Synthetase Yields Phosphotyrosine at the Ligation Site (Adenylylated Glutamine Synthetase/Micrococcal Nuclease) TODD M

Proc. Natl. Acad. Sci. USA Vol. 79, pp. 6458-6460, November 1982 Biochemistry

Micrococcal nuclease cleavage of nucleotide linked to glutamme synthetase yields phosphotyrosine at the ligation site (adenylylated glutamine synthetase/micrococcal nuclease) TODD M. MARTENSEN AND E. R. STADTMAN Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205 Contributed by Earl R. Stadtman, July 29, 1982 ABSTRACT The activity of micrococcal nuclease was studied tein containing a single residue ofphosphotyrosine, which can on a novel substrate, denatured adenylylated glutamine synthe- be utilized to study chemical techniques for tyrosine phosphate tase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2], quantification and protein phosphotyrosine phosphatase activ- which contains a unique tyrosyl residue linked through a phos- ities in cells and tissues. phodiester bond to 5'-AMP. The products of the digestion were adenosine and O-phosphotyrosylglutamine synthetase. The Km of MATERIALS AND METHODS the macromolecular substrate with the nuclease was 1/40 that of the synthetic substrate, nitrophenyl-pdT, which is commonly used Glutamine synthetase preparations with different adenylylation forassay ofthe enzyme. Native adenylylated glutamine synthetase states were obtained from E. coli cultured under various de- was not deadenosylated by micrococcal nuclease under the con- grees of nitrogen availability. The enzyme was purified by ditions that permit rapid deadenosylation ofdenatured glutamine Zn2+, acetone, and (NH4)2SO4 precipitation steps (9, 10). Ad- synthetase. Failure to attack native glutamine synthetase is prob- enylylation reactions were carried out with 1 mM [2,8,5'-3H]- ably not due to steric factors because the native enzyme is de- ATP or [a-32P]ATP (specific activity, 100-500 jiCi/tumol; 1 Ci adenylylated by snake venom phosphodiesterase under identical = 3.7 X 1010 becquerels) in 25 mM Tris HCI buffer (pH 7.6) conditions. The inability ofmicrococcal nuclease to deadenosylate containing 20 mM Mg2+, 12.7 mM Gln, 0.15 M KCI, 2 mM native glutamine synthetase may be due to the formation of an phosphoenolpyruvate, pyruvate kinase (=5 units/ml), and suf- inactive complex because the native protein inhibited the nuclease ficient adenylyltransferase to complete the reaction in 30 min. activity on the denatured protein. The modified protein was separated from the reaction components by chromatography on Sephadex G-25 (fine) in 10 mM Regulation of glutamine synthetase [L-glutamate:ammonia li- imidazole, pH 7.1. Carboxymethylation of glutamine synthe- gase (ADP-forming), EC 6.3.1.2] activity in Escherichia coli tase was carried out by a method similar to that ofShapiro and involves the covalent attachment ofan adenylyl group in phos- Stadtman (11). Prior to the addition ofiodoacetate (20 mM), the phodiester linkage to a single tyrosine residue in each Mr 50,000 enzyme was dialyzed against 6 M urea/i mM EDTA/0.2 M subunit (1). Both adenylylation and deadenylylation of the en- Tris HCI, pH 8.0, and then incubated with 10 mM dithiothrei- zyme are catalyzed by the same adenylyltransferase (2), whose tol for 30 min at 37°C. After carboxymethylation at 4°C (2 hr) activity is regulated by the interconversion ofa small regulatory in the dark, the sample was exhaustively dialyzed against 1.0 protein PI,, between uridylylated and nonuridylylated states mM sodium borate (pH 9.3). The sample