Evidence for a Tyrosine Residue at the Active Site of Phosphoglucomutase and Its Interaction with Vanadate (Enzyme Site) PORTER P

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Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 5010-5013, October 1979 Biochemistry Evidence for a tyrosine residue at the active site of phosphoglucomutase and its interaction with vanadate (enzyme site) PORTER P. LAYNE AND VICTOR A. NAJJAR Division of Protein Chemistry, Tufts University School of Medicine, Boston, Massachusetts 02111 Communicated by Sidney P. Colowick, July 16, 1979

ABSTRACT The rate of transfer of [32Plphosphate from acetylimidazole, tetranitromethane, sodium nitrite, and p- [32P-labeled phosphoglucomutase (a-D-glucose-1,6-bisphos- hydroxymercuribenzoate (Sigma); glucose 6-phosphate phate:a-D-glucose--phosphate phosphotransferase, EC 2.7.5.1) (Boehringer); sodium orthovanadate, (ICN); sodium arsenate to glucose increases dramatically between pH 8.5 and 10.5 with acid, (Fisher); ethyleneimine (K&K); [y32P]ATP a half maximal rate at pH 9.8. This suggests the participation and sulfanilic of a residue containing an ionizable group with a pK close to (25 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) (New England 10. The inhibition of enzyme activity obtained with tyrosine- Nuclear); ACS scintillant (Amersham); bovine serum albumin derivatizing reactions-iodination, nitration, acetylation, and (Miles). diazo coupling-is strongly indicative of tyrosine participation. The enzyme was crystallized from rabbit skeletal muscle (3). Thiol reagents, p-hydroxymercuribenzoate and ethyleneimine, 32P-Labeled enzyme was prepared as before by allowing ex- were without effect. Vanadate and arsenate augmented the change of glucose 6-[32P]phosphate and phosphorylated transfer reaction 200- and 2.5-fold, respectively, and lowered phosphoglucomutase according to the mechanism of the re- the pH optimum of the reaction. action (4, 5). The phosphate transfer reaction was carried out as described (2). The extent of reaction was measured by the The physiological function of phosphoglucomutase (a-D-glu- production of trichloroacetic acid-soluble radioactive organic cose-1,6-bisphosphate:a-D-glucose-l-phosphate phospho- phosphate. transferase, EC 2.7.5.1) is to mediate the interconversion of Chemical Modification of Enzyme with Tyrosine Re- glucose 1-phosphate and glucose 6-phosphate via the inter- agents. Iodination. The method of Azari and Feeney (6) was mediate glucose 1,6-bisphosphate. Both phospho- and de- used. [32P]Phosphoglucomutase (0.02 ,gmol) was incubated in phospho-enzyme forms participate in this interconversion. 0.1 M borate buffer at pH 9.5 with various dilutions of a stock We have recently shown that 32P-labeled phosphogluco- iodine/iodide solution of 0.05 M 12 in 0.24 M KI at 0C. The mutase ([32P]phosphoglucomutase) is capable of transferring iodine reagent was bleached within 15 s; however, the incu- the phosphate group to several nucleophiles (1) including glu- bation was continued for 10 mm. Final volume was 200 y1. The cose and its analogs (2). The rate of this transfer is several orders extent of inactivation with this reagent, as with all the other of magnitude slower than to glucose monophosphates. This derivatizing reagents discussed below, was determined by made possible a detailed study of the initial rate of the reaction, adding a 1000-fold excess of the substrate glucose 1-phosphate the effect of variations in reaction conditions, and the structural to the reaction. The amount of radioactive phosphate removed requirements of the acceptor molecule. In fact, with this ap- served as a measure of the remaining active enzyme. proach, we have made several observations that would have Acetylation. The reaction was carried out in a manner similar been unlikely otherwise and that are valid and relevant to the to that described by Simpson et al. (7). [32P]Phosphogluco- catalytic reaction. For example, we have previously been able mutase (0.02 .umol) was acetylated by incubation in 200 mM to define quite accurately several stringent structural re- N-acetylimidazole at 0C for 30 min in 40 mM 1,4-piperaz- quirements of the substrate for reaction, including the essential inediethanesulfonic acid (Pipes) buffer at pH 7.5 in a final orientations of the