Vol. 131, No. 3 Printed in U.S.A.
JOUtNAL or BAcTZioLoGY, Sept. 1977, p. 88-890 Copyright 0 1977 American Society for Microbiology
Regulation of the Neurospora crassa Assimilatory Nitrate Reductase PAUL A. KETCHUM,* DEBORAH D. ZEEB, AND MARTHA S. OWENS' Department of Biological Sciences, Oakland University, Rochester, Michigan 48063 Received for publication 11 March 1977
Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the K. of NADPHnitrate reductase for NADPH but did not significantly change that for nitrate. The K,. of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source. Assimilation of nitrate nitrogen in Neuro- reductase and nitrite reductase, exposure to spora crassa proceeds via the reductive activi- suitable levels of ammonium ions results in an ties of reduced nicotinamide adenine dinucleo- accelerated decrease in the activities of these tide phosphate (NADPH)-nitrate reductase enzymes (5, 21). Regulation of NADPH-nitrate reductase has (EC 1.6.6.2) and NADPH-nitrite reductase (EC 1.6.6.4). Both enzymes are synthesized in Neu- justifiably been studied, since this is the first rospora when the organism is grown on either enzyme in the pathway of nitrate assimilation nitrate or nitrite as the sole nitrogen source (5, and since the obvious repressor, ammonia, does 12). These enzymes are soluble electron trans- not affect purified NADPH-nitrate reductase port enzymes that contain heme iron and re- activity (8). Assuming that the organic prod: quire flavin (7, 8, 13). NADPH-nitrate reduc- ucts of ammonia assimilation serve as regulatase catalyzes the two-electron reduction of ni- tory metabolites in place of ammonia, we investrate to nitrite, and NADPH-nitrite reductase tigated the effects of metabolites on purified catalyzes the six-electron reduction of nitrite to nitrate reductase and found that certain amino ammonia (8, 13). The coupling ofelectron trans- acids modulate NADPH-nitrate reductase acport to high-energy phosphate bond formation tivity. by these soluble electron transfer enzymes has MATERIALS AND METHODS not been observed. and growth. N. crassa wild type (FGSC Reduction of nitrate to ammonia, therefore, 354)Cultures the Fungal Genetic Stock was obtained requires the expenditure of energy by utilizing Culture Collection,from Humboldt State University 4 mol of NADPH per mol of ammonia produced. Foundation, Arcata, Calif. Cultures were mainNeurospora is able to bypass this energy ex- tained and grown on Fries medium supplemented penditure if a reduced source of nitrogen (i.e., with 5 g of Casamino Acids (Difco Laboratories, ammonia, amino acids, etc.) is available in the Detroit, Mich.) per liter. Mycelia were derepressed medium. In the presence of these reduced nitro- for nitrate reductase by harvesting, washing with gen sources, Neurospora forms neither distilled water, and transferring 18 g (wet weight) of NADPH-nitrate reductase nor NADPH-nitrite mycelia into 800 ml of Fries medium containing 6.9 g NaNO, per liter, adjusted to pH 4.5 with H3PO4 reductase (5, 12, 19). Furthermore, when Neu- of (12). rospora contains derepressed levels of nitrate Enzyme asays. NADPH-nitrate reductase was I Present address Department of Biochemistry, Univerassayed by measuring (8) the amount of nitrite formed during 10 min of enzyme incubation in 0.5 ml
sity of Wyoming, Laramie, WY 82070.
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of assay mixture containing 10 i,mol of nitrate, 100 Results were analyzed by linear regression. The nmol of NADPH, 5 nmol of flavin adenine dinucleo- correlation coefficients of the linear regression analtide, and 30 ,umol of KH2PO4-K2HPO4 buffer (pH yses were >0.96. 7.3) at 23°C. One unit of NADPH-nitrate reductase equals 1 nmol of nitrite formed per 10 min. Protein RESULTS was determined by the modified Folin-Lowry procedure, with bovine serum albumin as a standard (15). Ammonia assimilation in Neurospora utiAmino acids and nucleotides were obtained from lizes metabolic precursors generated by the triSigma Chemical Co., St. Louis, Mo. Only the L- carboxylic acid cycle (24) and results in the forms of the amino acids were used. N-acetyl gluta- formation of glutamine metabolic products mate, N-acetyl-glutamine, and N-acetyl glycine (10, 11, 25, 26). The synthetic NADPH-specific were obtained from U.S. Biochemical Corp. Purification of NADPH-nitrate reductase. Crude glutamate dehydrogenase in N. crassa is reguextracts of N. crassa wild-type (FGSC 354) unfrozen lated by the precursors of ammonia assimilamycelia, derepressed for 3 h on Fries-NO3- medium, tion and is stimulated by EDTA (24). The abwere prepared by homogenizing 1 g (wet weight) of sence of this enzyme in mutants of N. crassa mycelia per 3 ml of preparation buffer: 0.1 M (manuscript in preparation) and Aspergillus K2HPO4-KH2PO buffer (pH 7.3) containing 5 x 10-4 nidulans (1, 9) results in unusual kinetics for M ethylenediaminetetraacetic acid (EDTA). The nitrate reductase formation. These observa20,000 x g supernatant fraction was treated with clupine sulfate by the Downey procedure (4). The tions led us to the following investigation of clupine sulfate-treated supemnatant fraction was nitrate reductase