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
The rate of transfer of [32Plphosphate from [32P-labeled phosphoglucomutase (a-D-glucose-1,6-bisphos-
ABSTRACT
phate:a-D-glucose--phosphate phosphotransferase, EC 2.7.5.1) to glucose increases dramatically between pH 8.5 and 10.5 with a half maximal rate at pH 9.8. This suggests the participation of a residue containing an ionizable group with a pK close to
10. The inhibition of enzyme activity obtained with tyrosinederivatizing reactions-iodination, nitration, acetylation, and diazo coupling-is strongly indicative of tyrosine participation. Thiol reagents, p-hydroxymercuribenzoate and ethyleneimine, were without effect. Vanadate and arsenate augmented the transfer reaction 200- and 2.5-fold, respectively, and lowered the pH optimum of the reaction.
The physiological function of phosphoglucomutase (a-D-glucose-1,6-bisphosphate:a-D-glucose-l-phosphate phosphotransferase, EC 2.7.5.1) is to mediate the interconversion of glucose 1-phosphate and glucose 6-phosphate via the intermediate glucose 1,6-bisphosphate. Both phospho- and dephospho-enzyme forms participate in this interconversion. We have recently shown that 32P-labeled phosphoglucomutase ([32P]phosphoglucomutase) is capable of transferring the phosphate group to several nucleophiles (1) including glucose and its analogs (2). The rate of this transfer is several orders of magnitude slower than to glucose monophosphates. This made possible a detailed study of the initial rate of the reaction, the effect of variations in reaction conditions, and the structural requirements of the acceptor molecule. In fact, with this approach, we have made several observations that would have been unlikely otherwise and that are valid and relevant to the catalytic reaction. For example, we have previously been able to define quite accurately several stringent structural requirements of the substrate for reaction, including the essential orientations of the hydroxyl functions of the glucose molecule (2). We have now obtained additional interesting findings, again through the use of the phosphate transfer reaction. In this article, we present evidence for the participation of a tyrosine residue at the active site of phosphoglucomutase. This is based on the behavior of the reaction rate with increasing pH and on the sensitivity of the enzyme to tyrosine derivatizing reagents. No residue other than tyrosine could account fully for the results observed. In addition, vanadate and arsenate, which stimulate the transfer reaction markedly, appear to manifest their activating effect by lowering the pKa of the active site tyrosine. MATERIALS AND METHODS All chemicals were reagent grade and used without further purification. These were obtained as follows: D-glucose, a-D-glucose 1-phosphate, myo-inositol 2-phosphate, NThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
acetylimidazole, tetranitromethane, sodium nitrite, and phydroxymercuribenzoate (Sigma); glucose 6-phosphate (Boehringer); sodium orthovanadate, (ICN); sodium arsenate and sulfanilic acid, (Fisher); ethyleneimine (K&K); [y32P]ATP (25 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) (New England Nuclear); ACS scintillant (Amersham); bovine serum albumin (Miles). The enzyme was crystallized from rabbit skeletal muscle (3). 32P-Labeled enzyme was prepared as before by allowing exchange of glucose 6-[32P]phosphate and phosphorylated phosphoglucomutase according to the mechanism of the reaction (4, 5). The phosphate transfer reaction was carried out as described (2). The extent of reaction was measured by the production of trichloroacetic acid-soluble radioactive organic phosphate. Chemical Modification of Enzyme with Tyrosine Reagents. Iodination. The method of Azari and Feeney (6) was used. [32P]Phosphoglucomutase (0.02 ,gmol) was incubated in 0.1 M borate buffer at pH 9.5 with various dilutions of a stock iodine/iodide solution of 0.05 M 12 in 0.24 M KI at 0C. The iodine reagent was bleached within 15 s; however, the incubation was continued for 10 mm. Final volume was 200 y1. The extent of inactivation with this reagent, as with all the other derivatizing reagents discussed below, was determined by adding a 1000-fold excess of the substrate glucose 1-phosphate to the reaction. The amount of radioactive phosphate removed served as a measure of the remaining active enzyme. Acetylation. The reaction was carried out in a manner similar to that described by Simpson et al. (7). [32P]Phosphoglucomutase (0.02 .umol) was acetylated by incubation in 200 mM N-acetylimidazole at 0C for 30 min in 40 mM 1,4-piperazinediethanesulfonic acid (Pipes) buffer at pH 7.5 in a final volume of 200 ,p. The extent of inactivation was determined as in the iodination reaction. Deacetylation with subsequent regeneration of enzyme activity was performed in two ways. The inactivated acetylated enzyme was incubated with 1 M hydroxylamine (pH 7) at room temperature for 20 min. Alternatively, deacetylation was effected