Upsala J M e d Sci 87: 251-258, 1982 Effects of Chemical Modification of Lysine, Tyrosine and Tryptophan Residues in Pea Seed Nucleoside Diphosphate Kinase and Inhibition of the Enzyme with Antibodies Bror Edlund Department of Clinical Chemistry, University Hospital, and Institute of Medical and Physiological Chemistry, University qf Uppsala, Uppsala, Sweden ABSTRACT 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 ) i s an o l i g o m e r i c , t e t r a m e r i c enzyme w h i c h has been shown t o be 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, presumably f o r m i n g an 1 - p h o s p h o h i s t i d i n e a t i t s a c t i v e s i t e . has a l s o a r e a c t i v e l y s i n e r e s i d u e been d e m o n s t r a t e d a t t h e a c t i v e s i t e . I n t h e p r e s e n t i n v e s t i g a t i o n n i t r a t i o n of a t y r o s i n e r e s i d u e i s shown t o i n - a c t i v a t e t h e enzyme. enzyme on m o d i f i c a t i o n o f t h e s e e s s e n t i a l l y s i n e and t y r o s i n e r e s i d u e s , s i n c e d o u b l e d i f f u s i o n e x p e r i m e n t s w i t h an i n h i b i t o r y a n t i s e r u m shows no d i f f e r e n c e i n r e a c t i v i t y between t h e n a t i v e enzyme and t h e m o d i f i e d enzyme. I t i s a l s o f o u n d t h a t m o d i f i c a t i o n o f a l l t r y p t o p h a n r e s i d u e s i n t h e enzyme r e d u c e s t h e enzyme a c t i v i t y o n l y t o a s m a l l degree, i n d i c a t i o n t h a t t h e s e h y d r o p h o b i c amino a c i d r e s i d u e s a r e n o t d i r e c t l y i n v o l v e d i n t h e c a t a l y t i c p r o c e s s . R e c e n t l y There seems o n l y t o be a s l i g h t s t r u c t u r a l change i n t h e INTRODUCTION ATP: 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 t r a n s p h o s p h o r y l a s e ( E C 2.7.4.6) (NDP k i n a s e ) f r o m pea seed i s an o l i g o m e r i c , t e t r a m e r i c enzyme w i t h a m o l e c u l a r w e i g h t o f 70 000 d a l t o n s ( 1 ) . s u b s t r a t e ATP, presumably f o r m i n g an 1 - p h o s p h o h i s t i d i n e r e s i d u e a t i t s a c t i v e s i t e ( 2 , 3 ) . The amino a c i d sequence a r o u n d t h i s p h o s p h o h i s t i d i n e r e s i d u e has been i n v e s t i g a t e d and f o u n d t o c o n t a i n a l y s i n e r e s i d u e ( 4 , 5 ) . s p e c i f i c f o r c e r t a i n amino a c i d r e s i d u e s , and i n a d d i t i o n t o a r e a c t i v e h i s t i d i n e r e s i d u e , has a r e a c t i v e l y s i n e r e s i d u e o f i m p o r t a n c e f o r enzyme a c t i v i t y been f o u n d ( 6 ) . sequence o f t w e n t y amino a c i d r e s i d u e s f r o m t h e a c t i v e s i t e o f pea seed NDP k j n a s e (51, and t h e r e i s n o e v i d e n c e f o r more t h a n one p o l y p e p t i d e c h a i n i n t h e enzyme ( 1 ) . s t i l l be a t y r o s i n e o r t r y p t o p h a n r e s i d u e i n t h e a c t i v e s i t e o f t h e enzyme I t i s 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 I n a r e c e n t r e p o r t t h e n a t i v e enzyme has been t r e a t e d , w i t h r e a g e n t s No a r o m a t i c amino a c i d r e s i d u e a p a r t f r o m h i s t i d i n e has been f o u n d i n t h e However, t h e p r e s e n t i n v e s t i g a t i o n was made s i n c e t h e r e m i g h t 25 1 at some distance in the primary structure from the histidine residue that is phosphorylated. benzyl bromide (HNB-bromide) and tetrani tromethane (TNM) to react with trypto- phan and tyrosine residues respectively (7,8). It also seemed to be of interest to study if antibodies