Nitrate reductase from Spinacea oleracea. FAD and the inactivation by NAD (P) H.

Treatment by p-hydroxymercuribenzoate of nitrate reductase from spinach leaves causes the disappearance of NADH-diaphorase activity and the appearance of an FAD-requirement for the inactivation by NAD(P)H of FNH₂-nitrate reductase. The diaphorase activity of the treated preparation is not affected by incubation with FAD or the addition of this nucleotide to the assay mixture. Conversely, filtration of the native preparation through a column of Sepharose 6B produces the appearance of an FAD-stimulation of the diaphorase activity, but no effect of FAD on the NADH-inactivation was observed. These differences between the FAD-requirement of NADH-diaphorase activity and NADH-inactivation agree with the postulated independence of the two processes.     

Nitrate reductase from Spinacea oleracea. Reversible inactivation by NAD(P)H and by thiols

The enzymatic complex nitrate reductase from Spinacea oleracea is inactivated by NADH or NADPH and by simple thiols. The inactivation affects FNH₂-nitrate reductase but not NADH-diaphorase. Reactivation can be achieved by addition of ferricyanide. The extent of inactivation by dithioerythritol is increased by NAD⁺, but not by NADP⁺. Nitrate protects against inactivation by NADH or NADPH, and abolishes the effect of NAD⁺ on the inactivation by dithioerythritol. The NAD(P)H-inactivation of nitrate reductase requires that the diaphorase moiety of the complex be functional. However, there is no proportionality between NADH-diaphorase or NADH-nitrate reductase activities and the susceptibility of the enzymatic preparation to NADH or NADPH. It seems likely that the nitrate reductase complex contains a specific regulatory site, different from the catalytic site, the reduction of which is accompanied by the production of an inactive form of the complex.     

Role of molybdenum in nitrate reduction by chlorella

Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The whole enzyme nicotinamide adenine dinucleotide nitrate reductase is formed by cells grown in the absence of added molybdate, but only its first activity (nicotinamide adenine dinucleotide diaphorase) is functional. The second activity of the complex, which subsequently participates also in the enzymatic transfer of electrons from nicotinamide adenine dinucleotide to nitrate (FNH₂-nitrate reductase), depends on the presence of molybdenum. Neither molybdate nor nitrate is required for nitrate reductase synthesis de novo, but ammonia acts as a nutritional repressor of the complete enzyme complex. Under conditions which exclude de novo synthesis of nitrate reductase, the addition of molybdate to molybdenum-deficient cells clearly increases the activity level of this enzyme, thus suggesting in vivo incorporation of the trace metal into the pre-existing inactive apoenzyme.     

In vitro stability of nitrate reductase from wheat leaves: I. Stability of highly purified enzyme and its component activities

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH₂)-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes—methylviologen-nitrate reductase and NADH-ferricyanide reductase—also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH₂-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH₂-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t_½ [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH₂ do not donate electrons to the same site of the nitrate reductase protein.     

Nitrate reductase-deficient mutants in barley: enzyme stability and peptide mapping

Thermal stability and pH optima of NADH-nitrate reductase-associated cytochrome c reductase and FMNH₂-nitrate reductase from wild type, cv Steptoe or Winer, and mutants nar 1d, nar 1g, nar 1h, Xno 18 and Xno 19 were compared to determine if structural differences in the nitrate reductase protein could be detected. Also, the nitrate reductase-associated cytochrome c reductase from nar 1d was purified and compared with the wild type by peptide mapping. The pH optimum for FMNH₂-nitrate reductase from Steptoe and nar 1h, and for NADH-cytochrome c reductase from Steptoe, nar 1d, nar 1g and nar 2a was 7.5. Thermal stabilities of the nitrate reductase-associated activities (FMNH₂-nitrate reductase or NADH-cytochrome c reductase) from nar mutants were less than the Steptoe wild type, while Xno mutants were equal to the Winer wild type. Cleveland peptide maps of nar 1d NADH-cytochrome c reductase and Steptoe nitrate reductase were identicalwhen digested with endoprotease lys-C but were distinctly different in one peptide when digested with Staphylococcus aureus endoprotease V8. These results provide evidence that nar 1 gene codes for the nitrate reductase polypeptide.     

Comparative studies on the induction and inactivation of nitrate reductase in corn roots and leaves

A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH₂-nitrate reductase and NO₃⁻-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO₃⁻ was supplied. WO₄⁼ suppressed the induction of NADH- and FMNH₂-nitrate reductase activities in root tips and leaves. The NO₃⁻-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO₄⁼. In leaves, on the other hand, there was a marked superinduction of the NO₃⁻-induced NADH-cytochrome c reductase in the presence of WO₄⁼.     