regulation by metabolites, 35% saturated with ammonium sulfate at 4°C and which are precursors to, or products of, ammocentrifuged at 20,000 x g for 20 min. This supemna- nia assimilation. tant fraction was then 50% saturated with ammoNADPH-nitrate reductase was assayed in nium sulfate and centrifuged, and the resulting pel- the presence of metabolites that derive at least let was suspended in the buffer described above. The one of their nitrQgen atoms from glutamine (26) 35 to 50% ammonium sulfate fraction was dialyzed (Table 1). The enzyme was extensively dialyzed against 0.001 M K2HPO4-KH2PO4 buffer (pH 7.3) containing 5 x 10-4 M EDTA and then placed on a against phosphate buffer to remove EDTA, Whatman DE-52 anion-exchange column (12 by 1.5 since EDTA stimulates nitrate reductase as cm) equilibrated in fresh dialysis buffer. NADPH- well as NADPH-glutamate dehydrogenase (24). nitrate reductase was eluted with a linear gradient Histidine, tryptophan, glycine, arginine, glutaof 0.001 and 0.5 M K2HPO4-KH2PO4 buffer (pH 7.3) mate, glutamine, and alanine activated containing 5 x 10-4 M EDTA. The peak tubes were NADPH-nitrate reductase 25- to 46-fold when pooled, and the nitrate reductase activity was con- assayed in the absence of EDTA under the concentrated by ammonium sulfate fractionation (0 to ditions reported in Table 1. Neither adenosine 50%). The resuspended pellet (1.0 to 2.0 ml) was 5'-monophosphate nor uridine had a significant chromatographed on a Sephadex G-200 column (2.5 effect on NADPH-nitrate reductase activity. by 40 cm) equilibrated in preparation buffer. The fractions containing NADPH-nitrate reductase ac- Adenosine 5'-diphosphate and cytidine 5'-tritivity were pooled and dialyzed against 0.1 M phosphate stimulated nitrate reductase activK2HPO4-KH2PO4 buffer (pH 7.3) before use in the ity, but this stimulation was not additive with described enzyme experiments. All procedures were the amino acid stimulation. 2-Oxoglutarate apperformed at 4°C. The total recovery and specific peared to inhibit the nitrate reductase even in activity of NADPH-nitrate reductase purified by the presence of stimulating amino acids. Inhibithis procedure varied with the effectors present in tion was also observed when nitrate reductase the assay, as explained in the text. However, when was assayed in the presence of both uridine and EDTA was used throughout, fold purification varied The additives listed in Table 1 did from 14 to 37, percent recovery varied from 14 to 1.8, glutamate. and specific activities varied from 3,400 to 9,360 U/ not affect the chemical assay for nitrite when the enzyme was absent. Stimulation of nitrate mg of protein. Enzyme kinetics. All additives were prepared in reductase activity by the amino acids occurred 0.1 M K2HPO4-KH2PO4 buffer (pH 7.3) and adjusted immediately and was not increased by preincuto pH 7.3 if necessary. Enzyme assays were per- bation with the enzyme at either 0 or 4°C for 5, formed at room temperature in a total volume of 0.5 10, or 20 min. EDTA apparently binds tightly to ml (unless otherwise stated). When NADPH was nitrate reductase, since extensive dialysis was used as the variable substrate, pyridine nucleotides required before attaining stimulating effects by were removed from the assay mixture by the barium these amino acids. precipitation procedure (5) before the nitrite conAmino acid analogs of glutamine, glutamate, centration was determined. The concentrations of NADPH used in the kinetic assays were 140, 90, 70, and glycine did not significantly stimulate ni60, 50, 40, 30, and 20 nmol per assay. The concentra- trate reductase. N-acetyl glutamate, N-acetyl tions of nitrate used in the kinetic assays were 200, glycine, and N-acetyl glutamine at a final con160, 100, 80, 60, 40, 30, and 20 nmol per assay. centration of 1 mM increased the nitrate reduc-
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TABLZ 1. Effects of metabolites on purified NADPH-nitrate reductasea nmol of nitrite formed/10 min Additiveb
Histidine Tryptophan Glycine Arginine Glutamate Glutamine Alanine 15.2 16.7 19.5 14.8 16.4 19.3 0.6 28.1 15.2 14.8 16.7 14.8 16.7 25.2 16.4 0.5 14.2 18.6 16.2 13.8 15.7 24.8 17.4 4.5 16.2 14.8 13.3 14.9 16.2 5.6 17.6 17.6 16.2 13.3 9.3 15.2 10.5 0.1 28.0 20.4 13.1 14.8 11.9 22.4 14.0 11.9 0.0 11.7 2-Oxoglutarate a Purified NADPH-nitrate reductase from wild-type N. crdssa was extensively dialyzed against 0.1 M KH2PO4-K2HPO4 buffer (pH 7.3), incubated with the compound(s) indicated for 10 min, and asayed for 10 min after the addition of NADPH, flavine adenine dinucleotide, and nitrate. Each tube contained 3 jAg of protein and 2.5 itmol of the compounds listed in a total volume of 0.5 ml. Each compound tested individually had no effect on the nitrite asay. The undialyzed enzyme preparation formed 2.56 nmol ofnitrite per 10 mi per 3 lsg of protein. The compounds tested derive at least one nitrogen from glutamine (26) or are involved in glutamine formation or metabolism. ' AMP, Adenosine 5'-monophosphate; ADP, adenosine 5'-diphosphate; CTP, cytidine 5'triphosphate. Control
Control .......... AMP ........... ADP ........... CTP ........... Uridine ......... ...