was stored at -20°C (3). Uridylylation of the PI, protein involves attachment of 5'- after removal ofan aliquot for protein (12), phosphate (13), and UMP through a phosphodiester bond to one of two tyrosine radioactivity measurements. Micrococcalnuclease and bacterial residues in each Mr 11,000 subunit ofthe protein (4). Recently, alkaline phosphatase were obtained from P-L Biochemicals. the modification of several other proteins has been shown to Snake venom phosphodiesterase was obtained from Boehringer involve the nucleotidylylation of tyrosine residues. The RNA Mannheim. The nuclease (1.0-5.0 mg) was dissolved in 1.0 ml genome of polio virus is ligated to a protein tyrosine residue of20 mM Tris HCI, pH 7.6/20% glycerol. Digestions ofnative through a phosphodiester bond (5, 6), and DNA is ligated to and carboxymethylated adenylyl glutamine synthetase were topoisomerases through a tyrosine residue (7, 8), probably as normally carried out at 37°C in 40-50 mM borate, pH 9.3/ a reaction intermediate. Characterization oftyrosine as the site 8-10 mM CaC12. Fixed time assays were carried out in 50 X of nucleotide ligation can be demonstrated by both enzymatic 6 mm culture tubes in 25- to 50-,ul volumes. Reactions were and chemical hydrolyses. Alkaline pH difference spectroscopy stopped by the addition of 100 IlI of0.1 M HCl containing bo- at 293 nm demonstrated that snake venom phosphodiesterase vine serum albumin as carrier (5 mg/ml), followed by 500 IlI treatment ofan adenylylated decapeptide produced a free phe- of10% trichloroacetic acid. The samples were centrifuged after nolic hydroxyl (1). Protein linked to 32P-labeled polynucleotide standing on ice for 15 min, and an aliquot of the supernatant was enzymatically digestedwith nucleases toyieldprotein-pUp; was removed for scintillation counting. Snake venom phospho- partial acid hydrolysis of the latter yielded, among several prod- diesterase assays of native adenylylated glutamine synthetase ucts, phosphotyrosine and tyrosine-pUp, which was further were carried out by using a similar technique. Thin-layer chro- degraded with micrococcal nuclease to tyrosine phosphate and matography was carried out on silica gel plates (Eastman) de- 3'-UMP (5, 6). We demonstrate here that micrococcal nuclease veloped with n-propanol:H20:0.1 M NH40H, 3:1:0.1 (vol/ treatment of denatured adenylylated glutamine synthetase vol). yields phosphotyrosyl glutamine synthetase and adenosine. The procedure allows the preparation ofmilligram quantities ofpro- RESULTS Cleavage of Adenylylated Glutamine Synthetase by Micro- The publication costs ofthis article were defrayed in part by page charge coccal Nuclease. When carboxymethylated [3H]adenylylglu- payment. This article must therefore be hereby marked "advertise- tamine synthetase (110 AM) was incubated with micrococcal ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. nuclease (0.16 mg/ml), 48%, and 98% of the tritium label was 6458 Downloaded by guest on September 25, 2021 Biochemistry: Martensen and Stadtman Proc. Natl. Acad. Sci. USA 79 (1982) 6459 released in 16 and 210 min, respectively. Protein from the nu- Table 1. Effect of pH and Ca2+ concentration on the release of clease digest was precipitated with 2 volumes of ethanol, and [5H]adenosine from carboxymethylated [3H]adenylylated the supernatant was subjected to thin-layer chromatography. glutamine synthetase by micrococcal nuclease During chromatography, the tritium label comigrated with car- [3H]adenosine Relative rier adenosine. When [3H, 