Upsala J Med Sci 87: 243-250, 1982 Effect of Chemical Modification of a Histidine and a Lysine Residue of Pea Seed Nucleoside Diphosphate Kinase Bror Edlund*, Carl Henrik Heldin and Lorentz Engstrom From the Institute of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsala, Sweden *Present address: Department of Clinical Chemistry, University Hospital, University of Uppsala, Sweden ABSTRACT Chemical m o d i f i c a t i o n o f a h i s t i d i n e and l y s i n e r e s i d u e i n a c t i v a t e s pea seed n u c l e o s i d e d i p h o s p h a t e k i n a s e (NDP k i n a s e ) . r e a c t i v e l y s i n e r e s i d u e , a t t h e a c t i v e s i t e o f pea seed NDP k i n a s e , i n a d d i - t i o n t o t h e h i s t i d i n e r e s i d u e p h o s p h o r y l a t e d b y t h e s u b s t r a t e ATP as a con- sequence o f t h e enzyme r e a c t i o n . The presence o f a r e a c t i v e l y s i n e a t t h e a c t i v e s i t e o f t h e enzyme c o u l d e x p l a i n why a s m a l l amount o f N-E-phospho- l y s i n e , as w e l l a s 1 - p h o s p h o h i s t i d i n e and 3 - p h o s p h o h i s t i d i n e Y i s formed on a l k a l i n e h y d r o l y s i s o f t h e enzyme. Thus t h e r e seems t o be a INTRODUCTION E a r l i e r s t u d i e s have i n d i c a t e d t h a t NDP k i n a s e ( n u c l e o s i d e d i p h o s p h a t e : ATP t r a n s p h o s p o r y l a s e EC 2.7.4.6) f r o m pea seed i s a t e t r a m e r i c p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 70 000, w i t h f o u r i d e n t i c a l s u b u n i t s , each c o n t a i n i n g an a c t i v e s i t e (1,2). i n t e r m e d i a t e l y p h o s p h o r y l a t e d b y i t s s u b s t r a t e ATP on a h i s t i d i n e r e s i d u e a t t h e a c t i v e s i t e , presumably as 1 - p h o s p h o h i s t i d i n e , t h e phosphoamino a c i d d o m i n a t i n g i n an a l k a l i n e h y d r o l y s a t e o f p h o s p h o r y l a t e d NDP k i n a s e f r o m baker’s y e a s t (3,4). The amino a c i d sequences o f t h e d o m i n a t i n g p h o s p h o p e p t i d e s f r o m t h e a c t i v e s i t e o f t h e pea seed enzyme i n a c t i v a t e d i n t w o d i f f e r e n t ways, 3. w i t h a l k a l i and a c i d , and degraded w i t h two d i f f e r e n t p r o t e a s e s , t r y p s i n and p e p s i n r e s p e c t i v e l y , a r e o v e r l a p p i n g (5,6) i n d i c a t i n g t h a t t h e p h o s p h o r y l g r o u p i s bound t o t h e same h i s t i d i n e r e s i d u e i n t h e enzyme i r r e s p e c t i v e of t h e i n a c t i v a t i o n method used. Two t r y p t i c p h o s p h o p e p t i d e s w i t h t h e same amino a c i d sequence b u t d i f f e r - Evidence has a l s o been o b t a i n e d , t h a t t h e enzyme i s i n g i n s t a b i l i t y t o a c i d o f t h e p h o s p h o r y l bond a r e o b t a i n e d , p r o b a b l y b y a p h o s p h o r y l g r o u p m i g r a t i o n f r o m 1 - p h o s p h o h i s t i d i n e t o 3 - p h o s p h o h i s t i d i n e ( 7 ) . A l k a l i n e h y d r o l y s i s o f p h o s p h o r y l a t e d pea seed NDP k i n a s e g i v e s m a i n l y p h o s p h o p e p t i d e s , and i n a d d i t i o n s m a l l amounts o f p h o s p h o h i s t i d i n e and N-E- 243 phospholysine (8,9). The present investigation was therefore focused on the possibility that there might exist, in addition to the reactive histidine residue, a lysine residue, essential for enzyme activity, in the active site of pea seed NDP kinase. the active site and more chemically reactive than