tase activity by no more than 15%, whereas the natural amino acids more than doubled it. Citrate was the only intermediate in the tricarboxylic acid cycle that increased NADPHnitrate reductase activity (Table 2). Citrate stimulated nitrate reductase approximately 10fold, whereas histidine and EDTA stimulated it 60- and 50-fold, respectively. Therefore, tricarboxylic acid cycle intermediates that activate NADPH-glutamate dehydrogenase in N. crassa (23) do not exert parallel control over nitrate reductase in N. crassa. Two possible mechanisms for the stimulation of nitrate reductase by these amino acids, EDTA, and citrate, are: chelation of heavy metals, which inhibit the enzyme; and/or activation ofnitrate reductase at multiple sites or at a common site by allosteric mechanisms. If the chelation mechanism were correct, then the removal of all inhibitory metals would prevent the observed decrease in nitrate reductase ac-
tivity when ED1A was removed by dialysis. This mechanism would not require physical interaction between the chelator and nitrate reductase. Alternatively, the allosteric mechanism would require the physical interaction between the enzyme and the activator. Experiments with Chelex resini (Bio-Rad Laboratories, Richmond, Calif.) were designed to physically remove the activators from the enzyme. EDTA-treated enzyme was dialyzed against a buffer that contained Chelex resin (Table 3). This resin contains imminodiacetate functional groups and chemically removes heavy metals from solution in a manner identical to that of EDTA. After dialysis of the enzyme against phosphate or phosphate-Chelex buffer, nitrate reductase activity was greatly reduced (Table 3). Therefore, maintenance of
TABLz 2. Activity of wild-type N. crassa nitrate reductase in the presence of tricarboxylic acid cycle intermediatesa Additive
Nitrate reductase
Expt Malate ............... Oxaloacetate Citrate ............... Succinate Isocitrate ............
0.47 0.47 8.1 0 0
.........
............
activity
Control 30.4 Histidine ............. 24.3 EDTA ............... 0.47 None ................ * Enzyme, dialyzed against 0.1 M KH2PO4K2HPO, buffer (pH 7.3), was incubated in this buffer containing 2.5 smol of the additive (pH 7.3) in a 0.3ml final volume for 10 min before the assay for NADPH-nitrate reductase. Each tube contained 7 ,Ag of protein. The activity is expressed as nanomoles of nitrite formed per 10 min.
enzymatic activity during dialysis requires the physical interaction of EDTA and the enzyme and is not solely dependent upon the chelating ability of EDTA. The activity lost during dialysis was restored when these enzyme preparations were assayed in the presence of EDTA (Table 4). Assay of these dialyzed enzyme preparations in the presence of amino acids (Table 4) demonstrated significant nitrate reductase activity. When the dialysis buffer contained only phosphate, the greatest stimulation was observed when EDTA was added to the assay mixture. When EDTA was present, adding amino acids during the assay did not result in stimula-
NADPH-NITRATE REDUCTASE REGULATION
VOL. 131, 1977
TABLz 3. Chelex dialysis experiment Aa Dialysis buffer Assay buffer
Phosphate Phosphate ......... Phosphate-EDTA . . Phosphate-Chelex..
7 82 12
Phosphate- PhosphateEDTA 94 100 93
Chelex 15 83 17
6 The buffer used was 0.1 M K2HPO4-KH2PO4 (pH 7.3) with or without the additive 0.5 mM EDTA or 2 mg of Chelex (2.9 meq/g [dry weight]) per ml. Enzyme, dialyzed against the indicated buffer solution, was diluted 40-fold in fresh buffer solution and then assayed in the indicated buffer. The figures reported are percentages of the maximum observed activity, which was 7,800 U/ml of dialyzed enzyme containing 12.32 mg of protein per ml.
TABLz 4. Chelex dialysis experiment Ba Dialysis and assay buffer Addition to assay
Phosphate
None .......... EDTA .........
Glutamate..... Glutamine
.....
Glycine ........ Alanine Arginine
.......
......
Tryptophan
....
10 63 45 53 43 44 47 38
PhosphateEDTA 95 91 92 95 87 92 94 90
PhosphateChelex 11 77 68 71 62 65 70 61
* The buffer used was 0.1 M K2HPO4-KH2PO4 (pH 7.3) with or without the additive 0.5 mM EDTA or 2 mg of Chelex (2.9 meq/g [dry weight]) per ml. The amino acid concentration in the assays was 5 mM, and EDTA was 0.5 mM. Enzyme, dialyzed against the indicated buffer solution, was diluted 40-fold in fresh buffer solution and then asayed in the indicated buffer solutions plus additions. The figures reported are percentages of the maximum observed activity, which was 7,800 U/ml of dialyzed enzyme containing 12.32 mg of protein per ml.