32P]adenylylglutamine synthetase Sample components released, cpm activity, % was treated with nuclease (Fig. 1), only the tritium label was released (as [3H]adenosine); the 32p label remained Ches, pH 9.4 0 0 bound to Ches, pH 9.4/0.1 mM Ca2+ 91 5.2 the protein. Upon treatment of the 32P-labeled protein with Ches, pH 9.4/1.0 mM Ca2+ 356 20 bacterial alkaline phosphatase, 93% ofthe 32P was released from Ches, pH 9.4/10 mM Ca2+ 1,617 95 the protein. It is evident from these results that adenosine and Tris, pH 9.4/10 mM Ca2+ 1,761 100 phosphotyrosyl glutamine synthetase are the major products Tris, pH 9.0/10 mM Ca2+ 1,459 83 when adenylylated glutamine synthetase is digested with mi- Tris, pH 8.4/10 mM Ca2+ 924 52 crococcal nuclease. Tris, pH 8.0/10 mM Ca2+ 654 37 Optimal Conditions for Nuclease Activity. Ca2+ was re- quired for the deadenosylation ofadenylylglutamine synthetase Assay mixtures contained 120 ,uM glutamine synthetase subunits (6.0 mg/ml), 84 mM Ches [2-(N-cyclohexylamino)ethanesulfonic acid] by nuclease, and the optimal pH was _9.4 (Table 1). It should or Tris as indicated, 83 pg of nuclease per ml, and Ca2+ as indicated. be noted that the small loss of 32p shown in Fig. 1 was the same Maximal substrate loss was 16% 10 whether 6 mM Ca2+, Mg2+, or Mn2+ was present in the assay per min. mixture; however, the release of [3H]adenosine with 6 mM tase to nuclease digestion is not likely due to burial of the ad- Mg2+ or Mn2+ was <3% of that obtained with 6 mM Ca2 . enylyltyrosine group in the native configuration. Physical stud- From double reciprocal plots ofvelocity versus glutamine syn- ies (14) indicate that the adenylyl moiety of native enzyme is thetase concentration (Fig. 2), it was estimated that the Km for exposed to the solvent. Moreover, the ability of snake venom glutamine synthetase is -50 X 10-6 M and the kcatalysis is 2.3 phosphodiesterase to release AMP from the adenylylated en- min-1. Therefore, the affinity for the macromolecular sub- zyme (1) and the fact that phosphorylytic cleavage of the ad- strate, glutamine synthetase, is good. enylyltyrosine bond is readily catalyzed by adenylyltransferase Unfortunately, the nuclease could not catalyze the deaden- (2) suggest that the adenylyltyrosine group is in an exposed po- osylation ofnative [3H]adenylylglutamine synthetase under any sition. However, these studies were all carried out in the pres- ofseveral conditions in which different cations, buffers, and pH ence ofeither Mn2+ or Mg2' and at a lower pH than that used values were varied. This resistance ofnative glutamine synthe- in the nuclease digestions. Therefore, we examined the ability ofphosphodiesterase to 100 hydrolyze native adenylylglutamine synthetase and the low molecular weight substrate (nitrophenyl-pdT) under the conditions thatsupportnucleasedigestionofdenatured enzyme-i.e., in the presence of Ca2+ at pH 9.0. Snake venom phosphodiesterase catalyzed cleavage ofboth substrates under these con- 80 ditions (Table 2). Therefore, it is unlikely that the failure to deadenosylate native glutamine synthetase is due to inaccess- ability ofthe adenylyltyrosine group to nuclease attack. In fact, the native enzyme must bind to the nuclease because native adenylylated glutamine synthetase strongly inhibited the -6 60 dead- cn 0 enosylation of denatured [3H]adenylylglutamine synthetase GS t303 H]Adenosine (Fig. 3). The inhibition was not due to digestion of the native 0~ p-O-[3 U enzyme because, in a parallel incubation with native [3H]adenylylglutamine synthetase, there was only 0.3-0.7% release of 0 r 40 0 GS O_32p_0+ [3H]Adenosine 1.6