against the enzyme were inhibitory to enzyme action and if chemical modification altered its antigenicity, as has been found with other enzymes, for a review see ( 9 ) . In the present investigation the effect of antibodies on enzyme activity has been studied as well a s the reactivity in an immunodiffusion test of native NDP kinase and enzyme modified by fluoro-2,4-dinitrobenzene (FDNB) and TNM. Thus the native enzyme has been treated with 2-OH-5-NO2- The effect of chemical modification and antibodies on enzyme activity are discussed. MATERIALS AND METHODS Pea seed NDP kinase was prepared as previously described ( 3 ) . Enzyme activity was measured according to Monrad and Parks, using dGDP for nucleoside diphosphate (10). Protein was measured by a Folin method, using human serum albumin as standard (11). Photometric measurements were made, using a Zeiss PMQII spectrophotometer, equipped with an automatic cell unit recorder. 2-OH-5-N02-benzyl bromide (HNB-bromide) and tetranitromethane (TNM) were from Sigma. Innnunodiffusion experiments were performed according to Ouchterlony (12). Agarose 1'Industrie Biologique Francaise (0,8% w/v) in a sodium diemal buffer (75 mmoles/l , pH 8.6) containing calcium lactate (2 mmoles/l) was used. After visible precipitation lines had formed, usually within 24 hours at room temperature, the agarose layers were extracted with NaCl (0,9% w/v) dried and stained with Coomassie Brilliant Blue R-250 essentially according to Laurel1 (13). 1 ml aliquots of a NDP kinase solution, 1 mg per ml of a phosphate buffer (0.1 moles/l, pH 7,4) were incubated with 25-50 pl-of HNB-bromide (0,2-0,4 moles/l) dissolved in acetone. The HNB-bromide solutions were made ex tempore using redistilled acetone. The incubation mixtures were then cooled in an ice-water bath and the HNB-hydroxide formed was separated by centrifugation at 4000 rpm for 5 min. The supernatant solutions were chromatographed at room temperature on 0,9x60 cm Sephadex 6-50 columns, equilibrated and eluted with sodium borate buffer (0,Ol moles/l, pH 8,5). absorbancy at 410 nm of the void volume fractions were determined. A control experiment was run in parallel with no HNB-bromide added. It was found that the enzyme activity was unaffected by the acetone concentrations used (up to 5% v/v). Reaction of tryptophan residues with HNB-bromide. The enzyme activity, protein concentration and The specific activity of each test sample was then compared to that 252 of the control experiment and the number of tryptophan groups modified per subunit was calculated using a molar absorbance value of 18 000 cm-l for the HNB-groups in the protein (7). moles/l) in sodium borate buffer (0,l moles/l, pH 8.5) were added 50 p1 o f a solution of TNM in ethanol (1 mole/l). mixture kept at 3OoC. ed in Fig. 2, and chromatographed on 0,9x60 cm Sephadex 6-50 columns, equili- brated and eluted at room temperature with sodium borate buffer (10 mmoles/l, pH 8.5). The absorbancy at 428 nm of the void volumes was determined, as well as protein concentration. subunit was calculated from the molar absorbance of 4 300 cm-l for a nitro- tyrosine group in a protein (14) and the protein content. activity was determined for each sample and compared to that of a control sample treated in the same way but without TNM added. rabbits by injecting subcutaneously 1 ml of a 1:l suspension of Freund's complete adjuvant (Difco, USA) and pea seed NDP kinase ( 2 mg/l) in a sodium phosphate buffer ( 0 , l moles/l, pH 7.4) containing NaCl (0,15 moles/l). One week later an intramuscular injection of the same amount of enzyme was made, but now using Freund's incomplete adjuvant (Difco, USA), and a booster dose was given in the same way after another week. The rabbits were starved over- night and bled through the ear vein to harvest serum. of the antiserum was 85 g/l. antiserum were added to 20 pg of pea seed NDP kinase in 100 111 o f a triethanol- amine-acetic acid buffer (10 mmoles/l, pH 7,4). kept at 37' for 30 min, followed by dilution in an ice-cold tris-acetic acid buffer (0,15 moles/l, pH 7,4). of a sample incubated at 37OC for 30 min without antiserum added. activity of the enzyme (1500 units/mg) was unaffected by this treatment. enzyme tests were performed in duplicate. treated with 14C-FDNB and TNM. ly as described before (5), except that only a twofold molar excess of 14C- FDNB (17 mCi/mmole) over NDP kinase was used. Samples were taken after a reaction time of 12 hours, when 0,8 moles of 14C-DNP-lysine residues had been formed per mole of subunit, and when 25% of the enzyme activity remained. sample was also taken before the addition of FDNB (control sample). immunological reactivity of the enzyme was tested as described in Fig. 4 A. NDP kinase was also inactivated by nitration with TNM as described above. Nitration of tyrosine residues with TNM. To 3,5 ml o f NDP kinase (31 After mixing was the incubation 0,5 ml samples were taken at time invervals, as indicat- The number of nitrotyrosine residues formed per The specific enzyme Production of antiserum. Antibodies against NDP kinase were rised in Protein concentration 25-200 ul of rabbit Inactivation of pea seed NDP kinase by antibodies. The incubation mixtures were The specific activities were compared to that All The specific Reactivity of inhibitory antibodies against the native enzyme and enzyme The enzyme was treated with I4C-FDNB essential- A The 17-822858 253 A control sample was taken before addition of TNM and treated in the same way. Samples were taken 95 min after the addition of TNM, when 1,O mole of tyrosine residues had reacted per mole of subunit o f the enzyme (21, 5% of the enzyme activity remaining. The samples were tested for immunological reactivity as described in Fig. 4 6. RESULTS AND DISCUSSION There seems to be a tyrosine residue more reactive to tetranitromethane than the other tyrosine residues in each subunit of the enzyme, and nitration of this tyrosine residue leads to inactivation of the enzyme, indicating that this tyrosine residue is of importance for enzyme activity as seen in Fig. 1. Q 50 100 Time I m i n l Fig. 1. Inactivation o f pea seed NDP kinase by nitration of tyrosine. Open circles indicate enzyme activity remaining, filled circles indicate number of nitrotyrosine residues formed per subunit. For details see text. The product ADP (4 moles/l) did not protect the enzyme from inactivation. This is in contrast to its protection of the enzyme from inactivation by 2,4- dinitrofluorobenzene (6) , ZY4,6-trinitrobenzene sulfonic acid (6) and diethyl- pyrocarbonate (6). enzyme on chromatography on Sephadex 6-200, indicating that the enzyme was not dissociated into its subunits on nitration. Fifty percent of the enzyme activity remained after reaction of all three tryptophan residues in each subunit by HNB-bromide (Fig. 2). Neither did the nitroenzyme separate from the native 254 ’C % 2 25 t \ hl A 1 I I Fig. 2. Filled number Modification of pea seed NDP-kinase with 2-OH-5-NO -benzyl bromide. triangel s indicate enzyme activity remaining after midification of the of tryptophan residues per subunit given on the abscissa. For details see text. Therefore, modification of the tryptophan residues does not seem to directly involve the active site. As seen in Fig. 3 , the native enzyme was inactivated in direct proportion to the relative amount of antiserum added, strongly indicating antigenic sites involving the active site of the enzyme. diffusion or trapping of active enzyme in antigen-antibody comp’lexes seem to have occurred since some enzyme activity was regained by using a large excess of anti- serum. Thus, 30% of enzyme activity remained using 850 pg of antiserum per pg o f A certain restriction o f substrate antiserum per ug o f NDP kinase. I I I I I 100 200 300 400 500 pg antiserum / g NDP k i n a s e t.’ Fig. 3 . Filled circles indicate enzyme activity after incubation of native enzyme with antiserum. For details see text. Inactivation of pea seed NDP kinase by specific rabbit antiserum. 255 9SZ Thus apart from the histidine residue phosphorylated during enzyme action (2,3) there seem to be a lysine (5) and a tyrosine residue essential for enzyme activity in pea seed NDP kinase. ACKNOWLEDGEMENTS This work was supported by the Swedish Medical Research Council (Project No. 13X-50). discussions and kind support of this work. I wish to thank professor Lorentz Engstrom for valuable The skilful technical assistance of 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 72. Mrs. Jill Ekstrom is gratefully acknowledged. REFERENCES Edlund, B.: Evidence for identical subunits in pea seed nucleoside di- phosphate kinase. FEBS Lett 38:222-224, 1973. Edlund, B., Rask, L., Olsson, P., Wblinder, O., Zetterqvist, U. & Engstrom, L.: yeast and purification of 1-phosphohistidine as the main phosphorylated product of an alkaline hydrolysate of enzyme incubated with adenosine ( triphosphate. Edlund, B.: and phosphorylation of the enzyme with adenosine ( Acta Chem Scand 25:1370-1376, 1971. Edlund, B.: phate kinase. Upsala J Med Sci 79:143-147, 1974. Edlund, B. & Engstrom, L.: pea seed nucleoside diphosphate kinase. FEBS Lett 47:279-283, 1974. Edlund, B., Heldin, C.H. & Engstrom, L.: Effect of chemical modification of a histidine and a lysine residue of pea seed nucleoside diphosphate kinase. Upsala J Med Sci (in press). Barman, T.E. & Koshland, D.E. J:r: A colorimetric procedure for the quantitative determination of tryptophan residues in proteins. J Biol Chem Riordan, J.F. , Sokolovsky, M. & Valler, B.L.: Tetranitromethane. A reagent for the nitration of tyrosine and tyrosyl residues of proteins. J Am Chem SOC 88:4104-4105, 1966. Arnon, R. in Current Topics in microbiology and immunology 54:47-61, 1971. Mourad, N. & Parks, J:r, R.E.: Erytrocytic nucleoside diphosphate kinase. 11. Isolation and kinetics. J Biol Chem 241:271-278, 1966. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J.: Protein measure- ments with the phenol reagent. J Biol Chem 193:265-275, 1951. Ouchterlony, U. in Handbook of Experimental Immunology fed. D.M. Weir) p. 655-706. Oxford and Edinburgh, Blackwell Sci Publ, 1967. Preparation of crystalline nucleoside diphosphate kinase from baker’s 32 P) Eur J Biochem 9:451-455, 1969. Purification of nucleoside diphosphate kinase from pea seed 32 P ) triphosphate. Active site phosphopeptides from pea seed nucleoside diphos- A peptic phosphopeptide from the active site of 242:5771-5776, 1968. 257 13. Laurell, C.-B.: Electroimmunoassay. Scand J Clin Lab Invest 29, suppl 124 14. Ness, S . , Schmidt, W . & Schneider, F.: Chemische Modifizierung und kata- 21-37, 1972. lytische Aktivitat der Carbonate-Hydro-Lyase B aus Rindererythrozyten. Modifizierung von Arginin, Histidin, Lysin, Tyrosin und Tryptophan. Hoppe-Seyler’s Z Physiol Chem 352:355-368, 1971. Address for reprints: Docent Bror Edlund Department o f C1 inical Chemistry University Hospital S-750 14 Uppsala Sweden 258