Phytochemisrry, Vol. 31, No. 7, pp. 2259 2262, 1992
0031.9422/‘92 S5.00+0.00
Printed in Great Britain.
Q 1992 Pcrgamon Press Ltd
ARGININE AND LYSINE RESIDUES AS NADH-BINDING SITES IN NADH-NITRATE REDUCTASE FROM SPINACH YUKIKO SATO,NAOMASASHIRAISHI, Department
of Agricultural
Chemistry.
TAKAHIDE
Faculty
SATO,NAGAO OGURA and
of Horticulture, Chiba
(Received in reoisedform Key Word I&x-Spinacia oleracea; Chenopodiaceae; residue; lysine residue; NADH binding site.
10 December
spinach;
chemical
University,
HIROKI NAKAGAWA
Matsudo,
Chiba
271, Japan
1991) modification;
nitrate
reductase;
arginine
Abstract-Chemical modifications of spinach leaf nitrate reductase, and its 28 Ooo M, 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 FMNH,-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities. NADH protects the inactivation of NADH-ferricyanide reductase activity of the 28000 M, 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.
RESULTS
Assimilatory nitrate reductase (NADH-nitrate oxido reductase, EC 1.6.6.1) catalyses the reduction of nitrate to nitrite which is a rate-limiting step in nitrate assimilation. Although the enzyme from higher plants has been extensively investigated Cl, 33, the role of the functional groups associated with the active sites of the enzyme has not yet received much attention. Chemical modification suggests that one sulphydryl group per subunit is required for NADH binding and catalytic activity [4]. However, it is probably not directly involved in electron transfer between prosthetic groups [4]. Nitrate reductase uses NADH as one of the substrates and this substrate possesses an anionic group. Such groups often form an anionic interaction with cationic amino acid residues such an arginine [S-8] and lysine [9], at the active site of an enzyme. Arginine residues have been implicated in the function of nitrate reductase from Amaranthus [IO], and Chlorella [l 11. In an effort to understand the functional groups associated with the NADH binding site, we have chosen to examine the role that cationic amino acid residues may play in NADH binding. In the present study, we have analysed modified nitrate reductase and the 28 000 M, fragment of nitrate reductase from spinach leaves, in order to gain information concerning the presence of arginine and lysine residues at the active site of the enzyme. NADH-dependent nitrate reductase activities which require NADH-binding sites were rapidly inactivated by either arginyl-specific reagents such as phenylglyoxal and 2,3-butanedione or lysyl-specific reagent, pyrydoxal phosphate. Also these arginyl- and lysyl-specific reagents greatly reduced the NADH-ferricyanide reductase activity of the 28 Ooo M, fragment from spinach nitrate reductase. The enzyme is protected from inactivation when NADH is present during the reaction.
Inactivation of native nitrate reductase with arginyl- or lysyl-specific reagents
The pathway of electron flow from NADH to nitrate through nitrate reductase in eukaryotes is generally accepted to be: NADH-+(FAD+cytochrome
b,,,+molybdopterin)
-nitrate. Besides the reduction of nitrate by NADH, nitrate reductase also catalyses several partial reactions which may be classified as either NADH dehydrogenase or terminal nitrate reductase activities. NADH dehydrogenase activities include NADH-dependent ferricyanide reductase and cytochrome c reductase activities, whereas terminal nitrate reducing activities include FMNH,-nitrate reductase and MVH-nitrate reductase activities. Treatment of the enzyme with 10 mM phenylglyoxal led to a rapid inactivation followed by a slower phase of inactivation of NADH-nitrate reductase and NADHdependent dehydrogenase activities (Fig. 1). By contrast, terminal nitrate reducing activities were less affected by the same treatment (Fig. 1). At IO mM phenylglyoxal, NADH-nitrate reductase was quickly inactivated (70%) followed by a time-dependent monophasic inactivation. The diaphorase activities were inactivated by 50% (cytochrome c reductase) and 70% (ferricyanide reductase) in 20 mM phenylglyoxal in 15 min (Fig. I). In comparison, the terminal nitrate reducing activities were much less sensitive, while FMNH,-nitrate reductase activity underwent a very slow time-dependent inactivation resulting in a loss of about 25% of the activity in 40 min. In contrast, MVH-nitrate reductase appears to be quite stable (Fig. 1). It appears that the inhibition of NADH-nitrate reductase by phenylglyoxal is primarily due to the inactivation of its
Y.
2260
SAT0et
al.
T 0
0
0
10
20 Time
30
40
(min)
Fig. 1. Inactivation of nitrate reductase activities by phenylglyoxal. The enzyme (20 fig) was incubated in the dark at 30” in 2 ml of 50 mM sodium borate buffer (pH 9.0) containing 0.5% BSA and 10mM phenylglyoxal. Aliquots were assayed for NADH-nitrate reductase ( X ), NADH-ferricyanide reductase (O), NADH-cytcchrome c reductase (A), FMNH,-nitrate reductase (m) and MVH-nitrate reductase (7).