hydroxyl functions of the glucose molecule volume of 200 ,p. The extent of inactivation was determined (2). We have now obtained additional interesting findings, again as in the iodination reaction. Deacetylation with subsequent through the use of the phosphate transfer reaction. regeneration of enzyme activity was performed in two ways. In this article, we present evidence for the participation of The inactivated acetylated enzyme was incubated with 1 M a tyrosine residue at the active site of phosphoglucomutase. This hydroxylamine (pH 7) at room temperature for 20 min. Al- is based on the behavior of the reaction rate with increasing pH ternatively, deacetylation was effected by incubation of the and on the sensitivity of the enzyme to tyrosine derivatizing enzyme in 250 mM Tris-HCl (pH 9.0) at room temperature for reagents. No residue other than tyrosine could account fully for 20 min. The extent of regeneration was then determined as the results observed. In addition, vanadate and arsenate, which before. stimulate the transfer reaction markedly, appear to manifest Nitration. This reaction was carried out according to the their activating effect by lowering the pKa of the active site method of Sokolovsky et al. (8). [32P]Phosphoglucomutase (0.02 tyrosine. ,gmol) was incubated with various concentrations of tetranitromethane at room temperature for 30 min in 0.05 M Tris-HCI MATERIALS AND METHODS (pH 8) in a final volume of 200 ,g. The desired dilutions were All chemicals were reagent grade and used without further made from a stock solution of 0.84 M tetranitromethane in purification. These were obtained as follows: D-glucose, methanol. The extent of inactivation was then determined. 2-phosphate, N- Diazo Coupling. This reaction was carried out according to a-D-glucose 1-phosphate, myo-inositol Riordan and Vallee (9). [32P]Phosphoglucomutase (0.02 ,mol) was incubated with 5 mM diazotized sulfanilic acid in 0.1 M The publication costs of this article were defrayed in part by page 5 and 30 min in charge payment. This article must therefore be hereby marked "ad- sodium bicarbonate buffer (pH 8.8) at 0°C for vertisement" in accordance with 18 U. S. C. §1734 solely to indicate a final volume of 200 ,l. The extent of inactivation was again this fact. determined by the addition of glucose 1-phosphate. 5010 Downloaded by guest on October 1, 2021 Biochemistry: Layne and Najjar Proc. Natl. Acad. Sci. USA 76 (1979) 5011 Chemical Modification of the Enzyme with Thiol Re- agents. Again, 0.02 Mrmol of the enzyme was used with tetWo reagents employed. p-Hydroxymercuribenzoate was used at a final concentration of 10 mM in 50 mM Tris-HCl at pH 8.0, and the reaction mixture was incubated for 5 and 30 min at 240C in a final volume of 200 ,ul. This reagent was shown previously to react rapidly with the enzyme (10). Ethyleneimine was used at 100 mM in Tris-HCI (pH 8.6) in a final volume of 200 pl for the same time intervals. The remaining activity with both thiol reagents was again assayed by adding an excess of glucose 1-phosphate. Amino acid analysis was done in a Beckman 119CL amino acid analyzer. RESULTS Inhibition of Phosphate Transfer Reaction by Inositol 2-Phosphate. As reported (2), glucose is phosphorylated by phosphoglucomutase at a very slow rate. Consequently, the duration of the reaction was such as to yield easily measurable 6 7 8 9 10 11 rates of phosphate transfer (2). Inositol 2-phosphate is an in- pH hibitor of this reaction with a Ki of 0.58 mM. Double reciprocal FIG. 2. Effect of pH on rate of phosphate transfer reaction. plots of initial rates at varying glucose concentrations were [32P]Phosphoglucomutase (0.02 Amol) in 5 mM Tris-HCl/2 mM made at inositol 2-phosphate concentrations of 0.1 and 0.5 mM. MgCl2 at pH 7.5 was added to a previously prepared solution com- The plot appears to represent a clear case of competitive inhi- posed of 55 Al of water, 100 gl of 20 mM glucose, and 20 Al of 1 M bition (Fig. 1). buffer at the appropriate pH. Final volume was 200 ,ul. The reaction Effect of pH on Rate of Phosphate Transfer. Fig. 2 shows mixture was incubated at 37°C for various time periods. Samples were increase in the initial reaction rate was measured after 10 s for vanadate and 60 s for arsenate and control. that a small, gradual All values are