tion. Presumably the nitrate reductase was maximally activated by the concentration of EDTA present. Dialysis of nitrate reductase against phosphate-Chelex buffer resulted in an 89% activity loms, which was significantly restored by adding either EDTA or one of the amino acids used in experiment B. A parallel experiment, in which all reagents were treated with Chelex before constructing the assay mixtures, was performed. Under these conditions, histidine, citrate, glutamate, and glutamine at 5 mM and EDTA at 1 mM restored nitrate reductase activity to the enzyme preparations that were dialyzed against Chelex. An analogous experiment was to purify nitrate reductase in the absence of EDTA. This preparation had a specific activity of 331, representing a 12% recovery, and was significantly stimulated when assayed in the presence of the amino acids listed in Table 1 or EDTA. These experi-
887
ments indicate that EDTA and the other effectors must be in physical contact with nitrate reductase to stimulate this enzyme. Therefore, amino acids, citrate, and EDTA do not stimulate nitrate reductase by chelation of heavy metals or other inhibitors. Kinetics. An alternative mechanism for activating nitrate reductase involves amino acid altering the Km of the NADPH-nitrate reductase for nitrate, NADPH, or both. The Km for each substrate was determined by holding one substrate in constant excess and varying the other. Before the kinetic assays, the enzyme was extensively dialyzed to remove EDTA and then assayed in the presence of different concentrations of a given amino acid. Three amino acid concentrations were chosen (usually falling at the bottom, middle, and top) from the sigmoidal curve obtained by plotting the concentration of the amino acid versus the nitrate reductase activity. Double-reciprocal plots of nitrate reductase activity versus NADPH concentration for glutamine, glutamate, tryptophan, and histidine were linear. Increasing concentrations of these amino acids increased the velocity of the reaction and also altered the Vmax. Histidine affected nitrate reductase activity at concentrations of 20 to 100 uM. These concentrations were two orders of magnitude lower than the effective concentrations of the other amino acids. The kinetics of nitrate reductase activity in the presence of alanine, glycine, and arginine were similar to those of glutamate and glutamine. Increasing concentrations from 0.5 to 2 mM increased the velocity of the reaction and increased the Vmax. Stimulation by increasing concentrations of arginine, alanine, and glycine was less than that for the other amino acids. Moreover, 2 mM concentrations of arginine and alanine resulted in maximal stimulation. The relative stimulation by different amino acids in these kinetic experiments is similar to the relative stimulation observed in Table 1. Finally, increasing concentrations of citrate stimulated nitrate reductase, though it is a poorer stimulator than the amino acids since high concentrations were required to in-
duce stimulation. The apparent Km values of nitrate reductase for NADPH in the presence of these amino acids are compiled in Table 5. These Km values varied from 14 to 360 ,uM when determined in the presence of 10 mM glutamine and 2 mM histidine, respectively. A 100-fold increase in the histidine concentration (20 ,uM to 2 mM) increased the Km by more than 10-fold (Table 5). Therefore, increasing the concentration in-
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creased the Km of nitrate reductase for NADPH. Tryptophan and glutamine had the opposite effect; i.e. increased concentrations of these amino acids decreased the apparent K. of nitrate reductase for NADPH (Table 5). Increasing the concentration of alanine, arginine, glutamate, or glycine by 10-fold resulted in less than a 2-fold increase in the apparent Km for NADPH. Apparent Km values of nitrate reductase for NADPH in the presence of amino acid mixtures were not determined. Similar kinetic experiments to determine the Km of NADPH-nitrate reductase for nitrate in the presence of amino acids were performed. The apparentKm for nitrate varied between 210 and 380 ,uM, when the seven amino acids were present individually in the assay (Table 6). The small variation in the enzyme's Km values for nitrate suggests that a significant control is not exerted by amino acids at the nitrate site. Attempts to measure the Km values of nitrate reductase for nitrate and NADPH in the absence of additives were unsuccessful. Low enzymatic activity and/or high protein concentrations resulted in unreliable results with low correlation coefficients. T&rLz 5. Apparent Km and Vm., values of NADPHnitrate reductase for NADPH in the presence of effectors Effector concn (mM) Alanine (2) ......... Glutamate (2) ...... Glutamine (2) ...... Glutamine (10) ..... Histidine (0.02) ..... Histidine (2) ........ Glycine (2) .........
Tryptophan (1) Tryptophan (4) Arginine (2)
......
......
........
K. value for NADH (!LM) 50 60 28
V
(U) 26.0 5.4 4.3 6.1 26.0 50.0 49.0 9.6 11.0 51.0
14
33 360 100 150 70 87
EDTA (0.5) .........
100
24.0
Citrate (1) ..........
100
7.7
TABLz 6. Apparent Km values of NADPH-nitrate reductase for nitrate in the presence of effectors Effector concn (mM)
Alanine (5) ................ Arginine (5) ............... Glutamate (5) ............. Glutamine (5) ............. Glycine (5) ................ Histidine (5)............... Tryptophan (5)............. EDTA (0.5)
................