2
0
l
4
6
8
10
12
Time (min) Fig. 3. Inactivatton of NADH ferricyanide reductase activity of 28000 M, fragment of spinach nitrate reductase by phenylglyoxal. The 28 000 M, fragment (20 pg) of nitrate rcductase was preincubatcd with (0, 0) or without (0, l ) the addttion of 2 mM NADH to the reaction mixture (2 ml) for 10 min prior to the addition of the IOmM phenylglyoxal and 1mM pyridoxal phosphate, respectively. Aliquots were assayed for NADHferricyanide reductase activity at the incubated times.
an initial decrease of 30% (Fig. 2). It appears that the inhibition of NADH-nitrate reductase by pyridoxal phosphate is primarily due to the inactivation of its NADH-dehydrogenase activities. The pyridoxal phosphate also yielded biphasic inactivation curves similar to those shown in Fig. I. Inactivation of NADH-ferricyanide reductase acticit) qf 28 000 M,fiagment with arginyl- or Iysyl-specific reagents
101 0
5
10 15 20 25 Time (min)
30
Fig. 2. Inactivation of nitrate reductase activities by pyridoxal phosphate. The enzyme (20/1g) was incubated in the dark at 30” in 2ml of 5OmM HEPES buffer (pH 7.8) and 1mM pyridoxal
phosphate. Aliquots were assayed for NADH-nitrate reduetase. ( X ), NADH-ferricyanide reductase ( l ), NADH-cytechrome c reductase (A), FMNH,-nitrate nductase (m) and MVH-nitrate reductase (Cl). NADH-dehydrogenase activities. Studies using 57 mM 2,3-butanedione which preferentially modifies arginine gave results which were similar to those obtained when using phenylglyoxal (data not shown). Treatment of purified nitrate reductase with I mM PLP led to an extensive loss of enzymatic activities (Fig. 2). A rapid concomitant loss of both NADH-nitrate reductase activity and NADH-diaphorase activities occurs, with less than 30% of either activity remaining after 15 min. In contrast, terminal nitrate reductase activities, FMNH,and MVH-nitrate reductase activities, remain essentially constant over a 20 min period after
Treatment of the purified 28000 M, fragment with 10 mM phenylglyoxal led to an extensive loss of NADHferricyanide reductase activity (Fig. 3) which fell to about 40% of the control after 10 min of treatment. The rate of loss of activity corresponded to a pseudo-exponential process of decay. NADH-ferricyanide reductase activity without phenylglyoxal was stable and in all experiments reported herein controls were run to ensure that there was no unaccountable loss of activity during the reactions, The data for inactivation by the other arginyl-specific reagent, 2,3-butanedione was similar to that found with phenylglyoxal (data not shown). The treatment of purtfied 28000 M, fragment with lysyl-specific reagent, pyridoxal phosphate, led to a loss of NADH-ferricyanide reductase activity (Fig. 3). Protection by substrates against inactication The ability of NADH to protect NADH-ferricyanide reductase from inactivation by arginyl- or lysyl-specific reagents was examined. The data for both arginyl-specific reagents, phenylglyoxal and 2,3-butanedione were similar, so only the phenylglyoxal inactivation data arc shown graphically in Fig. 3. The data in Fig. 3 clearly demonstrate that NADH provides protection against inactivation by phenylglyoxal. With 1 mM pyridoxal phosphate, the NADH-ferricyanide reductase activity of the 28 000 M, fragment was inhibited by about 50%. Preincubation with NADH. however, provided substantial protection (Fig. 3).
NADH-binding sites in NADH-nitrate
2261
Spi~cia olemcea Arabidopsis thaliana Nicotiana tabacum
Lycopersicon esculentum Cucurbita maxima Phasedus vulgaris
Bell&l pendda Hordeum vu&are NADH Hordeurn vu&are NAD(P)H
Otyze saliva Zea mays Neurospora crassa Aspergillus niakiulonce cytochrome b5 Fig. 4. Sequence comparison of the putative NADH-binding sites of nitrate reductase and of human cytochrome b, reductase. Nitrate reductase sequences from Spinacia oleracea [13, 141, Arabidopsis thaliana [lS], Nicotiana tabacum [16]. Lycopersicon esculentum [UJ, Cucwbila maxim0 [18], Phaseolus uulgaris 1193, Be&la pendula 1183, Hordeum uulgare [ZO, 213, Oryze satiua [22], Zea mays [23], Neurospora crassa [24], Aspergillus nidulance [25] were composed by introducing gap positions, as indicated by dashes, to optimize the alignment. The lysine and
arginine residues corresponding to the NADH-binding are showed by bold form and consensus motifs are boxed.