adjusted to cpm transferred. Where appropriate, 2 JAI obtained with increasing pH from pH 5 to pH 8.5. However, of 100 mM sodium arsenate or sodium vanadate was included. The beyond pH 8.5 a rapid increase in rate began with a maximum buffers used were 4-morpholineethanesulfonic acid (Mes) for pH 6 at pH 10.5 and a half-maximal rate at pH 9.8. Beyond this, the and 7, Tris-HCl for pH 8 and 9, and sodium carbonate for pH 10 and transfer activity decreased. This segment of the pH curve 11. Because vanadate caused a considerable rate increase, it was suggests the presence of an ionizable group having a pKa of necessary to decrease the final glucose concentration from 10 mM as the involvement of one of in the control (glucose alone) to 1 mM for accurate measurement of about 10.0. This would signal any initial rates. Under all conditions, the reaction was stopped and assayed as in Fig. 1. A, Glucose alone; 0, glucose with arsenate; 0, glucose with vanadate. 4000 three functional groups: the hydroxyl of tyrosine, the E-amino 3500 of lysine, or the thiol of cysteine. It must be noted here that under the conditions of our experiments spontaneous dephos- 3000 phorylation at elevated pH values in the absence of glucose was 4-O negligible. The figure also shows that with 1 mM arsenate or . 25000 1 mM vanadate a shift occurred in the activity curve toward E more acidic pH values. M The effect of vanadate was much more dramatic than that E 2000 _/ of arsenate. Vanadate caused a shift of the half-maximal rate 0o 1500 from pH 9.8 to 7.7, whereas the shift with arsenate was only .C slight with a half-maximal rate obtained at pH 9.5. In this connection, it is of interest that both anions are also activators -° 1000- of the phosphate transfer reaction (11). Fig. 3 shows that arsenate at 1 mM stimulated the reaction approximately 2.5-fold with 1 mM glucose. Under the same conditions, 1 mM vanadate stimulated the transfer reaction by a factor greater than 200. 0 0.01 0.02 0.03 Other anions such as tungstate, molybdate, and columbate were 1/Glucose, mM-1 ineffective. FIG. 1. Double reciprocal plot of reaction of glucose with phos- Inactivation of Phosphoglucomutase by lodination. Table phoglucomutase in presence of inositol 2-phosphate. Glucose (100 gl 1 shows that inactivation of this enzyme under the conditions of 200, 160, 120, and 80 mM) in 80 mM imidazole/2 mM MgCl2 at pH 7.5 was incubated for 5, 10, 15, and 20 min at 370C with 0.02 ,tmol of detailed above was very rapid, approximately 15 s, over a range l:12Pjphosphoglucomutase previously dialyzed against 5 mM Tris-HCl, of concentrations (0.25-1 mM 12 and, correspondingly, 1.2-4.8 pH 7.5. Final volume was 200 ,l. Where appropriate, 5 ,ul of 20 mM mM KI). Enzyme concentration was kept at 0.1 mM through- or 4 mM inositol 2-phosphate was added to yield a final concentration out. Complete inactivation of the enzyme was obtained at ap- of 0.5 and 0.1 mM, respectively. Fifty microliters of 6% bovine serum proximately 1 mM 12 in KI. albumin was added followed immediately by 100 ul of 25% trichlo- The addition of 10 mM inositol 2-phosphate to the enzyme roacetic acid. After centrifugation 200 yl of supernatant was assayed for radioactivity. Controls containing only buffer yield negligible before the iodination reagent considerably diminished the in- ~roduction of inorganic phosphate (12% per hr). 0, Glucose alone; activating effect. The results shown in Table 1 represent the 0, glucose with 0.1 mm inositol 2-phosphate; A, glucose with 0.5 mM percentage inhibition obtained with iodination, as well as with inositol 2-phosphate. the diazo coupling reaction, nitration, and acetylation. Included Downloaded by guest on October 1, 2021 5012 Biochemistry: Layne and Najjar Proc. Natl. Acad. Sci. USA 76 (1979) acetylimidazole was carried out at 0WC. After 30 min only 45% E 16- 40/ of the enzyme activity remained (Table 1). Inclusion of 5mM E inositol 2-phosphate in the acetylation reaction medium af- 14- forded good protection of the enzyme to the extent of 58% of the lost activity. Incubation of the derivatized enzyme in 1 M 7612 -30- E hydroxylamine (pH 7) or 250mM Tris-HCl (pH 9) at 250C for C 0 20 min was found to regenerate 90 or 52% of the inhibited activity, respectively. 8 20- Nitration of Phosphoglucomutase by Tetranitromethane.