Arginine and lysine residues as NADH-binding sites in NADH-nitrate reductase from spinach.

Chemical modifications of spinach leaf nitrate reductase, and its 28,000 M(r) fragment with phenylglyoxal, 2,3-butanedione and pyridoxal phosphate reduce the catalytic activity of the enzyme. The kinetics of the modification indicate a rapid inactivation followed by a slower rate of inactivation. NADH-nitrate reductase, NADH-cytochrome c reductase and NADH-ferricyanide reductase activities of the nitrate reductase complex are inactivated at a faster rate when compared to the loss of FMNH2-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities. NADH protects the inactivation of NADH-ferricyanide reductase activity of the 28,000 M(r) fragment of nitrate reductase. These data suggest that nitrate reductase contains active sites of arginine and lysine residues that are involved in the NADH binding site of the enzyme.     

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora.

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 muM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH2-nitrate reductase of Neurospora suggests some differences in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 mu M) markedly enhanced the action of NAD(P)H on the maize enzyme; Cyanide at a higher level (6 mu M) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no effect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.     

Inhibition of the nitrate reductase complex from spinach by oxylamines

The nitrate reductase complex from spinach (Spinacia oleracea) was found to be inhibited by oxylamine compounds such as aminooxyacetate, hydroxylamine and O-methoxylamine. These compounds appear to interact with reduced cytochrome b₅₅₇ during catalysis of the enzyme. However, if the enzyme is maintained in a reduced state by NADH in the absence of nitrate, an additional component involved in FMNH₂-nitrate reductase is also affected by them. The binding of the oxylamines with the enzyme is non-covalent in nature as the inhibition can be reversed by treatment with 2-oxoglutarate.     

A nitrate reductase inactivating enzyme from the maize root

The nitrate reductase in the mature root extract of 3-day maize (Zea mays) seedlings was relatively labile in vitro. Insoluble polyvinylpyrrolidone used in the extraction medium produced only a slight increase in the stability of the enzyme. Mixing the mature root extract with that of the root tip promoted the inactivation of nitrate reductase in the latter. The inactivating factor in the mature root was separated from nitrate reductase by (NH₄)₂SO₄ precipitation. Nitrate reductase was found in the 40% (NH₄)₂SO₄ precipitate, while the inactivating factor was largely precipitated by 40 to 55% (NH₄)₂SO₄. The latter fraction of the mature root inactivated the nitrate reductase isolated from the root tip, mature root, and scutellum. The inactivating factor, which has a Q₁₀ 15 to 25 C of 2.2, was heat labile, and hence has been designated as a nitrate reductase inactivating enzyme. The reduced flavin mononucleotide nitrate reductase was also inactivated, while an NADH cytochrome c reductase in nitrate-grown seedlings was inactivated but at a slower rate. The inactivating enzyme had no influence on the activity of nitrite reductase, glutamate dehydrogenase, xanthine oxidase, and isocitrate lyase. The activity of the nitrate reductase inactivating enzyme was not influenced by nitrate and was also found in the mature root of minus nitrate-grown seedlings.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg²⁺. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent K_m of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent K_m and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent K_m for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high K_m. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent K_m for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent K_m of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.     

Characterization of the Reversible Inactivation of Ankistrodesmus braunii Nitrate Reductase by Hydroxylamine

The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH₂OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH₂OH. To demonstrate that the terminal activity was readily inactivted by NH₂OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH₂OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH₂OH appears to be competitive versus NO₃⁻. Since NO₃⁻, as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO₃⁻), protect the enzyme from NH₂OH inactivation, it is suggested that NH₂OH binds to the nitrate active site. The NH₂OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN⁻. NH₂OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.     

Further Characterization of Nitrate and Nitrite Reductases from Chlamydomonas reinhardii

The enzymes responsible for nitrate reduction in Chlamydomonas reinhardii, namely NADH-nitrate reductase and ferredoxin-nitrite reductase, have been further characterized. The first activity of the nitrate reducing complex, NADH-diaphorase, is protected by FAD against thermic inactivation. This fact suggests an important structural and functional role for this nucleotide in the first moiety of the nitrate reductase complex. The effect of p-hydroxymercuribenzoate on the diaphorase activity and the protection by NADH against its inactivation indicate that some—SH groups participate in the electron transfer mediated by diaphorase. Radioactive labelling of nitrate reductase with ⁹⁹Mo and ¹⁸⁵W as well as competition experiments between Mo and W indicate that molybdenum is an essential component of terminal nitrate reductase activity. Iron seems to participate in the redox processes mediated by both nitrate and nitrite reductases as suggested by experiments performed at physiological level. Finally a tentative mechanism for the whole process of nitrate assimilation in Chlamydomonas is proposed.     