DISCUSSION
The inhibitory pattern exhibited by phenylglyoxal, 2,3-butanedione and pyridoxal phosphate on the activities associated with the spinach nitrate reductase complex (Figs 1 and 2) and the 28OtXl M, fragment (Fig. 3) indicates the involvement of one or more relatively accessible arginine or lysine residues in the NADHbinding site which exists in the 28 000 M, domain of the nitrate reductase molecule. Of the five reactions studied, NADH-nitrate reductase was inactivated to the greatest extent. Terminal nitrate reductase activities such as FMNH,and reduced viologen-nitrate reductase activities were less affected, while NADH-dehydrogenase activities such as NADHferricyanide reductase and NADH-cytochrome c reductase activities were preferentially affected. This suggests that the inhibition of NADH-nitrate reductase may be primarily due to inactivation of its NADH-ferricyanide reductase. By contrast, Baijal and Sane have found that NADH-nitrate reductase and the FMNH,-nitrate reductase activities of the nitrate reductase from Amaranthus dubious are inactivated with either phenylglyoxal or 2,3-butanedione at a faster rate compared to the NADHdehydrogenase activity [lo]. This suggests that the arginine residue(s) in the vicinity of the flavin site of Amaranthus nitrate reductase is less reactive. Biphasic inactivation patterns have been obtained for NADH-dependent spinach nitrate reductase activities. Such curves have been obtained for FMNH,-nitrate reductase activity of Amaranthus nitrate reductase [lo] and also for other enzymes [7,83. Treatment of the native nitrate reductase complex with arginyl-specific or lysylspecific reagents showed a rapid inactivation followed by a slower phase of inactivation of NADH-nitrate reductase and NADH-dehydrogenase activities. However, treatment of the 28 000 M, fragment with chemical modifying reagents led to an extensive loss of NADH-ferricyanide reductase activity in a monophasic manner. These observations lead us to speculate that the nitrate reductase contains at least two arginine and lysine re-
sidues that are important for the NADH-dependent activities of the enzyme. These are distinguished by different rates of reactivity with phenylglyoxal or pyridoxal phosphate. It is proposed that the fast initial rate of inactivation may be due to a location of some of the arginine or lysine residues at the periphery of the 28 000 M, domain of the nitrate reductase complex and that these residues are also important for the NADH-binding of the enzyme. The protection by NADH suggests that arginine and lysine residues may be involved in the binding of NADH. This argument is also supported by the data on protection of the NADH-ferricyanide reductase activity of the 28 000 M, fragment. This protection by NADH, against inactivation of NADH-requiring enzyme activity of the 28OCKlM, fragment, suggests that arginine and lysine residues may be located at the active site of the fragment that binds NADH. The protection by NADH is afforded only against the initial phase of inactivation by these reagents. Nitrate provides no protection against inactivation indicating that the nitrate binding site of the enzyme does not contain functional and exposed arginine and lysine residues. Lysine residue has been shown to be involved in NADH binding by cytochrome bs reductase [12]. The lysine residue has been localized in cytochrome b, reductase and a corresponding lysine is present in all nitrate reductasc sequences 1113255 (Fig. 4; position 741 in spinach nitrate reductase). Its position falls within a wellconserved segment of the sequence. An arginine residue may also be involved in NADH binding, although its position is still unknown. Only one arginine residue, however, is found to be common to nitrate reductase sequences from plants [13-231, fungi [24, 253 and cytochrome b5 reductase [12] (Fig. 4; position 722 in spinach nitrate reductase). The authors therefore suppose the arginine residue found in the 28 000 M, fragment sequences from plant and fungi to be a good candidate. Again this residue falls within a very wellconserved region of the sequence.
Y. SAT0 et al.
2262 EXPERIMENTAL
2. Kleinhofs,
V8 protease was obtained from Boehringer Mannheim Yamanouchi Co., Ltd. Phenylglyoxal, 2,3-butanedione and pyridoxal phosphate were obtained from Sigma Chemical Co. Purification oJ nitrate reduclase. Nitrate reductase from spinach leaves was purified as previously described by Nakagawa et a/. [26]. Enzyme assays. NADH-nitrate reductase. activity was assayed as described previously [26]. NADH-ferricyanide reductase activity was assayed by monitoring ferricyanide-dependent NADH-oxidation at 340 nm [27]. NADH-cytochrome c reductase activity was assayed by monitoring the increase in absorbance at 550 nm due to the reduction of cytochrome c by NADH [28]. FMNH,-nitrate reductase or MVH-nitrate reductase activities were assayed by measuring the formation of nitrite due to the reduction of nitrate by FMNH, or MVH [28]. One unit of nitrate reductase activity produced 1pmol of nitrite per min. Protein was determined by the method of Bradford [29] using BSA as the standard. Isolation of the 28000 M, fragment containing NADHferricyanide reductase activity was performed as described previously [30]. Proteolytic products of purified nitrate reductase were separated by HPLC on a TSK G3ooOSW column as described previously [30]. Muteriuls.
Staphylococcus
Enzymaric
inacriratlon
aureus
with
chemical
modgying
reagents.