4- Incubation of the enzyme for 30 min at 250C in 0.75-6 mM 0~~ ~ ~ ~ ~ ~ tetranitromethane, under the conditions described above, re- sulted in a loss of 41-92% of the phosphate transfer activity. The extent of nitration at 3 mM for 20 min, under these conditions, was determined by amino acid analysis. Seven tyrosine residues

0 20 40 60 80 100 120 1 4 out of a total of 20(5) were nitrated. Time, min Time, min Inositol 2-phosphate (3mM) afforded considerable protection FIG. 3. Relative stimulatory activity of arsenate and vanadate. and reduced inactivation caused by 3 mM tetranitromethane (Left) One hundred microliters of 2mM glucose in 80mM imidazole/2 from 83% of the original activity to 15% (Table 1). mM MgCl2 at pH 7.5 was added to 100 ,l containing 0.02 ,gmol of The derivatizing reagents so far utilized, although highly [32P]phosphoglucomutase in the presence and absence of 1 mM so- reactive with the tyrosine residues, nevertheless react to some dium arsenate. The reaction mixture was incubated at 370C for 15, degree also with cysteine. Consequently, the enzyme was tested 30, 60, 90, and 120 min. (Right) Same as Left except that 1 mM so- for sensitivity to two cysteine reagents-namely, p-hydroxy- dium vanadate was used and the incubation was for 1, 2, 3, 4, and 5 min. In all cases, the reaction was stopped and assayed as in Fig. 1. mercuribenzoate and ethyleneimine. Neither reagent caused 0, Glucose alone; A, glucose with arsenate; 0, glucose with vana- any loss of activity. This excludes cysteine as a participant in date. phosphoglucomutase catalysis, as was shown earlier (10). Coupling of Sulfanilic Acid Diazonium Salt to Phospho- also is the protective effect of inositol 2-phosphate. It was not glucomutase. Reaction between 0.1 mM enzyme and 3, 6, and feasible to attempt protection of the enzyme with either of the 12 mM diazonium salt of sulfanilic acid in 0.1 M bicarbonate hexose monophosphate substrates, glucose 1-phosphate or buffer at pH 8.8 and 0°C yielded a maximal inactivation of 40% glucose 6-phosphate, because under these conditions there (Table 1). However, no inhibition was observed when 6 mM would be an instantaneous transfer of the enzyme phosphate inositol 2-phosphate was added before the diazonium salt. to the substrate at a far more rapid rate than the rate of iodination at 0WC. DISCUSSION Acetylation of Phosphoglucomutase with N-Acetylim- The kinetics of the enzyme reaction with increasing pH and the idazole. Acetylation of 0.02 ,umol of enzyme in 200 mM N- derivatization reactions that lead to inactivation establish the importance of the tyrosine residue in the catalytic activity of Table 1. Inactivation of phosphoglucomutase by derivatizing the enzyme. agents and protective effect of substrate analog The kinetics of inhibition of glucose phosphorylation by inositol 2-phosphate inositol 2-phosphate, as shown in Fig. 1, exhibit competitive % inactivation characteristics. This indicates that inositol 2-phosphate binds With Without at the active site of the enzyme. It also explains the protection inositol inositol afforded the enzyme against inactivation caused by the deri- Derivatizing reaction 2-phosphate 2-phosphate vatization of a residue at the active site. The data obtained by the chemical modification of the en- Diazo coupling (00C, 30 min) at elevated pH Diazosulfanilate (3 mM) 0 7 zyme and the profile of glucose phosphorylation Diazosulfanilate (6 mM) 0 16 values indicate the presence of a tyrosine residue at the active Diazosulfanilate (12 mM) 0 34 site. The compelling evidence for this conclusion includes the Iodination (00C, 10 min) inactivation by nitration, iodination, coupling with diazonium Iodine (0.5 mM) 0 41 salt, and acetylation. All the modifying reagents are known to Iodine (1.0 mM) 15 100 possess both a high reactivity and a good degree of specificity Iodine (2.0 mM) 55 100 for the tyrosine residue. Other amino acid residues that might Nitration (250C, 30 min) possess some reactivity with these reagents include cysteine, Tetranitromethane (0.75 mM) 41 lysine, and histidine. However, the activity of the enzyme is not Tetranitromethane (1.50 mM) - 57 inhibited by the cysteine derivatizing reagents p-hydroxy- Tetranitromethane (3.00 mM) 15 82 mercuribenzoate and ethyleneimine. Lysine, on the other hand, Acetylation (00C, 30 min) is readily eliminated from consideration because tetranitro- N-acetylimidazole (100 mM) 35 methane and I2 plus I- do not react with this residue under the N-acetylimidazole (200 mM) 23 