Apparent K,. value of nitrate (AtM) 250 380 210 250 225 210 260 310
DISCUSSION Assimilation of nitrate nitrogen is a major biological process in plants, fungi, and some bacteria. The key enzyme to be regulated in this process is NADPH-nitrate reductase, since it is the first enzyme in the assimilatory process. Regulation of the de novo synthesis of nitrate reductase has been extensively investigated (12, 14, 19, 22). Once formed, the enzyme requires further regulation, since it is energy utilizing, and recent reports (3, 18) suggest that it has an extremely high turnover number. Nitrate reductase from N. crassa at specific activities of 80 to 100 Amol of nitrite formed per min per mg of protein (S. S. Pan et al., Fed. Proc., abstr. 2636, 34:682, 1975) and from Escherichia coli at 229 ymol of nitrite formed per min per mg of protein (3) have turnover numbers of 153 and 440 mol/s per gram atom of molybdenum (assuming 2 mol of molybdenum per enzyme). These numbers are significantly higher than those for nitrogenase (2), which were reported as 1 mol/s per gram atom of molybdenum. Therefore, N. crassa requires minute quantities of nitrate reductase and must control the rate of nitrate reduction lest it consume energy (NADPH) unnecessarily. Other investigators (8, 20) have not observed any effect of amino acids on nitrate reductase. We attribute this apparent conflict with our results to the presence of EDTA in their preparations and/or to the use of a different strain of N. crassa. EDTA, at concentrations of
JOUtNAL or BAcTZioLoGY, Sept. 1977, p. 88-890 Copyright 0 1977 American Society for Microbiology
Regulation of the Neurospora crassa Assimilatory Nitrate Reductase PAUL A. KETCHUM,* DEBORAH D. ZEEB, AND MARTHA S. OWENS' Department of Biological Sciences, Oakland University, Rochester, Michigan 48063 Received for publication 11 March 1977
Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the K. of NADPHnitrate reductase for NADPH but did not significantly change that for nitrate. The K,. of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source. Assimilation of nitrate nitrogen in Neuro- reductase and nitrite reductase, exposure to spora crassa proceeds via the reductive activi- suitable levels of ammonium ions results in an ties of reduced nicotinamide adenine dinucleo- accelerated decrease in the activities of these tide phosphate (NADPH)-nitrate reductase enzymes (5, 21). Regulation of NADPH-nitrate reductase has (EC 1.6.6.2) and NADPH-nitrite reductase (EC 1.6.6.4). Both enzymes are synthesized in Neu- justifiably been studied, since this is the first rospora when the organism is grown on either enzyme in the pathway of nitrate assimilation nitrate or nitrite as the sole nitrogen source (5, and since the obvious repressor, ammonia, does 12). These enzymes are soluble electron trans- not affect purified NADPH-nitrate reductase port enzymes that contain heme iron and re- activity (8). Assuming that the organic prod: quire flavin (7, 8, 13). NADPH-nitrate reduc- ucts of ammonia assimilation serve as regulatase catalyzes the two-electron reduction of ni- tory metabolites in place of ammonia, we investrate to nitrite, and NADPH-nitrite reductase tigated the effects of metabolites on purified catalyzes the six-electron reduction of nitrite to nitrate reductase and found that certain amino ammonia (8, 13). The coupling ofelectron trans- acids modulate NADPH-nitrate reductase acport to high-energy phosphate bond formation tivity. by these soluble electron transfer enzymes has MATERIALS AND METHODS not been observed. and growth. N. crassa wild type (FGSC Reduction of nitrate to ammonia, therefore, 354)Cultures the Fungal Genetic Stock was obtained requires the expenditure of energy by utilizing Culture Collection,from Humboldt State University 4 mol of NADPH per mol of ammonia produced. Foundation, Arcata, Calif. Cultures were mainNeurospora is able to bypass this energy ex- tained and grown on Fries medium supplemented penditure if a reduced source of nitrogen (i.e., with 5 g of Casamino Acids (Difco Laboratories, ammonia, amino acids, etc.) is available in the Detroit, Mich.) per liter. Mycelia were derepressed medium. In the presence of these reduced nitro- for nitrate reductase by harvesting, washing with gen sources, Neurospora forms neither distilled water, and transferring 18 g (wet weight) of NADPH-nitrate reductase nor NADPH-nitrite mycelia into 800 ml of Fries medium containing 6.9 g NaNO, per liter, adjusted to pH 4.5 with H3PO4 reductase (5, 12, 19). Furthermore, when Neu- of (12). rospora contains derepressed levels of nitrate Enzyme asays. NADPH-nitrate reductase was I Present address Department of Biochemistry, Univerassayed by measuring (8) the amount of nitrite formed during 10 min of enzyme incubation in 0.5 ml
sity of Wyoming, Laramie, WY 82070.
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of assay mixture containing 10 i,mol of nitrate, 100 Results were analyzed by linear regression. The nmol of NADPH, 5 nmol of flavin adenine dinucleo- correlation coefficients of the linear regression analtide, and 30 ,umol of KH2PO4-K2HPO4 buffer (pH yses were >0.96. 