Inactivations were carried out on protein (20 fig) in 2 ml of 50 mM sodium borate buffer (pH 9.0) containing 0.5% BSA and the indicated amount of phenylglyoxal. When 2.3-butanedione was used, 50 mM sodium borate buffer (pH 7.5) containing 0.5% BSA was used. The mixtures were incubated for the time indicated at 30” in the dark to minimize light-induced decomposition [3lJ. At designated intervals, aliquots were withdrawn and diluted 20-fold in the assay mixture containing 0.5% BSA. Control experiments have shown that the enzyme was stable without the modifying reagent. Inactivations with pyridoxal phosphate were carried out according to the method of Basu er a/. [32]. Nitrate reductase (20 pg) in 2 ml of 50 mM HEPES buffer (pH 7.8) was incubated with various concentrations of pyridoxal phosphate in the same huger at 30 and adjusted to pH 7.8. The time course of inactivation was followed by removing aliquots of the incubation mixture at mtervals and assaying for enzymatic activities. The effects on the inactivation of chemical modifymg reagents in the presence of substrates were also Investigated. authors thank Prof. A. Oaks, Guelph University, for reading the manuscript. This work was supported in part by Grant-in-Aid for Scientific Research (No, 01540558) from the Ministry of Education, Science and Culture, Japan. Acknowledgements-The
Rec. PIant
M. G., Vega, J. M. and Losada, Physiol. 32, 169.
A., Waner,
Surueys qf P/ant
R. L. and Narayanan, Molecular
K. R. (1985) 2, 91.
and Cell Biology,
3. Solomonson,
L. P. and Barber, M. J. (1990) Ann. Res. Plant Eiol. 41, 225. Barber, M. J. and Solomonson, L. P. (1986) J. Biol. Chem. 261. 44562. Lange, L. G., Riordan, J. F. and Vallee. B. L. (1964) Biochemistry 13, 4361. Bleile, D. M., Foster, M., Brady, J. W. and Harrison, J. H. (1975) .I. Biol. Chem. 250, 6222. Zakim. D., Hochman. Y. and Kenney. W. C. (1983) J. Biol. Physiol.
4. 5. 6. 7.
Mol.
Chem. 248, 6430.
8. Dailey,
H. A. and Fleming,
J. E. (1986) J. Biol. Chem. 261.
7902.
9. Hackett, C. S., Novoa. W. B., Kensil, C. R. and Strittmatter, P. (1986) J. Biol. Chem. 263, 7539. 27. 1969. 10. Baijal, M. and Sane, P. V. (1988) Phytochemistry 11. Solomonson. L. P. and Barber, M. J. (1989) J. Biol. Chem. ICn.844.
S.. Tamura, M. and 12. Yubisui, T., Miyata, T., Iwanaga, Takeshita, M. (1986) J. Biochem. 94, 407. 13. Presser, 1. M. and Lazarus. C. M. (1990) PIanr Mol. Biol. 15, 187.
14. Shiraishi, N., Kubo, Y., Takeba, G., Kiyota, S., Sakano, K. and Nakagawa. H. (1991) Planf Cell Physiol. 32, 1031. 15. Crawford, N. M., Smith, M., Bellissimo, D. and Davis, R. W. (1988) Proc. Natn. Acad. Sci. U.S.A. 85, 5006. J., Caboche, M. 16. Vaucheret, H.. Vincentz, M., Kronenberger, and Rouze, P. (1989) Mol. Gen. Genet. 216. IO. 17. Daniel-Vedele, F., Dorbe, M. F., Caboche. M. and Rouze. P. (1989) Gene 85. 371. K. and Hachtel, W. (1991) Mol. 18. Friemann, A., Brinkmann, Gen. Genet. 227, 97. 19. Ho& T., Stummann, B. M. and Henningsen, K. W. (1991) Physiol. Plum. 82. 197. 20. Cheng, C.-L., Dewdeny, J., Nam, H. G. and den Boer, B. G. W. (1988) EMBO J. 7, 3309. 21. Miyazaki, J., Juricek, M., Angelis, K., Schnorr, K. M., Kleinhofs, A. and Wamner, R. L. (1991) Mol. Gen. Gener. 22& 329. 22. Choi, H. K., Kleinhofs, A. and An, G. (1989) Plant Mol. Biol. 13, 731. 23. Gowri, G. and Campbell, W. H. (1989) Plant Physiol: 90,792. 24. Okamoto. P. M.. Fu, Y. H. and Marzluf. G. A. (1991) Mol. Gen. Genet. 227, 213. 25. Johnstone, I. L., McCabe, P. C.. Greaves, P., Cole, G. E., Brow, M. A. D.. Gurr, S. J.. Unkies, S. E.. Clutterbuck. A. J. Kinghorn. J. R. and Innis, M. (1990) Gene 90, 181. 26. Nakagawa, H., Yonemura, Y., Yamamoto, H., Sato, T., Ogura, N. and Sato, R. (1985) P/am Physiol. 77, 124. 27. OJi, Y. and Izawa, G. (1969) Plant Cell Physiol. 10, 743. 28. Wray, J. L. and Filner, P. (1970) Biochem. J. 119, 715. 29. Bradford. M. M. (1974) Analyr. Biochem. 72, 248. 30. Kubo, Y., Ogura, N. and Nakagawa, H. (1988)5. Biol. Chem. 263, 19684.
REFERENCES I. Guerrero,
Oxford
M. (1981) Ann.
31. Gripton, J. C. and Hofmann, T. (1981) Biochem. J. 193, 55. 32. Basu, A., Kedae, P., Wilson, S. H. and Modak, M. J. (1989) Biochemistry
Zs, 6305.