the rest of the amino acid residues of the enzyme. modification of one lysine and one histidine residue were to inactivate the enzyme and if substrate and product protected the enzyme from inactivation. The enzyme was therefore treated with 1 -fluoro-2,4-dinitrobenzene (FDNB) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) which are known to react preferenti- ally with lysine residues (10,11,12) and diethylpyrocarbonate (DPC) which reacts with histidine residues (13,14). was studied in relation to the degree of chemical modification obtained, also in the presence of the substrate ATP and product ADP. If so, these amino acid residues should be exposed in It was considered that support for this view would be obtained if chemical The activity of the treated enzyme MATERIALS AND METHODS The enzyme was prepared as previously described (8). An absorbance value of E:iO = 14.1 was used for the purified enzyme (8). The enzyme activity was determined by a coupled assay according to Mourad and Parks, using dGDP as nucleoside diphosphate (15). FDNB was obtained from Pierce, TNBS from Sigma, DPC from Eastman and sodium dodecylsulfate (SDS (hydroxymethyl )-methyl -2-amino-ethanol sulfonic British Drughouse Ltd. All chemicals were of h Spectroscopic measurements were made with a PMQ using a recorder for registration. Reaction of NDP kinase with FDNB. To 20 m from Pierce. N-tris- acid (TES) was purchased from ghest quality available. I1 Zeiss spectrophotometer, of NDP kinase (15 pmoles/l) in TES buffer (0.2 moles/l, pH 8.5) 1.6 ml of a solution of FDNB in ethanol (0.1 moles/l) were added. The solution was kept at room temperature (23OC). Aliquots (1 ml) were taken at fixed intervals and chromatographed at 23OC on a Sephadex 6-50 column (1.4~16 cm) equilibrated and eluted with sodium phosphate buffer (0.05 moles/l, pH 6.5). fractions eluted with the void volume were measured and the molar amount of incorporated dinitrophenyl groups was calculated. A molar absorbance of 16 000 cm-l at 365 nm was used for the dinitrophenyl groups (10) and the number of modified lysine residues per subunit was calculated. activities were determined and compared with that of a sample chromatographed without previous addition of FDNB. reacting with DPC were determined by difference spectroscopy at 240 nm, using a molar absorbance of 3200 cm-' (13). The absorbance at 280 nm of the The enzyme Treatment of NDP kinase with DPC. The number of histidyl residues The carbetoxylation of the enzyme was 244 carried out in sodium phosphate buffer (0.05 moles/l, pH 6.5) or in triethanol- amine-acetic acid buffer (0.05 moles/l, pH 7.4). were prepared ex tempore. The final concentration of DPC was 1 mmole/l and that of the enzyme 15 umoles/l. calculated in relation to that of the enzyme treated in the same way but without addition of DPC. in sodium hydrogen carbonate (0.25 moles/l), 1 ml of TNBS (1.7 mmoles/l) were added at zero time. A blank solution without NDP kinase was also prepared. The reaction mixtures were kept at 4OoC. Aliquots were taken for enzyme assay at fixed intervals. At the same time 100 u1 of sample and blank solutions were diluted with 100 u1 of 10% (w/v) SDS in water followed immediately by 50 111 of HC1 (1 mole/l) and 3 ml of HC1 (0.01 moles/l) (12). The difference in absorb- ance between sample and blank solutions at 344 nm was determined and the number of trinitrophenyl groups incorporated was calculated from the molar absorbance of 11 000 cm-l at 344 nm given for a trinitrophenyl group in a protein (12). activity remaining after the experimental