0 1.2 20 -4 .- 0 C- 1 2 3 4 Time, hr FIG. 1. Time course for digestion of carboxymethyl [3H, 32p]- adenylylated glutamine synthetase with micrococcal nuclease. Car- 0.8 1.2 boxymethylated [3H, 32Pladenylylated glutamine synthetase (5.8 mg/ ml) was digested in 48 mM sodium borate/9.5 mM CaCl2, pH 9.3, with S-I X 10, .4M-1 95 pg of micrococcal nuclease per ml at 37°C. Aliquots were removed at the indicated times and precipitated with trichloroacetic acid (10%, FIG. 2. Double-reciprocal plotofdeadenosylation rate (V-1) versus wt/vol). The supernatants were carefully removed and added to scin- carboxymethylated [3H]adenylylated glutamine synthetase (S-1) con- tillation vials for assay of 3p (0) and 3H (e). The washed precipitate centration. Assays at 37°C contained 50 mM borate (pH 9.3), 10 mM was solubilized in 1 M NaOH and also assayed. CaCl2, and 33 pg of micrococcal nuclease per ml. Downloaded by guest on September 25, 2021 6460 Biochemistry: Martensen and Stadtman Proc. Natl. Acad. Sci. USA 79 (1982) Table 2. Effectors of snake venom phosphodiesterase activity on 1100 native [32P]adenylylated glutamine synthetase and nitrophenyl-dTMP Effector v (nmol ml-' min-') % maximum

. - Substrate: [32P]adenylylated glutamine synthetase .>e, 3.3 mM Mg0i2, pH 6.9 6.6 35 e- .o4 3.3 mM MgCi2, pH 8.0 13 69 sQ6 mM 9.0 16 82 3.3 MgCi2, pH .,-j.!5 . 10 mM MgCi2 15 80 4-3 3.3 mM MgCi2/6.7 mM MnCi2 14 75 --t4Q 0 >" 3.3 mM MgC12/6.7 mM CaCi2 13 68 Substrate: nitrophenyl-dTMP 5 mM MgCi2 17.8 92 10 mM MgCi2 19.4 ± 0.4 100 15 mM Mg0i2 18.4 95 10 mM MgC12/10 mM MnCi2 20.4 105 0.5 1.0 1.5 2.0 10 mM MgCi2/10 mM CaCi2 16.8 87 10 mM Mg0i2/0.1 M KCl 16.3 84 Native adenylylated GS, mg/ml Native [32P]adenylylated glutamine synthetase (5.3 mg/ml) was FIG. 3. Inhibition of the digestion of carboxymethylated [3H]- digested with snake venom phosphodiesterase (6.7 units/ml) for 5 min adenylylated glutamine synthetase by native [3H]adenylylated glu- at 370C in 33 mM Tris-HCI (pH 9.0) unless specified otherwise. (The tamine synthetase (GS). Carboxymethylated [3H]adenylylated gluta- heading "% maximum" refers to the percentage of total substrate ra- mine synthetase (0.82 mg/ml) digestions with micrococcal nuclease dioactivity solubilized during assay.) Nitrophenyl-pdT (0.96 mM) was (17 ,ug/ml) were carried out for 5 min at 3700 (W). Native [3H]ade- digested in 0.1 M Tris-HCI (pH 9.0) at 370C with snake venom phos- nylylated glutamine synthetase was added to three samples at assay phodiesterase (28 milliunits/ml). (In this case, the heading "% maxi- concentrations of 0.44 mg (4,200 cpm), 0.88 mg/ml (8,500 cpm), and mum" is the activity relative to 10 mM MgCi2.) 1.76 mg/ml (17,000 cpm). Digestion of native enzyme alone under the same conditions is shown (o). (Inset) Dixon plot of the inhibition. radioactivity, whereas there was 30% release of radioactivity glutamine synthetase has enabled the preparation of milligram from the 3H-labeled denatured enzyme. A Dixon plot ofthe data quantities ofa 32P-radiolabeled phosphotyrosine protein for use (Fig. 3 Inset) showed that the inhibition was linear with in- in developing analytical methods for measuring tyrosine phos- creasing concentrations of native enzyme. This suggests (but phate and protein phosphotyrosine phosphatase activities in does not prove) that an inactive complex is formed between the cells (16). The nuclease also could be used as a diagnostic test nuclease and the native glutamine