0031.9422/‘92 S5.00+0.00
Printed in Great Britain.
Q 1992 Pcrgamon Press Ltd
ARGININE AND LYSINE RESIDUES AS NADH-BINDING SITES IN NADH-NITRATE REDUCTASE FROM SPINACH YUKIKO SATO,NAOMASASHIRAISHI, Department
of Agricultural
Chemistry.
TAKAHIDE
Faculty
SATO,NAGAO OGURA and
of Horticulture, Chiba
(Received in reoisedform Key Word I&x-Spinacia oleracea; Chenopodiaceae; residue; lysine residue; NADH binding site.
10 December
spinach;
chemical
University,
HIROKI NAKAGAWA
Matsudo,
Chiba
271, Japan
1991) modification;
nitrate
reductase;
arginine
Abstract-Chemical modifications of spinach leaf nitrate reductase, and its 28 Ooo M, 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 FMNH,-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities. NADH protects the inactivation of NADH-ferricyanide reductase activity of the 28000 M, 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.
RESULTS
Assimilatory nitrate reductase (NADH-nitrate oxido reductase, EC 1.6.6.1) catalyses the reduction of nitrate to nitrite which is a rate-limiting step in nitrate assimilation. Although the enzyme from higher plants has been extensively investigated Cl, 33, the role of the functional groups associated with the active sites of the enzyme has not yet received much attention. Chemical modification suggests that one sulphydryl group per subunit is required for NADH binding and catalytic activity [4]. However, it is probably not directly involved in electron transfer between prosthetic groups [4]. Nitrate reductase uses NADH as one of the substrates and this substrate possesses an anionic group. Such groups often form an anionic interaction with cationic amino acid residues such an arginine [S-8] and lysine [9], at the active site of an enzyme. Arginine residues have been implicated in the function of nitrate reductase from Amaranthus [IO], and Chlorella [l 11. In an effort to understand the functional groups associated with the NADH binding site, we have chosen to examine the role that cationic amino acid residues may play in NADH binding. In the present study, we have analysed modified nitrate reductase and the 28 000 M, fragment of nitrate reductase from spinach leaves, in order to gain information concerning the presence of arginine and lysine residues at the active site of the enzyme. NADH-dependent nitrate reductase activities which require NADH-binding sites were rapidly inactivated by either arginyl-specific reagents such as phenylglyoxal and 2,3-butanedione or lysyl-specific reagent, pyrydoxal phosphate. Also these arginyl- and lysyl-specific reagents greatly reduced the NADH-ferricyanide reductase activity of the 28 Ooo M, fragment from spinach nitrate reductase. The enzyme is protected from inactivation when NADH is present during the reaction.
Inactivation of native nitrate reductase with arginyl- or lysyl-specific reagents
The pathway of electron flow from NADH to nitrate through nitrate reductase in eukaryotes is generally accepted to be: NADH-+(FAD+cytochrome
b,,,+molybdopterin)
-nitrate. Besides the reduction of nitrate by NADH, nitrate reductase also catalyses several partial reactions which may be classified as either NADH dehydrogenase or terminal nitrate reductase activities. NADH dehydrogenase activities include NADH-dependent ferricyanide reductase and cytochrome c reductase activities, whereas terminal nitrate reducing activities include FMNH,-nitrate reductase and MVH-nitrate reductase activities. Treatment of the enzyme with 10 mM phenylglyoxal led to a rapid inactivation followed by a slower phase of inactivation of NADH-nitrate reductase and NADHdependent dehydrogenase activities (Fig. 1). By contrast, terminal nitrate reducing activities were less affected by the same treatment (Fig. 1). At IO mM phenylglyoxal, NADH-nitrate reductase was quickly inactivated (70%) followed by a time-dependent monophasic inactivation. The diaphorase activities were inactivated by 50% (cytochrome c reductase) and 70% (ferricyanide reductase) in 20 mM phenylglyoxal in 15 min (Fig. I). In comparison, the terminal nitrate reducing activities were much less sensitive, while FMNH,-nitrate reductase activity underwent a very slow time-dependent inactivation resulting in a loss of about 25% of the activity in 40 min. In contrast, MVH-nitrate reductase appears to be quite stable (Fig. 1). It appears that the inhibition of NADH-nitrate reductase by phenylglyoxal is primarily due to the inactivation of its
Y.
2260
SAT0et
al.
T 0
0
0
10
20 Time
30
40
(min)
Fig. 1. Inactivation of nitrate reductase activities by phenylglyoxal. The enzyme (20 fig) was incubated in the dark at 30” in 2 ml of 50 mM sodium borate buffer (pH 9.0) containing 0.5% BSA and 10mM phenylglyoxal. Aliquots were assayed for NADH-nitrate reductase ( X ), NADH-ferricyanide reductase (O), NADH-cytcchrome c reductase (A), FMNH,-nitrate reductase (m) and MVH-nitrate reductase (7).