period. experiments the final NDP kinase concentration was about 0.2 umoles/l. enzyme solutions contained magnesium acetate (1.5 mmoles/l) in order to obtain the nucleotides in the magnesium form. or ADP (0.8 mmoles/l) were incubated at 3OoC with DPC (1 mmole/l) in tri- ethanolamineacetic acid buffer (40 mmoles/l, pH 7.4) for 10 min, TNBS (2 mmoles/ 1) in sodium hydrogen carbonate (10 mmoles/l) for 30 min or FDNB (20 mmoles/l) in sodium dihydrogen carbonate (40 mmoles/l) for 30 min. activity was compared with that of a control solution in which the modifying agent was omitted. was dissolved in ethanol, the same amount of ethanol was included in the control experiment (5% to 10% (v/v)). these ethanol concentrations. Solutions of DPC in ethanol The extent of inactivation of the enzyme was Trinitrophenylation of lysine residues. To 1 ml of NDP kinase (30 pmoles/) The extent of inhibition was calculated as per cent o f enzyme Inactivation of NDP kinase in the presence of ATP and ADP. In all The Samples containing ATP (0.8 moles/l) The residual enzyme In the experiments with DPC and FDNB, where the reagent The enzyme activity was not affected by RESULTS AND DISCUSSION The reaction of pea seed NDP kinase with FDNB indicates that there was one lysine residue per subunit which reacted faster than the other nine (2). All enzyme activity was lost when this residue had reacted, but the enzyme activity seemed to decrease more rapidly than the dinitrophenylation of the lysine residue, indicating that another kind of amino acid residue was also reacting. at 365 nm and would therefore not have been detected by the method used for measuring the degree of dinitrophenylation of the enzyme in the present work Imidazole-dinitrophenyl -histidine does not show an absorbance peak 245 (16). It is therefore possible that a histidine residue was also blocked by FDNB, leading to inactivation of the enzyme (Fig. 1 ) . - - I - - - - I - 1.0 0.5 TIME I MIN Fig. 1. Filled circles represent enzyme activity remaining and open circles represent DNP groups incorporated per mole of subunit. Inhibition of pea seed NDP kinase by FDNB at pH 8.5. The carbetoxylation at pH 7.4 of one histidine residue per subunit o f the enzyme out o f three (2) leads to inactivation - 100 s c 2 z # 5 0 t L 6 x >- I- V 6 Y > 8 pH 7.4 H pH 6.5 D--O n 1 2 L w MOLES OF HISTIDINE RESIDUES MODIFIED PER MOLE OF SUBUNIT of enzyme (Fig. 2). 1 Fig. 2. enzyme activity remaining at pH 7.4 and open squares represent enzyme activity remaining at pH 6.5 after modification of the indicated number of histidine residues per subunit. Modification of histidine residues of pea seed NDP kinase by carbet- 'oxylation with DPC and its effect on enzyme activity. Filled squares represent 246 At pH 6.5 two histidine residues reacted before the enzyme was completely inactivated. The enzyme from beef heart cytosol has also been inactivated by carbetoxylation (17). tion of the enzyme, supporting the view that the enzyme contains a lysine residue which is essential for its activity (Fig. 3 ) . incubation, all ten lysine residues in each subunit were modified, showing that all of them became accessible to TNBS. The trinitrophenylation of pea seed NDP kinase leads to rapid inactiva- However, on prolonged - 1001 1 s I c3 z 5 I W cr t t 1 a l- 0 W I t N z w 50 100 TIME ( MIN 1 v) W Fig. 3 . Inhibition and modification of pea seed NDP kinase by TNBS. Filled circles represent enzyme activity remaining and open circles trinitrophenyl groups incorporated per subunit after the indicated incubation period. The fact that the substrate