Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.2 (nitrate reductase)
3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.
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PMID:[Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)]. 615 80

A study has been made of e.p.r. signals due to Mo(V) in reduced sulphite oxidase (EC 1.8.3.1) from chicken liver. Reduction by SO3(2-), or photochemically in the presence of a deazaflavin derivative, produces spectra indistinguishable from one another. Three types of spectra from the enzyme were distingusihed and shown to correspond to single chemical species, since they could be simulated at both 9 and 35 GHz by using the same parameters. These were the low-pH form of the enzyme, with gav. 1.9805, the high-pH form, with gav. 1.9681 and a phosphate complex, with gav. 1.9741. The low-H form shows interaction with a single exchangeable proton, with A(1H)av. (hyperfine coupling constant) = 0.98 mT, probably in the form of an MoOH group. Parameters of the signals are compared with those for signals from xanthine oxidase and nitrate reductase. The signal from the phosphate complex of sulphite oxidase in unique among anion complexes of Mo-containing enzymes in showing no hyperfine coupling to protons. There is no evidence for additional weakly coupled protons or nitrogen nuclei in the sulphite oxidase signals. The possibility is considered that the enzymic mechanism involves abstraction of a proton and two electrons from HSO3- by a Mo = O group in the enzyme.
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PMID:Electron-paramagnetic-resonance parameters of molybdenum(V) in sulphite oxidase from chicken liver. 624 54

The three enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa strain PAO were induced strongly (50- to 100-fold) by a shift from aerobic growth conditions to very low oxygen tension. Arginine in the culture medium was not essential for induction, but increased the maximum enzyme levels twofold. The induction of the three enzymes arginine deiminase (EC 3.5.3.6), catabolic ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3) appeared to be coordinate. Catabolic ornithine carbamoyltransferase was studied in most detail. Nitrate and nitrite, which can replace oxygen as terminal electron acceptors in P. aeruginosa, partially prevented enzyme induction by low oxygen tension in the wild-type strain, but not in nar (nitrate reductase-negative) mutants. Glucose was found to exert catabolite repression of the deiminase pathway. Generally, conditions of stress, such as depletion of the carbon and energy source or the phosphate source, resulted in induced synthesis of catabolic ornithine carbamoyltransferase. The induction of the deiminase pathway is thought to mobilize intra- and extracellular reserves of arginine, which is used as a source of adenosine 5'-triphosphate in the absence of respiration.
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PMID:Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa. 625 88

Chlorella nitrate reductase catalyzes the reduction of nitrate to nitrite by NADH. Initial velocity studies showed that the kinetic mechanism is sequential, indicating that both substrates must bind to the enzyme before any products are released. Product inhibition with NAD and nitrite showed that competitive inhibition was observed when the inhibitor was similar to the varied substrate, while noncompetitive inhibition was observed when the inhibitor was dissimilar to the varied substrate. Likewise, dead-end inhibition with adenosine 5'-diphosphoribose and thiocyanate showed competitive inhibition when the inhibitor was similar to the varied substrate and noncompetitive inhibition when the inhibitor was dissimilar to the varied substrate. These results indicate that Chlorella nitrate reductase follows a random bi bi kinetic mechanism. Phosphate was found to stimulate NADH:nitrate reductase activity and 2-fold. The NADH:cytochrome c reductase activity associated with nitrate reductase was not affected by phosphate suggesting the effect of phosphate is on the nitrate-reducing moiety of the enzyme. Phosphate increases Vmax but has no effect on the apparent Km for nitrate.
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PMID:Kinetic mechanism of assimilatory NADH:nitrate reductase from Chlorella. 627 5