synthetase. for the ligation site of a protein nucleic acid bond. 32P-Radio- labeled nucleotide linked to protein tyrosine residues through DISCUSSION a 5' phosphodiester bond should be cleaved by snake venom Micrococcal nuclease cleavage of adenylylated glutamine syn- diesterase and resistant to phosphatase before but not after thetase (carboxymethylated) to form adenosine and phospho- micrococcal nuclease digestion. tyrosyl glutamine synthetase is in agreement with the cleavage pattern obtained with synthetic substrates (15) having the struc- T.M.M. was supported by a grant from Merck Sharp, & Dohme. ture RpN. The R is a nitrophenyl group and pN is 5'-dTMP; 1. Shapiro, B. M. & Stadtman, E. R. (1968) J. BioL Chem. 243, the nuclease cleavage products are p-nitrophenyl phosphate 3769-3771. and deoxythymidine. The Km and with this substrate 2. Anderson, W. B., Hennig, S. B., Ginsburg, A. & Stadtman, E. kcatalysis R. (1970) Proc. Nati Acad. Sci. USA 67, 1417-1424. are 2.2 x 10-3 M and 9.1 min-1, respectively. The affinity of 3. Brown, M. S., Segal, A. & Stadtman, E. R. (1971) Proc. NatL the nuclease for synthetic substrates is dependent on the size Acad. Sci. USA 68, 2949-2953. and charge of substituents esterified to the 3' hydroxyl group 4. Adler, S. P., Purich, D. & Stadtman, E. R. (1975)J. BioL Chem. ofthe base; nitrophenyl-pdTp was bound 200 times more tightly 250, 6264-6272. than nitrophenyl-pdT (15). The nature of the base was consid- 5. Rothberg, P. G., Harris, T. J. R., Nomoto, A. & Wimmer, E. ered to be ofminor importance. When the R group is glutamine (1978) Proc. Nati Acad. Sci. USA 75, 4868-4872. x 6 2.3 6. Ambros, V. & Baltimore, D. (1978)J. BioL Chem. 254, 5263-5266. synthetase, the Km is -50 10 M and the kcadYsjs is 7. Tse, Y.-C., Kirkegaard, K. & Wang, J. C. (1980) J. BioL Chem. min-'. This suggests that esterification of the 5'-phosphoryl 255, 5560-5565. moiety ofa nucleoside substrate to a protein promotes its bind- 8. Champoux, J. J. (1981)J. BioL Chem. 256, 4805-4809. ing affinity. Ifthe Km value is a true measure ofthe dissociation 9. Miller, R., Shelton, E. & Stadtman, E. R. (1974) Arch. Biochem. constant, as was shown for simple synthetic substrates (15), then Biophys. 163, 155-171. adenylylated glutamine synthetase binds to the nuclease with 10. Woolfolk, C. A., Shapiro, B. M. & Stadtman, E. R. (1966) Arch. Biochem. Biophys. 116, 177-192. 40-fold greater affinity than does the low molecular weight 11. Shapiro, B. M. & Stadtman, E. R. (1967) J. BioL Chem. 242, analog. 5069-5079. The kcatysis of the carboxymethylated macromolecular sub- 12. Lowry, 0. H., Rosebrough, M. J., Farr, A. L. & Randall, R. J. strate is 25% of that of the low molecular weight analog. This (1951)J. BioL Chem. 193, 265-275. may result from the macromolecular substrate restricting the 13. Ames, B. N. (1966) Methods EnzymoL 8, 115-118. conformational freedom of catalytic site groups. Furthermore, 14. Chock, P. B., Villafranca, J. J., Rhee, S. G., Ubom, G. A. & Stadtman, E. R. (1979) in NMR and Biochemistry, eds. Opella, with native glutamine synthetase, strong interactions may occur S. J. & Lu, P. (Dekker, New York), pp. 405-418. such that turnover is prevented or reduced to a small level. 15. Cuatrecasas, P., Wilchek, M. & Anfinsen, C. B. (1969) Biochem- The novel catalytic specificity ofmicrococcal nuclease to pro- istry 8, 2277-2284. duce a phosphotyrosyl residue at the nucleotide ligation site of 16. Martensen, T. M. (1982) J. BioL Chem. 257, 9648-9652. Downloaded by guest on September 25, 2021