by the purified enzyme preparation, is NADP(+)-specific and yields methenyltetrahydrofolate and the reduced pyridine nucleotide. K(m) values for NADP(+) and for 5,10-methylenetetrahydrofolate (prepared as the formaldehyde adduct of biologically synthesized l,l-tetrahydrofolate) were calculated to be 0.021 and 0.026 mm, respectively. Neither purine bases and their derivatives nor serine inhibited the reaction. In growing cultures, the differential rate of synthesis of the methylenetetrahydrofolate dehydrogenase was dependent upon the composition of the medium. A medium which contained acid-hydrolyzed casein, and thus an exogenous source of serine, was repressive for this enzyme. In a serine-free, completely defined medium, the amount of folate added (for serine synthesis de novo) affected the duration of the initial exponential growth phase. At the termination of this phase, which primarily reflected the onset of a decreased rate of serine biosynthesis, synthesis of the methylenetetrahydrofolate dehydrogenase was derepressed. Exogenous serine in the completely defined medium prevented the derepression. Furthermore, physiological concentrations of l-serine were repressive not only for the dehydrogenase but also for the methenyltetrahydrofolate cyclohydrolase and the serine hydroxymethyl-transferase. Concomitantly, the differential rate of synthesis of the formyltetrahydrofolate synthetase of S. faecium var. durans A(k) was increased. Apparently, serine regulates the differential rates of syntheses of these enzymes.