7.3) at 23°C. One unit of NADPH-nitrate reductase equals 1 nmol of nitrite formed per 10 min. Protein RESULTS was determined by the modified Folin-Lowry procedure, with bovine serum albumin as a standard (15). Ammonia assimilation in Neurospora utiAmino acids and nucleotides were obtained from lizes metabolic precursors generated by the triSigma Chemical Co., St. Louis, Mo. Only the L- carboxylic acid cycle (24) and results in the forms of the amino acids were used. N-acetyl gluta- formation of glutamine metabolic products mate, N-acetyl-glutamine, and N-acetyl glycine (10, 11, 25, 26). The synthetic NADPH-specific were obtained from U.S. Biochemical Corp. Purification of NADPH-nitrate reductase. Crude glutamate dehydrogenase in N. crassa is reguextracts of N. crassa wild-type (FGSC 354) unfrozen lated by the precursors of ammonia assimilamycelia, derepressed for 3 h on Fries-NO3- medium, tion and is stimulated by EDTA (24). The abwere prepared by homogenizing 1 g (wet weight) of sence of this enzyme in mutants of N. crassa mycelia per 3 ml of preparation buffer: 0.1 M (manuscript in preparation) and Aspergillus K2HPO4-KH2PO buffer (pH 7.3) containing 5 x 10-4 nidulans (1, 9) results in unusual kinetics for M ethylenediaminetetraacetic acid (EDTA). The nitrate reductase formation. These observa20,000 x g supernatant fraction was treated with clupine sulfate by the Downey procedure (4). The tions led us to the following investigation of clupine sulfate-treated supemnatant fraction was nitrate reductase regulation by metabolites, 35% saturated with ammonium sulfate at 4°C and which are precursors to, or products of, ammocentrifuged at 20,000 x g for 20 min. This supemna- nia assimilation. tant fraction was then 50% saturated with ammoNADPH-nitrate reductase was assayed in nium sulfate and centrifuged, and the resulting pel- the presence of metabolites that derive at least let was suspended in the buffer described above. The one of their nitrQgen atoms from glutamine (26) 35 to 50% ammonium sulfate fraction was dialyzed (Table 1). The enzyme was extensively dialyzed against 0.001 M K2HPO4-KH2PO4 buffer (pH 7.3) containing 5 x 10-4 M EDTA and then placed on a against phosphate buffer to remove EDTA, Whatman DE-52 anion-exchange column (12 by 1.5 since EDTA stimulates nitrate reductase as cm) equilibrated in fresh dialysis buffer. NADPH- well as NADPH-glutamate dehydrogenase (24). nitrate reductase was eluted with a linear gradient Histidine, tryptophan, glycine, arginine, glutaof 0.001 and 0.5 M K2HPO4-KH2PO4 buffer (pH 7.3) mate, glutamine, and alanine activated containing 5 x 10-4 M EDTA. The peak tubes were NADPH-nitrate reductase 25- to 46-fold when pooled, and the nitrate reductase activity was con- assayed in the absence of EDTA under the concentrated by ammonium sulfate fractionation (0 to ditions reported in Table 1. Neither adenosine 50%). The resuspended pellet (1.0 to 2.0 ml) was 5'-monophosphate nor uridine had a significant chromatographed on a Sephadex G-200 column (2.5 effect on NADPH-nitrate reductase activity. by 40 cm) equilibrated in preparation buffer. The fractions containing NADPH-nitrate reductase ac- Adenosine 5'-diphosphate and cytidine 5'-tritivity were pooled and dialyzed against 0.1 M phosphate stimulated nitrate reductase activK2HPO4-KH2PO4 buffer (pH 7.3) before use in the ity, but this stimulation was not additive with described enzyme experiments. All procedures were the amino acid stimulation. 2-Oxoglutarate apperformed at 4°C. The total recovery and specific peared to inhibit the nitrate reductase even in activity of NADPH-nitrate reductase purified by the presence of stimulating amino acids. Inhibithis procedure varied with the effectors present in tion was also observed when nitrate reductase the assay, as explained in the text. However, when was assayed in the presence of both uridine and EDTA was used throughout, fold purification varied The additives listed in Table 1 did from 14 to 37, percent recovery varied from 14 to 1.8, glutamate. and specific activities varied from 3,400 to 9,360 U/ not affect the chemical assay for nitrite when the enzyme was absent. Stimulation of nitrate mg of protein. Enzyme kinetics. All additives were prepared in reductase activity by the amino acids occurred 0.1 M K2HPO4-KH2PO4 buffer (pH 7.3) and adjusted immediately and was not increased by preincuto pH 7.3 if necessary. Enzyme assays were per- bation with the enzyme at either 0 or 4°C for 5, formed at room temperature in a total volume of 0.5 10, or 20 min. EDTA apparently binds tightly to ml (unless otherwise stated). When NADPH was nitrate reductase, since extensive dialysis was used as the variable substrate, pyridine nucleotides required before attaining stimulating effects by were removed from the assay mixture by the barium these amino acids. precipitation procedure (5) before the nitrite conAmino acid analogs of glutamine, glutamate, centration was determined. The concentrations of NADPH used in the kinetic assays were 140, 90, 70, and glycine did not significantly stimulate ni60, 50, 40, 30, and 20 nmol per assay. The concentra- trate reductase. N-acetyl glutamate, N-acetyl tions of nitrate used in the kinetic assays were 200, glycine, and N-acetyl glutamine at a final con160, 100, 80, 60, 40, 30, and 20 nmol per assay. centration of 1 mM increased the nitrate reduc-
886
KETCHUM, ZEEB, AND OWENS
J. BACTZRIOL.
TABLZ 1. Effects of metabolites on purified NADPH-nitrate reductasea nmol of nitrite formed/10 min Additiveb
Histidine Tryptophan Glycine Arginine Glutamate Glutamine Alanine 15.2 16.7 19.5 14.8 16.4 19.3 0.6 28.1 15.2 14.8 16.7 14.8 16.7 25.2 16.4 0.5 14.2 18.6 16.2 13.8 15.7 24.8 17.4 4.5 16.2 14.8 13.3 14.9 16.2 5.6 17.6 17.6 16.2 13.3 9.3 15.2 10.5 0.1 28.0 20.4 13.1 14.8 11.9 22.4 14.0 11.9 0.0 11.7 2-Oxoglutarate a Purified NADPH-nitrate reductase from wild-type N. crdssa was extensively dialyzed against 0.1 M KH2PO4-K2HPO4 buffer (pH 7.3), incubated with the compound(s) indicated for 10 min, and asayed for 10 min after the addition of NADPH, flavine adenine dinucleotide, and nitrate. Each tube contained 3 jAg of protein and 2.5 itmol of the compounds listed in a total volume of 0.5 ml. Each compound tested individually had no effect on the nitrite asay. The undialyzed enzyme preparation formed 2.56 nmol ofnitrite per 10 mi per 3 lsg of protein. The compounds tested derive at least one nitrogen from glutamine (26) or are involved in glutamine formation or metabolism. ' AMP, Adenosine 5'-monophosphate; ADP, adenosine 5'-diphosphate; CTP, cytidine 5'triphosphate. Control
Control .......... AMP ........... ADP ........... CTP ........... Uridine ......... ...