ATP and the product ADP protect, at least partially, the enzyme from inactivation by the reagents used here, indicates that the reactions involve the active site of the enzyme (Table 1). Nucleo- tides have also been shown to protect some NDP kinases from inactivation by p-chloromercuribenzoat (pCMB) (18,19,20,21). It has been discussed that such a sulfhydryl group may be essential for the qurtenary structure of the enzyme and not a part of the active site of the enzyme (18,211. Pea seed NDP kinase does not contain any SH-group at all (2). lysine residue in addition to a reactive histidine residue in the active site of pea seed nucleoside diphosphate kinase. Thus there seems to be a reactive 247 Table 1. FDNB, DPC and TNBS. addition of nucleotides. same specific activity (1 200 units/mg) as when the enzyme was kept in tri- ethanolamine-acetic acid buffer (0.01 moles/l , pH 7.4). obtained in duplicate experiments are given below. Effect of ATP and ADP on the inactivation of pea seed NDP kinase by In each series a control experiment was made with no In the control experiments the enzyme showed the The mean values For details see text. Additions ATP (control) FDNB FDNB + ATP ADP (control ) FDNB + ADP ATP (control ) DPC DPC + ATP ADP (control) DPC + ADP ATP (control TNBS TNBS + ATP ADP (control TNBS + ADP Residual enzyme activity (per cent) 100 47 80 100 84 100 4 39 100 75 100 58 81 100 75 ACKNOWLEDGEMENTS This work was supported by the Swedish Medical Research Council (Project No. 13X-50). The skilful technical assistance by Mrs. Jill Ekstrom i s gratefully acknowledged. REFERENCES 1. Edlund, B.: Tetrameric structure of nucleoside diphosphate kinase from 2. Edlund, B.: Evidence for identical subunits in pea seed nucleoside 3. Edlund, B. & Wdlinder, 0.: Evidence for an intermediary phosphorylation pea seed. FEBS Letters 13:56-58, 1971. diphosphate kinase. of nucleoside diphosphate kinase from pea seed. 1974. FEBS Letters 38:222-224, 1974. FEBS Letters 38:225-228, 248 4. Edlund, B., Rask, L., Olsson, P., Wblinder, O., Zetterqvist, U. & Engstrom, L.: from baker’s yeast and purification of 1-phosphohistidine as the main phosphorylated product of an a1 kal ine hydrolysate of enzyme incubated with adenosine ( PI-triphosphate. Eur J Biochem 9:451-455, 1969. 5. Edlund, B.: Active site phosphopeptides from pea seed nucleoside diphosphate kinase. Uppsala J Med Sci 79:143-147, 1974. 6. Edlund, B. & Engstrom, L.: A peptic phosphopeptide from the active site of pea seed nucleoside diphosphate kinase. FEBS Lett 47:279-283, 1974. 7. Hultquist, D.E.: The preparation and characterization of phosphorylated derivatives of histidine. Biochim Biophys Acta 153:329-340, 1968. 8. 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J Biol Chem 246:2258-2264, 1971. 249 19. Palmieri, R., Yue, R.H., Jacobs, H.K.,Maland, L., Wu, L. & Kuby, S.A.: Nucleoside triphosphate-nucleoside diphosphate transphosphorylase (Nucleoside diphosphokinase). 111. Subunit structure of the crystalline enzyme from brewer’s yeast. J Biol Chem 248:4486-4499, 1973. 20. Colomb, M.G., Chernuy, A. & Vignais, P.V.: Adenosine diphosphate as a regulatory ligand in beef heart cytosol nucleoside. Biochemistry 13:2269-2277, 1974. 21. Minh DUC, D., Lascu, I . , Porumb, H., Gozia, O., Schell, H.D. & Barzu, 0.: Differential sensitivity to p-chloromercuribenzoate and urea o f soluble and Sepharose-bound pig heart nucleoside diphosphate kinase. FEBS Lett 127: 281 -284, 1981. Address for reprints: Docent Bror Ed1 und Department o f C1 inical Chemistry University Hospital S-750 14 Uppsala 14 Sweden 250