The carbon monoxide oxidases (COXs) purified from the carboxydotrophic bacteria Pseudomonas carboxydohydrogena and Pseudomonas carboxydoflava were found to be molybdenum hydroxylases, identical in cofactor composition and spectral properties to the recently characterized enzyme from Pseudomonas carboxydovorans (O. Meyer, J. Biol. Chem. 257:1333-1341, 1982). All three enzymes exhibited a cofactor composition of two flavin adenine dinucleotides, two molybdenums, eight irons and eight labile sulfides per dimeric molecule, typical for molybdenum-containing iron-sulfur flavoproteins. The millimolar extinction coefficient of the COXs at 450 nm was 72 (per two flavin adenine dinucleotides), a value similar to that of milk xanthine oxidase and chicken liver xanthine dehydrogenase at 450 nm. That molybdopterin, the novel prosthetic group of the molybdenum cofactor of a variety of molybdoenzymes (J. Johnson and K. V. Rajagopalan, Proc. Natl. Acad. Sci. U.S.A. 79:6856-6860, 1982) is also a constituent of COXs from carboxydotrophic bacteria is indicated by the formation of identical fluorescent cofactor derivatives, by complementation of the nitrate reductase activity in extracts of Neurospora crassa nit-l, and by the presence of organic phosphate additional to flavin adenine dinucleotides. Molybdopterin is tightly but noncovalently bound to the protein. COX, sulfite oxidase, xanthine oxidase, and xanthine dehydrogenase each contains 2 mol of molybdopterin per mol of enzyme. The presence of a trichloroacetic acid-releasable, so-far-unidentified, phosphorous-containing moiety in COX is suggested by the results of phosphate analysis.
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PMID:Molybdopterin in carbon monoxide oxidase from carboxydotrophic bacteria. 658 59

The molybdenum cofactor common to a variety of molybdoenzymes has been shown to contain a novel pterin. The pterin has been isolated from sulfite oxidase from several sources, xanthine-oxidizing enzymes from milk and chicken liver, and nitrate reductase of Chlorella vulgaris after denaturation of the proteins in the presence of I2. Investigation of the anionic nature of the isolated pterin has revealed that it is a monophosphate ester susceptible to cleavage by alkaline phosphatase. Quantitative analyses have shown that one molecule of the pterin phosphate is associated with each molybdenum atom in sulfite oxidase. Studies to date have shown that the pterin is present in a reduced form in sulfite oxidase and xanthine dehydrogenase, and that in situ oxidation of the pterin leads to inactivation of sulfite oxidase.
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PMID:The pterin of the molybdenum cofactor. 695 16

Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems. The two enzymes are encoded by distinct operons located within two different loci on the E. coli chromosome. The narGHJI operon, encoding nitrate reductaseA, is located in the chlC locus at 27 minutes, along with several functionally related genes: narK, encoding a nitrate/nitrite antiporter, and the narXL operon, encoding a nitrate-activated, two component regulatory system. The narZYWV operon, encoding nitrate reductase Z, is located in the chlZ locus located at 32.5 minutes, a region which includes a narK homologue, narU, but no apparent homologue to the narXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both the narGHJI operon and the narK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while the narZYWV operon and the narU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase in E. coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.
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PMID:Nitrate reductases in Escherichia coli. 774 40

Assimilatory nitrate reductase from Chlorella vulgaris catalyzes the rate-limiting step, the conversion of nitrate to nitrite, in nitrate assimilation. Initial rate studies of nitrate reductase activity, performed under optimum conditions of constant ionic strength (mu = 0.2) and pH (8.0) and using NADH as reductant, indicated the absence of substrate inhibition at NADH concentrations below 300 microM and NO3- concentrations less than 3 mM. Chlorella nitrate reductase exhibited a marked preference for NADH (Vmax = 9.2 mumol NADH/min/nmol heme and Km = 2.3 microM) as the physiological electron donor but could also utilize alpha-NADH (Vmax = 5.6 mumol NADH/min/nmol heme and Km = 131 microM) and NADPH (Vmax = 0.6 mumol NADPH/min/nmol heme and Km = 910 microM) though with significantly decreased efficiency. Examination of various NADH-analogs indicated that reduced nicotinamide hypoxanthine dinucleotide (NHDH) was used most efficiently (Vmax = 9.3 mumol NHDH/min/nmol heme and Km = 7.9 microM), while reduced nicotinamide mononucleotide (NMNH) was utilized least efficiently (Vmax = 0.07 mumol NMNH/min/nmol heme and Km = 676 microM). Overall, modifications to the nicotinamide moiety or the addition of a phosphate group were observed to result in the most significant decreases in Vmax, indicating poor reducing substrates. Product inhibition studies indicated both NAD+ (Ki = 2.2 mM) and NADP+ (Ki = 10.5 mM) to be competitive inhibitors of Chlorella NR. A variety of NAD+ analogs were also determined to act as competitive inhibitors with varying degrees of efficiency. 3-Pyridinealdehyde adenine dinucleotide was the most efficient inhibitor (Ki = 0.74 mM) while nicotinamide was the least efficient (Ki = 18.1 mM). Overall, changing substituents on the nicotinamide ring or its complete deletion produced the most effective inhibitors compared to NAD+. In contrast, changes in the adenine or ribose moieties produced less effective inhibitors when compared to NAD+. These results represent the most comprehensive analysis of the effect of modifications of the physiological reductant (NADH) and product (NAD+) on nitrate reductase activity.
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PMID:Assimilatory nitrate reductase: reduction and inhibition by NADH/NAD+ analogs. 797 4