tase activity by no more than 15%, whereas the natural amino acids more than doubled it. Citrate was the only intermediate in the tricarboxylic acid cycle that increased NADPHnitrate reductase activity (Table 2). Citrate stimulated nitrate reductase approximately 10fold, whereas histidine and EDTA stimulated it 60- and 50-fold, respectively. Therefore, tricarboxylic acid cycle intermediates that activate NADPH-glutamate dehydrogenase in N. crassa (23) do not exert parallel control over nitrate reductase in N. crassa. Two possible mechanisms for the stimulation of nitrate reductase by these amino acids, EDTA, and citrate, are: chelation of heavy metals, which inhibit the enzyme; and/or activation ofnitrate reductase at multiple sites or at a common site by allosteric mechanisms. If the chelation mechanism were correct, then the removal of all inhibitory metals would prevent the observed decrease in nitrate reductase ac-
tivity when ED1A was removed by dialysis. This mechanism would not require physical interaction between the chelator and nitrate reductase. Alternatively, the allosteric mechanism would require the physical interaction between the enzyme and the activator. Experiments with Chelex resini (Bio-Rad Laboratories, Richmond, Calif.) were designed to physically remove the activators from the enzyme. EDTA-treated enzyme was dialyzed against a buffer that contained Chelex resin (Table 3). This resin contains imminodiacetate functional groups and chemically removes heavy metals from solution in a manner identical to that of EDTA. After dialysis of the enzyme against phosphate or phosphate-Chelex buffer, nitrate reductase activity was greatly reduced (Table 3). Therefore, maintenance of
TABLz 2. Activity of wild-type N. crassa nitrate reductase in the presence of tricarboxylic acid cycle intermediatesa Additive
Nitrate reductase
Expt Malate ............... Oxaloacetate Citrate ............... Succinate Isocitrate ............
0.47 0.47 8.1 0 0
.........
............
activity
Control 30.4 Histidine ............. 24.3 EDTA ............... 0.47 None ................ * Enzyme, dialyzed against 0.1 M KH2PO4K2HPO, buffer (pH 7.3), was incubated in this buffer containing 2.5 smol of the additive (pH 7.3) in a 0.3ml final volume for 10 min before the assay for NADPH-nitrate reductase. Each tube contained 7 ,Ag of protein. The activity is expressed as nanomoles of nitrite formed per 10 min.
enzymatic activity during dialysis requires the physical interaction of EDTA and the enzyme and is not solely dependent upon the chelating ability of EDTA. The activity lost during dialysis was restored when these enzyme preparations were assayed in the presence of EDTA (Table 4). Assay of these dialyzed enzyme preparations in the presence of amino acids (Table 4) demonstrated significant nitrate reductase activity. When the dialysis buffer contained only phosphate, the greatest stimulation was observed when EDTA was added to the assay mixture. When EDTA was present, adding amino acids during the assay did not result in stimula-
NADPH-NITRATE REDUCTASE REGULATION
VOL. 131, 1977
TABLz 3. Chelex dialysis experiment Aa Dialysis buffer Assay buffer
Phosphate Phosphate ......... Phosphate-EDTA . . Phosphate-Chelex..
7 82 12
Phosphate- PhosphateEDTA 94 100 93
Chelex 15 83 17
6 The buffer used was 0.1 M K2HPO4-KH2PO4 (pH 7.3) with or without the additive 0.5 mM EDTA or 2 mg of Chelex (2.9 meq/g [dry weight]) per ml. Enzyme, dialyzed against the indicated buffer solution, was diluted 40-fold in fresh buffer solution and then assayed in the indicated buffer. The figures reported are percentages of the maximum observed activity, which was 7,800 U/ml of dialyzed enzyme containing 12.32 mg of protein per ml.
TABLz 4. Chelex dialysis experiment Ba Dialysis and assay buffer Addition to assay
Phosphate
None .......... EDTA .........
Glutamate..... Glutamine
.....
Glycine ........ Alanine Arginine
.......
......
Tryptophan
....
10 63 45 53 43 44 47 38
PhosphateEDTA 95 91 92 95 87 92 94 90
PhosphateChelex 11 77 68 71 62 65 70 61
* The buffer used was 0.1 M K2HPO4-KH2PO4 (pH 7.3) with or without the additive 0.5 mM EDTA or 2 mg of Chelex (2.9 meq/g [dry weight]) per ml. The amino acid concentration in the assays was 5 mM, and EDTA was 0.5 mM. Enzyme, dialyzed against the indicated buffer solution, was diluted 40-fold in fresh buffer solution and then asayed in the indicated buffer solutions plus additions. The figures reported are percentages of the maximum observed activity, which was 7,800 U/ml of dialyzed enzyme containing 12.32 mg of protein per ml.
