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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)

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...
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PMID:Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism. 679 May 45

The reduction of nitrate by reduced nicotinamide-adenine dinucleotides, catalysed by extract of Candida utilis, exhibits an apparent high degree of stereospecificity for the 'B' methylene hydrogen atom of NADPH and mixed stereospecificity for the methylene hydrogen atoms of NADH. Purified nitrate reductase, on the other hand, exhibits 'A' stereospecificity for NADH and NADPH. The apparent switch of stereospecificity from the 'B' to the 'A' side of NADPH, which occurs after purification of the enzyme, is partly explained by the fact that in crude extracts nitrate is reduced completely to ammonia. Nitrite does not accumulate but is reduced to ammonia by nitrite dehydrogenase, which is 'B'-specific, so that up to 75% of hydrogen removed from NADPH during the reduction of nitrate could occur from the 'B' side. A further increase in the removal of hydrogen from the 'B' side of NADPH could be the kinetic isotope effect that is observed when ['A'-3H]NADPH is the reductant, the H--C bond being cleaved 2.3 times faster than the 3H--C bond. The mixed stereospecificity observed with NADH has been traced to an uncharacterized enzyme that catalyses a 'B'-specific exchange between NAD+ and NADH. This reaction is discussed in relation to the possibility that it may explain other cases of apparent mixed stereospecificity that have been reported.
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PMID:The stereospecificity of the reduction of nitrate by reduced nicotinamide-adenine dinucleotides catalysed by Candida utilis preparations. 689 Aug 12

The inhibition of the activity of xanthine oxidase by vanadate was strikingly similar to vanadate inhibition of another molybdoprotein nitrate reductase. Although the main catalytic activity of both enzymes was inhibited, the partial NADH oxidase activity associated with these enzymes was stimulated several fold. It appears that the metal ion binds at multiple site in both enzymes. In the absence of any enzymes a combination of vanadium (V) and molybdenum (V) in air was found to oxide NADH rapidly.
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PMID:Effects of vanadate on the molybdoproteins xanthine oxidase and nitrate reductase: kinetic evidence for multiple site interaction. 689 79

Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through in vitro activation of hormone synthesis in callus cells.
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PMID:Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus. 695 74

Two fractions of nitrate reductase inhibitor activities were found in extracts of primary and regenerated roots of nitrate-grown rice seedlings. The inhibitor was proteinaceous in nature and specific to nitrate reductase. The main site of action of the inhibitor was the NADH: cytochrome c reductase component of nitrate reductase. NADH was able to protect the NADH:nitrate reductase against the inhibitor.
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PMID:Nitrate reductase inhibitor of rice plants. 718 23

Fluoride had no effect on in vitro nitrate reductase activity in rice leaves, but in vivo activity was strongly inhibited. It is suggested that fluoride brings about this inhibition by adversely affecting the physiological NADH generating system required for in vivo nitrate reduction.
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PMID:Effect of fluoride on in vivo nitrate reduction in the rive leaves (Oryza sativa L.). 719 73

Demolybdo-nitrate reductase (cytochrome c reductase) (NADH: acceptor oxidoreductase, EC 1.6.99.3) of Chlorella vulgaris can be activated in vitro to nitrate reductase by insertion of Mo from molybdate into the apoprotein. Evidence is here presented that reduction of the enzyme by reduced pyridine nucleotides inhibits the process of molybdenum insertion. This report also describes the effect of molybdate and tungstate concentration on the activation process. The activation is sigmoidally related to molybdate concentration with a calculated Hill coefficient of NH = 3. At suboptimal molybdate concentrations, tungstate stimulates enzyme activation by molybdate; but at saturating molybdate concentrations, tungstate is inhibitory. These facts are regarded as an indication that molybdate and tungstate are both positive effectors of molybdenum incorporation, but that they are competitors for the active Mo center.
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PMID:Effect of reduced pyridine nucleotides and tungstate on the in vitro insertion of molybdenum into demolybdo-nitrate reductase of Chlorella vulgaris. 720 57

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.
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PMID:The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase. 730 57

Based on Lineweaver-Burk plots of the initial velocities, at different concentrations of NADH and nitrate, and product inhibition patterns, an Iso Ping Pong Bi Bi steady state kinetic mechanism is proposed for the spinach nitrate reductase. This mechanism incorporates the concept that the oxidized enzyme is present in two isomeric forms.
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PMID:Studies on the kinetic mechanism of nitrate reductase from spinach (Spinacea oleracea). 743 51

Spectroscopic and kinetic studies comparing the behavior of the recombinant cytochrome b reductase fragment of corn leaf nitrate reductase and a mutant in which cysteine 242 is replaced with a serine residue (C242S) have been carried out. The visible and circular dichroism spectra of the wild-type and mutant protein are virtually identical and compare well with those reported for nitrate reductases from other sources. The reduced wild-type protein forms a charge-transfer complex with NAD+ that has an absorption envelope that extends into the near infrared, with a maximum around 800 nm. The C242S mutant forms a similar charge-transfer complex with NAD+ but to a lesser extent than the wild-type. The reduction potential of the flavin for the wild-type protein is -287 mV, and that for the mutant is -279 mV. The rate of reduction by NADH of the C242S mutant is 7-fold slower than that for the wild-type protein, and the Kd is larger by a factor of 2. These results indicate that the cysteine 242 residue plays a role principally in facilitating electron transfer from NADH to the flavin rather than in binding of NADH to the enzyme.
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PMID:Spectroscopic and kinetic characterization of the recombinant wild-type and C242S mutant of the cytochrome b reductase fragment of nitrate reductase. 759 6


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