2
0
l
4
6
8
10
12
Time (min) Fig. 3. Inactivatton of NADH ferricyanide reductase activity of 28000 M, fragment of spinach nitrate reductase by phenylglyoxal. The 28 000 M, fragment (20 pg) of nitrate rcductase was preincubatcd with (0, 0) or without (0, l ) the addttion of 2 mM NADH to the reaction mixture (2 ml) for 10 min prior to the addition of the IOmM phenylglyoxal and 1mM pyridoxal phosphate, respectively. Aliquots were assayed for NADHferricyanide reductase activity at the incubated times.
an initial decrease of 30% (Fig. 2). It appears that the inhibition of NADH-nitrate reductase by pyridoxal phosphate is primarily due to the inactivation of its NADH-dehydrogenase activities. The pyridoxal phosphate also yielded biphasic inactivation curves similar to those shown in Fig. I. Inactivation of NADH-ferricyanide reductase acticit) qf 28 000 M,fiagment with arginyl- or Iysyl-specific reagents
101 0
5
10 15 20 25 Time (min)
30
Fig. 2. Inactivation of nitrate reductase activities by pyridoxal phosphate. The enzyme (20/1g) was incubated in the dark at 30” in 2ml of 5OmM HEPES buffer (pH 7.8) and 1mM pyridoxal
phosphate. Aliquots were assayed for NADH-nitrate reduetase. ( X ), NADH-ferricyanide reductase ( l ), NADH-cytechrome c reductase (A), FMNH,-nitrate nductase (m) and MVH-nitrate reductase (Cl). NADH-dehydrogenase activities. Studies using 57 mM 2,3-butanedione which preferentially modifies arginine gave results which were similar to those obtained when using phenylglyoxal (data not shown). Treatment of purified nitrate reductase with I mM PLP led to an extensive loss of enzymatic activities (Fig. 2). A rapid concomitant loss of both NADH-nitrate reductase activity and NADH-diaphorase activities occurs, with less than 30% of either activity remaining after 15 min. In contrast, terminal nitrate reductase activities, FMNH,and MVH-nitrate reductase activities, remain essentially constant over a 20 min period after
Treatment of the purified 28000 M, fragment with 10 mM phenylglyoxal led to an extensive loss of NADHferricyanide reductase activity (Fig. 3) which fell to about 40% of the control after 10 min of treatment. The rate of loss of activity corresponded to a pseudo-exponential process of decay. NADH-ferricyanide reductase activity without phenylglyoxal was stable and in all experiments reported herein controls were run to ensure that there was no unaccountable loss of activity during the reactions, The data for inactivation by the other arginyl-specific reagent, 2,3-butanedione was similar to that found with phenylglyoxal (data not shown). The treatment of purtfied 28000 M, fragment with lysyl-specific reagent, pyridoxal phosphate, led to a loss of NADH-ferricyanide reductase activity (Fig. 3). Protection by substrates against inactication The ability of NADH to protect NADH-ferricyanide reductase from inactivation by arginyl- or lysyl-specific reagents was examined. The data for both arginyl-specific reagents, phenylglyoxal and 2,3-butanedione were similar, so only the phenylglyoxal inactivation data arc shown graphically in Fig. 3. The data in Fig. 3 clearly demonstrate that NADH provides protection against inactivation by phenylglyoxal. With 1 mM pyridoxal phosphate, the NADH-ferricyanide reductase activity of the 28 000 M, fragment was inhibited by about 50%. Preincubation with NADH. however, provided substantial protection (Fig. 3).
NADH-binding sites in NADH-nitrate
2261
Spi~cia olemcea Arabidopsis thaliana Nicotiana tabacum
Lycopersicon esculentum Cucurbita maxima Phasedus vulgaris
Bell&l pendda Hordeum vu&are NADH Hordeurn vu&are NAD(P)H
Otyze saliva Zea mays Neurospora crassa Aspergillus niakiulonce cytochrome b5 Fig. 4. Sequence comparison of the putative NADH-binding sites of nitrate reductase and of human cytochrome b, reductase. Nitrate reductase sequences from Spinacia oleracea [13, 141, Arabidopsis thaliana [lS], Nicotiana tabacum [16]. Lycopersicon esculentum [UJ, Cucwbila maxim0 [18], Phaseolus uulgaris 1193, Be&la pendula 1183, Hordeum uulgare [ZO, 213, Oryze satiua [22], Zea mays [23], Neurospora crassa [24], Aspergillus nidulance [25] were composed by introducing gap positions, as indicated by dashes, to optimize the alignment. The lysine and
arginine residues corresponding to the NADH-binding are showed by bold form and consensus motifs are boxed.