tion. Presumably the nitrate reductase was maximally activated by the concentration of EDTA present. Dialysis of nitrate reductase against phosphate-Chelex buffer resulted in an 89% activity loms, which was significantly restored by adding either EDTA or one of the amino acids used in experiment B. A parallel experiment, in which all reagents were treated with Chelex before constructing the assay mixtures, was performed. Under these conditions, histidine, citrate, glutamate, and glutamine at 5 mM and EDTA at 1 mM restored nitrate reductase activity to the enzyme preparations that were dialyzed against Chelex. An analogous experiment was to purify nitrate reductase in the absence of EDTA. This preparation had a specific activity of 331, representing a 12% recovery, and was significantly stimulated when assayed in the presence of the amino acids listed in Table 1 or EDTA. These experi-
887
ments indicate that EDTA and the other effectors must be in physical contact with nitrate reductase to stimulate this enzyme. Therefore, amino acids, citrate, and EDTA do not stimulate nitrate reductase by chelation of heavy metals or other inhibitors. Kinetics. An alternative mechanism for activating nitrate reductase involves amino acid altering the Km of the NADPH-nitrate reductase for nitrate, NADPH, or both. The Km for each substrate was determined by holding one substrate in constant excess and varying the other. Before the kinetic assays, the enzyme was extensively dialyzed to remove EDTA and then assayed in the presence of different concentrations of a given amino acid. Three amino acid concentrations were chosen (usually falling at the bottom, middle, and top) from the sigmoidal curve obtained by plotting the concentration of the amino acid versus the nitrate reductase activity. Double-reciprocal plots of nitrate reductase activity versus NADPH concentration for glutamine, glutamate, tryptophan, and histidine were linear. Increasing concentrations of these amino acids increased the velocity of the reaction and also altered the Vmax. Histidine affected nitrate reductase activity at concentrations of 20 to 100 uM. These concentrations were two orders of magnitude lower than the effective concentrations of the other amino acids. The kinetics of nitrate reductase activity in the presence of alanine, glycine, and arginine were similar to those of glutamate and glutamine. Increasing concentrations from 0.5 to 2 mM increased the velocity of the reaction and increased the Vmax. Stimulation by increasing concentrations of arginine, alanine, and glycine was less than that for the other amino acids. Moreover, 2 mM concentrations of arginine and alanine resulted in maximal stimulation. The relative stimulation by different amino acids in these kinetic experiments is similar to the relative stimulation observed in Table 1. Finally, increasing concentrations of citrate stimulated nitrate reductase, though it is a poorer stimulator than the amino acids since high concentrations were required to in-
duce stimulation. The apparent Km values of nitrate reductase for NADPH in the presence of these amino acids are compiled in Table 5. These Km values varied from 14 to 360 ,uM when determined in the presence of 10 mM glutamine and 2 mM histidine, respectively. A 100-fold increase in the histidine concentration (20 ,uM to 2 mM) increased the Km by more than 10-fold (Table 5). Therefore, increasing the concentration in-
888
KETCHUM, ZEEB, AND OWENS
J. BACTZRIOL.
creased the Km of nitrate reductase for NADPH. Tryptophan and glutamine had the opposite effect; i.e. increased concentrations of these amino acids decreased the apparent K. of nitrate reductase for NADPH (Table 5). Increasing the concentration of alanine, arginine, glutamate, or glycine by 10-fold resulted in less than a 2-fold increase in the apparent Km for NADPH. Apparent Km values of nitrate reductase for NADPH in the presence of amino acid mixtures were not determined. Similar kinetic experiments to determine the Km of NADPH-nitrate reductase for nitrate in the presence of amino acids were performed. The apparentKm for nitrate varied between 210 and 380 ,uM, when the seven amino acids were present individually in the assay (Table 6). The small variation in the enzyme's Km values for nitrate suggests that a significant control is not exerted by amino acids at the nitrate site. Attempts to measure the Km values of nitrate reductase for nitrate and NADPH in the absence of additives were unsuccessful. Low enzymatic activity and/or high protein concentrations resulted in unreliable results with low correlation coefficients. T&rLz 5. Apparent Km and Vm., values of NADPHnitrate reductase for NADPH in the presence of effectors Effector concn (mM) Alanine (2) ......... Glutamate (2) ...... Glutamine (2) ...... Glutamine (10) ..... Histidine (0.02) ..... Histidine (2) ........ Glycine (2) .........
Tryptophan (1) Tryptophan (4) Arginine (2)
......
......
........
K. value for NADH (!LM) 50 60 28
V
(U) 26.0 5.4 4.3 6.1 26.0 50.0 49.0 9.6 11.0 51.0
14
33 360 100 150 70 87
EDTA (0.5) .........
100
24.0
Citrate (1) ..........
100
7.7
TABLz 6. Apparent Km values of NADPH-nitrate reductase for nitrate in the presence of effectors Effector concn (mM)
Alanine (5) ................ Arginine (5) ............... Glutamate (5) ............. Glutamine (5) ............. Glycine (5) ................ Histidine (5)............... Tryptophan (5)............. EDTA (0.5)
................
Apparent K,. value of nitrate (AtM) 250 380 210 250 225 210 260 310
DISCUSSION Assimilation of nitrate nitrogen is a major biological process in plants, fungi, and some bacteria. The key enzyme to be regulated in this process is NADPH-nitrate reductase, since it is the first enzyme in the assimilatory process. Regulation of the de novo synthesis of nitrate reductase has been extensively investigated (12, 14, 19, 22). Once formed, the enzyme requires further regulation, since it is energy utilizing, and recent reports (3, 18) suggest that it has an extremely high turnover number. Nitrate reductase from N. crassa at specific activities of 80 to 100 Amol of nitrite formed per min per mg of protein (S. S. Pan et al., Fed. Proc., abstr. 2636, 34:682, 1975) and from Escherichia coli at 229 ymol of nitrite formed per min per mg of protein (3) have turnover numbers of 153 and 440 mol/s per gram atom of molybdenum (assuming 2 mol of molybdenum per enzyme). These numbers are significantly higher than those for nitrogenase (2), which were reported as 1 mol/s per gram atom of molybdenum. Therefore, N. crassa requires minute quantities of nitrate reductase and must control the rate of nitrate reduction lest it consume energy (NADPH) unnecessarily. Other investigators (8, 20) have not observed any effect of amino acids on nitrate reductase. We attribute this apparent conflict with our results to the presence of EDTA in their preparations and/or to the use of a different strain of N. crassa. EDTA, at concentrations of