Affinity labelling of the eukaryotic elongation factor EF-2 with the guanosine nucleotide analogue 5′-p-fluorosulfonylbenzoylguanosine

During the translocation of the nascent peptide chain from the ribosomal aminoacyl-site to the peptidyl-site, GTP is hydrolyzed by a mechanism dependent on both ribosomes and the elongation factor EF-2. For insight into the mechanism of GTP hydrolysis, we studied the ability of the GTP analogue 5′-p-fluorosulfonylbenzoylguanosine (FSO₂BzGuo) to act as an affinity label of the guanine-specific site. Pre-incubation of EF-2 with FSO₂BzGuo at increasing concentrations progressively inactivated the EF-2 and ribosome-dependent GTPase activity. Up to 0.5 mM FSO₂BzGuo, the inactivation of the GTPase activity was stoichiometrically correlated with the covalent binding of [³H]FSO₂BzGuo. Thus, one molecule of covalently bound FSO₂BzGuo completely inactivated the GTPase activity of EF-2. Ribosomes or 60-S ribosomal subunits pre-incubated with FSO₂BzGuo were not inactivated, consistent with the idea that the GTP hydrolysis involved in the ribosomal translocation takes place on EF-2.     

Presence of one essential arginine that specifically binds the 2′-phosphate of NADPH on each of the ketoacyl reductase and enoyl reductase active sites of fatty acid synthetase

Two arginine modifying reagents, phenylglyoxal and 2,3-butanedione, inactivated fatty acid synthetase from goose uropygial gland. This inactivation could be partially prevented by NADP, 2′-AMP, and 2′,5′-ADP, whereas acetyl-CoA and/or malonyl-CoA provided very little protection. Ketoacyl reductase and enoyl reductase activities of fatty acid synthetase showed similar inactivation by phenylglyoxal and butanedione and protection by only NADP and its 2′-phosphate-containing analogs. Furthermore, 2′-AMP was found to be a competitive inhibitor of overall fatty acid synthetase, ketoacyl reductase, and enoyl reductase with apparent K_i values of 1.4, 0.2, and 14 mm, respectively. These results suggest that binding of NADPH to fatty acid synthetase involves specific interaction of the 2′-phosphate with the guanidino group of arginine residues at the active site of the two reductases. Quantitation of the number of arginine residues modified revealed that 4 out of 106 arginine residues per subunit of the synthetase showed high reactivity toward phenylglyoxal. Scatchard analysis showed that two rapidly reacting arginine residues had no effect on the catalytic activity, while modification of two additional arginine residues resulted in complete loss of enzyme activity. Under these conditions, of the seven partial reactions of fatty acid synthetase, only the ketoacyl reductase and enoyl reductase activities were inhibited by phenylglyoxal. The differential reversal of inhibition of the two reductases and the overall activity of fatty acid synthetase, resulting from dialysis of the modified enzyme, suggested that both ketoacyl reductase sites and enoyl reductase sites are required for the full expression of fatty acid synthetase activity. The results of the present chemical modification studies are consistent with the hypothesis that each subunit of fatty acid synthetase contains one ketoacyl reductase and one enoyl reductase and suggest that one essential arginine is present at each of these active sites.     

Hydroxymethylglutaryl coA reductase (NADPH) in the latex of Hevea brasiliensis

The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent K_m for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet.     

Molecular Asymmetry in Pigeonpea Urease: pH Inactivation Studies

Pigeonpea (Cajanus cajan) urease was inactivated by incubating it in buffer of low pHs i.e., 4.8 and 4.5. The pattern of inactivation at both pHs was found to be biphasic, in which half of the activity was destroyed more rapidly than the remaining half. This distribution of active site into two categories is suggestive of site-site heterogeneity, or more specifically, the half-site reactivity of the enzyme moiety. Our pH studies on the rate of reaction showed the presence of two ionizable groups of pK _a values 6.2 ± 0.1 and 8.8 ± 0.1, respectively (Srivastava PK & Kayastha AM, J Mol Catal B: Enz, 16 (2001) 81-89). The later group corresponds to the pK _a value of cysteine group. Here we correlate the loss of urease activity by low pH treatment is due to the effect on essential thiol residues.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).