DISCUSSION
The inhibitory pattern exhibited by phenylglyoxal, 2,3-butanedione and pyridoxal phosphate on the activities associated with the spinach nitrate reductase complex (Figs 1 and 2) and the 28OtXl M, fragment (Fig. 3) indicates the involvement of one or more relatively accessible arginine or lysine residues in the NADHbinding site which exists in the 28 000 M, domain of the nitrate reductase molecule. Of the five reactions studied, NADH-nitrate reductase was inactivated to the greatest extent. Terminal nitrate reductase activities such as FMNH,and reduced viologen-nitrate reductase activities were less affected, while NADH-dehydrogenase activities such as NADHferricyanide reductase and NADH-cytochrome c reductase activities were preferentially affected. This suggests that the inhibition of NADH-nitrate reductase may be primarily due to inactivation of its NADH-ferricyanide reductase. By contrast, Baijal and Sane have found that NADH-nitrate reductase and the FMNH,-nitrate reductase activities of the nitrate reductase from Amaranthus dubious are inactivated with either phenylglyoxal or 2,3-butanedione at a faster rate compared to the NADHdehydrogenase activity [lo]. This suggests that the arginine residue(s) in the vicinity of the flavin site of Amaranthus nitrate reductase is less reactive. Biphasic inactivation patterns have been obtained for NADH-dependent spinach nitrate reductase activities. Such curves have been obtained for FMNH,-nitrate reductase activity of Amaranthus nitrate reductase [lo] and also for other enzymes [7,83. Treatment of the native nitrate reductase complex with arginyl-specific or lysylspecific reagents showed a rapid inactivation followed by a slower phase of inactivation of NADH-nitrate reductase and NADH-dehydrogenase activities. However, treatment of the 28 000 M, fragment with chemical modifying reagents led to an extensive loss of NADH-ferricyanide reductase activity in a monophasic manner. These observations lead us to speculate that the nitrate reductase contains at least two arginine and lysine re-
sidues that are important for the NADH-dependent activities of the enzyme. These are distinguished by different rates of reactivity with phenylglyoxal or pyridoxal phosphate. It is proposed that the fast initial rate of inactivation may be due to a location of some of the arginine or lysine residues at the periphery of the 28 000 M, domain of the nitrate reductase complex and that these residues are also important for the NADH-binding of the enzyme. The protection by NADH suggests that arginine and lysine residues may be involved in the binding of NADH. This argument is also supported by the data on protection of the NADH-ferricyanide reductase activity of the 28 000 M, fragment. This protection by NADH, against inactivation of NADH-requiring enzyme activity of the 28OCKlM, fragment, suggests that arginine and lysine residues may be located at the active site of the fragment that binds NADH. The protection by NADH is afforded only against the initial phase of inactivation by these reagents. Nitrate provides no protection against inactivation indicating that the nitrate binding site of the enzyme does not contain functional and exposed arginine and lysine residues. Lysine residue has been shown to be involved in NADH binding by cytochrome bs reductase [12]. The lysine residue has been localized in cytochrome b, reductase and a corresponding lysine is present in all nitrate reductasc sequences 1113255 (Fig. 4; position 741 in spinach nitrate reductase). Its position falls within a wellconserved segment of the sequence. An arginine residue may also be involved in NADH binding, although its position is still unknown. Only one arginine residue, however, is found to be common to nitrate reductase sequences from plants [13-231, fungi [24, 253 and cytochrome b5 reductase [12] (Fig. 4; position 722 in spinach nitrate reductase). The authors therefore suppose the arginine residue found in the 28 000 M, fragment sequences from plant and fungi to be a good candidate. Again this residue falls within a very wellconserved region of the sequence.
Y. SAT0 et al.
2262 EXPERIMENTAL
2. Kleinhofs,
V8 protease was obtained from Boehringer Mannheim Yamanouchi Co., Ltd. Phenylglyoxal, 2,3-butanedione and pyridoxal phosphate were obtained from Sigma Chemical Co. Purification oJ nitrate reduclase. Nitrate reductase from spinach leaves was purified as previously described by Nakagawa et a/. [26]. Enzyme assays. NADH-nitrate reductase. activity was assayed as described previously [26]. NADH-ferricyanide reductase activity was assayed by monitoring ferricyanide-dependent NADH-oxidation at 340 nm [27]. NADH-cytochrome c reductase activity was assayed by monitoring the increase in absorbance at 550 nm due to the reduction of cytochrome c by NADH [28]. FMNH,-nitrate reductase or MVH-nitrate reductase activities were assayed by measuring the formation of nitrite due to the reduction of nitrate by FMNH, or MVH [28]. One unit of nitrate reductase activity produced 1pmol of nitrite per min. Protein was determined by the method of Bradford [29] using BSA as the standard. Isolation of the 28000 M, fragment containing NADHferricyanide reductase activity was performed as described previously [30]. Proteolytic products of purified nitrate reductase were separated by HPLC on a TSK G3ooOSW column as described previously [30]. Muteriuls.
Staphylococcus
Enzymaric
inacriratlon
aureus
with
chemical
modgying
reagents.