The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulatory system responsible for control of the anaerobic respiratory pathway genes in Escherichia coli, including nitrate reductase (narGHJI), dimethyl sulfoxide/trimethylamine-N-oxide reductase (dmsABC), and fumarate reductase (frdABCD) operons among others. The two membrane-bound proteins NarX and NarQ can independently sense the presence of nitrate and transfer this signal to the DNA-binding regulatory protein NarL, which controls gene expression by transcriptional activation or repression. To establish the role of protein phosphorylation in this process and to determine whether the NarX and NarQ proteins differ in their interaction with NarL, the cytoplasmic domains of NarX and NarQ were overproduced and purified. Both proteins were autophosphorylated in the presence of [gamma-32P]ATP and MgCl2 but not with [alpha-32P]ATP. Whereas these autophosphorylation reactions were unaffected by the presence of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor. The phosphorylated forms of 'NarX and 'NarQ were stable for hours at room temperature. Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at an apparently much faster rate than did 'NarX-phosphate. In addition, NarL was autophosphorylated with acetyl phosphate but not with ATP as a substrate. NarL-phosphate remained phosphorylated for at least 3 h. However, addition of 'NarX resulted in rapid dephosphorylation of NarL-phosphate. In contrast, 'NarQ exhibited a much slower phosphatase activity with NarL-phosphate. These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ability to interact with NarL.
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PMID:Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli. 805 Oct 11

The narL gene product is a nitrate-responsive activator and repressor of anaerobic respiratory gene expression. Mutational studies and sequence comparisons have suggested that NarL protein binding sites contain heptameric sequences related to the consensus, TACYNMT (where Y = C or T, M = A or C, and N = any nucleotide). There are four NarL heptamers in the -105 region of the fdnGHI (formate dehydrogenase-N) operon, and mutational analysis supports the role of these heptamers in nitrate induction. To examine NarL-DNA interactions, we purified the NarL protein as a maltose binding protein (MBP) fusion protein (MBP-NarL). A constitutive mutant form with a single substitution (V88A) in the amino-terminal (response regulator) region was used. The MBP-NarL (V88A) protein protected all four heptamers in the fdnG operon control region from DNase I cleavage. Identical footprints were observed with NarL (V88A) protein that had been proteolytically cleaved free from the MBP domain. Binding of MBP-NarL (V88A) protein to the four heptamers in the -105 region of the fdnG operon appeared to be cooperative, and occupancy of the central heptamers was necessary for occupancy of the flanking heptamers. In addition to the V88A substitution, a low molecular weight phosphodonor, such as acetyl phosphate, was required for observable footprints. This indicates that phosphorylation of the NarL protein enhances its affinity for its multiple DNA targets in the fdnG operon, perhaps by increasing protein-protein interactions rather than protein-DNA interactions. We also performed footprinting studies at the narGHJI (nitrate reductase), narK (nitrite efflux), and frdABCD (fumarate reductase) operon control regions. Extensive areas of each control region were protected from DNase I attack by phosphorylated MBP-NarL (V88A) protein. The narG operon control region was protected from positions -50 to -110, and, at higher protein concentrations, also around position -200. Mutational analysis indicates that the NarL heptamer centered at position -89, in addition to the previously-identified -200 region, is involved in nitrate induction. Comparisons of the four operon control regions studied indicate that the NarL heptamers are arranged with diverse orientations and spacing.
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PMID:In vitro interaction of nitrate-responsive regulatory protein NarL with DNA target sequences in the fdnG, narG, narK and frdA operon control regions of Escherichia coli K-12. 805 56


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