55 conditions used. Furthermore, the facile reactivation of the Derivatizing reagent (100 ul) was added to 0.02 Amol of [32PJ- acetylated enzyme with hydroxylamine or at high pH renders phosphoglucomutase in the presence or absence of inositol 2-phos- the lysine residue an unlikely candidate because the N-acet- phate in a final volume of 200 pl. The concentrations of inositol 2- yllysine amide bond would not be cleaved under these condi- phosphate used were as follows: 6 mM for the diazo coupling reaction, tions. By contrast, the O-acetyl ester bond, such as would occur 10 mM for iodination, 3 mM for nitration, and 5 mM for acetylation. at tyrosine, is easily cleaved by high concentrations of hy- Each concentration of iodine listed in the table contains 4.8 molar ion. excess of KI. A 1000-fold excess of glucose 1-phosphate was added at droxylamine and hydroxyl the end of the incubation period. The reaction was stopped with tri- The possibility that histidine might be involved should be chloroacetic acid and assayed as described in Fig. 1. considered because it is derivatized in the iodination and diazo Downloaded by guest on October 1, 2021 Biochemistry: Layne and Najjar Proc. Natl. Acad. Sci. USA 76 (1979) 5013

coupling reactions. However, other reasons negate its in- already been shown to have a tyrosine at the active site- volvement. The pH profile of the transfer reaction indicates a namely, carboxypeptidase A (7) and arginine kinase (14). maximum rate at pH 11. This is far removed from the pK of histidine. The acetylation of histidine in proteins by N-acetyl- This work was supported by Public Health Service Grant 5R01 imidazole is presumably unlikely because it has not been con- AL09116, National Science Foundation Grant PCM76-23008, and the sidered by others (12, 13). The most important fact that singles National Foundation March of Dimes Grant 1-556. out tyrosine as the derivatized residue is the nitration reaction. Tetranitromethane has been shown not to nitrate histidine or 1. Layne, P. P. & Najjar, V. A. (1975) J. Biol. Chem. 250, 966- N-acetylhistidine (8). 972. Fig. 2 depicts the relationship between activity and pH. As 2. Layne, P. P. & Najjar, V. A. (1978) Biochim. Biophys. Acta 526, noted above, the pattern is reminiscent of a titration curve of 429-439. 3. Najjar, V. A. (1955) Methods Enzymol. 1, 294-299. a residue with a pKa of about 10. This value corresponds very 4. Najjar, V. A. & Pullman, M. (1954) Science 119, 631-634. to pKa of 10.07 group ty- well the for the hydroxyl of free 5. Najjar, V. A. (1962) in The Enzymes, ed. Boyer, P. D. (Academic, rosine. New York), Vol. 6, pp. 161-178. In the presence of vanadate and arsenate, the phosphate 6. Azari, P. R. & Feeney, R. E. (1961) Arch. Biochem. Biophys. 92, transfer reaction is strongly stimulated. More importantly, the 44-52. pH optimum of this reaction is shifted to lower values by these 7. Simpson, R. T., Riordan, J. F. & Vallee, B. L. (1963) Biochemistry anions. Vanadate shifts the pH at which a half-maximal reaction 2,616-622. rate is obtained from pH 10 to pH 7.7. On the other hand, the 8. Sokolovsky, M., Riordan, J. F. & Vallee, B. L. (1966) Biochemistry corresponding shift in pH caused by arsenate is only from pH 5,3582-3589. 10 to pH 9.5. The obvious conclusion is that vanadate and ar- 9. Riordan, J. F. & Vallee, B. L. (1972) Methods Enzymol. 25, senate bind to the enzyme in such a way as to promote the 521-531. transfer reaction. This might occur by assisting in the ionization 10. Bocchini, V., Alioto, M. R. & Najjar, V. A. (1967) Biochemistry of the hydroxyl function of the tyrosine at the active site. The 6,313-322. 11. Layne, P. P. & V. A. Am. manner by which such an ionized residue functions is uncertain. Najjar, (1979) Fed. Proc. Fed. Soc. Exp. Biol. 38, in press. It a proton, a positive or might abstract neutralize charge, 12. Riordan, J. F., Wacher, W. E. C. & Vallee, B. L. (1965) Bio- participate or assist in a nucleophilic attack on a susceptible chemistry 4, 1758-1765. bond. Whatever the mechanism, it is certain that the tyrosine 13. Means, G. E. & Feeney, R. E. (1971) Chemical Modification of residue plays an essential role. The possibility of the involve- Proteins (Holden-Day, San Francisco), pp. 72-74. ment of O-phosphoryltyrosine is worthy of consideratior. 14. Roustan, C., Prudel, L. A., Kassab, R., Fattoum, A. & Thoai, N. This report adds yet another enzyme to several that have V. (1970) Biochim. Biophys. Acta 206, 369-379. Downloaded by guest on October 1, 2021