Inactivations were carried out on protein (20 fig) in 2 ml of 50 mM sodium borate buffer (pH 9.0) containing 0.5% BSA and the indicated amount of phenylglyoxal. When 2.3-butanedione was used, 50 mM sodium borate buffer (pH 7.5) containing 0.5% BSA was used. The mixtures were incubated for the time indicated at 30” in the dark to minimize light-induced decomposition [3lJ. At designated intervals, aliquots were withdrawn and diluted 20-fold in the assay mixture containing 0.5% BSA. Control experiments have shown that the enzyme was stable without the modifying reagent. Inactivations with pyridoxal phosphate were carried out according to the method of Basu er a/. [32]. Nitrate reductase (20 pg) in 2 ml of 50 mM HEPES buffer (pH 7.8) was incubated with various concentrations of pyridoxal phosphate in the same huger at 30 and adjusted to pH 7.8. The time course of inactivation was followed by removing aliquots of the incubation mixture at mtervals and assaying for enzymatic activities. The effects on the inactivation of chemical modifymg reagents in the presence of substrates were also Investigated. authors thank Prof. A. Oaks, Guelph University, for reading the manuscript. This work was supported in part by Grant-in-Aid for Scientific Research (No, 01540558) from the Ministry of Education, Science and Culture, Japan. Acknowledgements-The
Rec. PIant
M. G., Vega, J. M. and Losada, Physiol. 32, 169.
A., Waner,
Surueys qf P/ant
R. L. and Narayanan, Molecular
K. R. (1985) 2, 91.
and Cell Biology,
3. Solomonson,
L. P. and Barber, M. J. (1990) Ann. Res. Plant Eiol. 41, 225. Barber, M. J. and Solomonson, L. P. (1986) J. Biol. Chem. 261. 44562. Lange, L. G., Riordan, J. F. and Vallee. B. L. (1964) Biochemistry 13, 4361. Bleile, D. M., Foster, M., Brady, J. W. and Harrison, J. H. (1975) .I. Biol. Chem. 250, 6222. Zakim. D., Hochman. Y. and Kenney. W. C. (1983) J. Biol. Physiol.
4. 5. 6. 7.
Mol.
Chem. 248, 6430.
8. Dailey,
H. A. and Fleming,
J. E. (1986) J. Biol. Chem. 261.
7902.
9. Hackett, C. S., Novoa. W. B., Kensil, C. R. and Strittmatter, P. (1986) J. Biol. Chem. 263, 7539. 27. 1969. 10. Baijal, M. and Sane, P. V. (1988) Phytochemistry 11. Solomonson. L. P. and Barber, M. J. (1989) J. Biol. Chem. ICn.844.
S.. Tamura, M. and 12. Yubisui, T., Miyata, T., Iwanaga, Takeshita, M. (1986) J. Biochem. 94, 407. 13. Presser, 1. M. and Lazarus. C. M. (1990) PIanr Mol. Biol. 15, 187.
14. Shiraishi, N., Kubo, Y., Takeba, G., Kiyota, S., Sakano, K. and Nakagawa. H. (1991) Planf Cell Physiol. 32, 1031. 15. Crawford, N. M., Smith, M., Bellissimo, D. and Davis, R. W. (1988) Proc. Natn. Acad. Sci. U.S.A. 85, 5006. J., Caboche, M. 16. Vaucheret, H.. Vincentz, M., Kronenberger, and Rouze, P. (1989) Mol. Gen. Genet. 216. IO. 17. Daniel-Vedele, F., Dorbe, M. F., Caboche. M. and Rouze. P. (1989) Gene 85. 371. K. and Hachtel, W. (1991) Mol. 18. Friemann, A., Brinkmann, Gen. Genet. 227, 97. 19. Ho& T., Stummann, B. M. and Henningsen, K. W. (1991) Physiol. Plum. 82. 197. 20. Cheng, C.-L., Dewdeny, J., Nam, H. G. and den Boer, B. G. W. (1988) EMBO J. 7, 3309. 21. Miyazaki, J., Juricek, M., Angelis, K., Schnorr, K. M., Kleinhofs, A. and Wamner, R. L. (1991) Mol. Gen. Gener. 22& 329. 22. Choi, H. K., Kleinhofs, A. and An, G. (1989) Plant Mol. Biol. 13, 731. 23. Gowri, G. and Campbell, W. H. (1989) Plant Physiol: 90,792. 24. Okamoto. P. M.. Fu, Y. H. and Marzluf. G. A. (1991) Mol. Gen. Genet. 227, 213. 25. Johnstone, I. L., McCabe, P. C.. Greaves, P., Cole, G. E., Brow, M. A. D.. Gurr, S. J.. Unkies, S. E.. Clutterbuck. A. J. Kinghorn. J. R. and Innis, M. (1990) Gene 90, 181. 26. Nakagawa, H., Yonemura, Y., Yamamoto, H., Sato, T., Ogura, N. and Sato, R. (1985) P/am Physiol. 77, 124. 27. OJi, Y. and Izawa, G. (1969) Plant Cell Physiol. 10, 743. 28. Wray, J. L. and Filner, P. (1970) Biochem. J. 119, 715. 29. Bradford. M. M. (1974) Analyr. Biochem. 72, 248. 30. Kubo, Y., Ogura, N. and Nakagawa, H. (1988)5. Biol. Chem. 263, 19684.
REFERENCES I. Guerrero,
Oxford
M. (1981) Ann.
31. Gripton, J. C. and Hofmann, T. (1981) Biochem. J. 193, 55. 32. Basu, A., Kedae, P., Wilson, S. H. and Modak, M. J. (1989) Biochemistry
Zs, 6305.