by incubation of the enzyme in 250 mM Tris-HCl (pH 9.0) at room temperature for 20 min. The extent of regeneration was then determined as before. Nitration. This reaction was carried out according to the method of Sokolovsky et al. (8). [32P]Phosphoglucomutase (0.02 of tetrani,gmol) was incubated with various concentrations tromethane at room temperature for 30 min in 0.05 M Tris-HCI (pH 8) in a final volume of 200 ,g. The desired dilutions were made from a stock solution of 0.84 M tetranitromethane in methanol. The extent of inactivation was then determined. Diazo Coupling. This reaction was carried out according to Riordan and Vallee (9). [32P]Phosphoglucomutase (0.02 ,mol) was incubated with 5 mM diazotized sulfanilic acid in 0.1 M sodium bicarbonate buffer (pH 8.8) at 0°C for 5 and 30 min in a final volume of 200 ,l. The extent of inactivation was again determined by the addition of glucose 1-phosphate. 5010
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Layne and Najjar
5011
Chemical Modification of the Enzyme with Thiol Reagents. 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 rates of phosphate transfer (2). Inositol 2-phosphate is an inhibitor of this reaction with a Ki of 0.58 mM. Double reciprocal plots of initial rates at varying glucose concentrations were made at inositol 2-phosphate concentrations of 0.1 and 0.5 mM. The plot appears to represent a clear case of competitive inhibition (Fig. 1). Effect of pH on Rate of Phosphate Transfer. Fig. 2 shows that a small, gradual increase in the initial reaction rate was obtained with increasing pH from pH 5 to pH 8.5. However, beyond pH 8.5 a rapid increase in rate began with a maximum at pH 10.5 and a half-maximal rate at pH 9.8. Beyond this, the transfer activity decreased. This segment of the pH curve suggests the presence of an ionizable group having a pKa of about 10.0. This would signal the involvement of any one of
6
7
8
pH
9
10
11
FIG. 2. Effect of pH on rate of phosphate transfer reaction. [32P]Phosphoglucomutase (0.02 Amol) in 5 mM Tris-HCl/2 mM MgCl2 at pH 7.5 was added to a previously prepared solution composed of 55 Al of water, 100 gl of 20 mM glucose, and 20 Al of 1 M buffer at the appropriate pH. Final volume was 200 ,ul. The reaction mixture was incubated at 37°C for various time periods. Samples were
measured after 10 s for vanadate and 60 s for arsenate and control. All values are adjusted to cpm transferred. Where appropriate, 2 JAI of 100 mM sodium arsenate or sodium vanadate was included. The buffers used were 4-morpholineethanesulfonic acid (Mes) for pH 6 and 7, Tris-HCl for pH 8 and 9, and sodium carbonate for pH 10 and 11. Because vanadate caused a considerable rate increase, it was necessary to decrease the final glucose concentration from 10 mM as in the control (glucose alone) to 1 mM for accurate measurement of 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 3500 3000
4O . M
25000
E E 2000 _/
0o 1500 .C
-° 1000-
0
0.01
0.02 1/Glucose, mM-1
0.03
FIG. 1. Double reciprocal plot of reaction of glucose with phosphoglucomutase in presence of inositol 2-phosphate. Glucose (100 gl 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 l:12Pjphosphoglucomutase previously dialyzed against 5 mM Tris-HCl, pH 7.5. Final volume was 200 ,l. Where appropriate, 5 ,ul of 20 mM or 4 mM inositol 2-phosphate was added to yield a final concentration of 0.5 and 0.1 mM, respectively. Fifty microliters of 6% bovine serum albumin was added followed immediately by 100 ul of 25% trichloroacetic acid. After centrifugation 200 yl of supernatant was assayed for radioactivity. Controls containing only buffer yield negligible ~roduction of inorganic phosphate (12% per hr). 0, Glucose alone; 0, glucose with 0.1 mm inositol 2-phosphate; A, glucose with 0.5 mM inositol 2-phosphate.
three functional groups: the hydroxyl of tyrosine, the E-amino of lysine, or the thiol of cysteine. It must be noted here that under the conditions of our experiments spontaneous dephosphorylation at elevated pH values in the absence of glucose was negligible. The figure also shows that with 1 mM arsenate or 1 mM vanadate a shift occurred in the activity curve toward more acidic pH values. The effect of vanadate was much more dramatic than that of arsenate. Vanadate caused a shift of the half-maximal rate from pH 9.8 to 7.7, whereas the shift with arsenate was only slight with a half-maximal rate obtained at pH 9.5. In this connection, it is of interest that both anions are also activators 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. Other anions such as tungstate, molybdate, and columbate were ineffective. Inactivation of Phosphoglucomutase by lodination. Table 1 shows that inactivation of this enzyme under the conditions detailed above was very rapid, approximately 15 s, over a range of concentrations (0.25-1 mM 12 and, correspondingly, 1.2-4.8 mM KI). Enzyme concentration was kept at 0.1 mM throughout. Complete inactivation of the enzyme was obtained at approximately 1 mM 12 in KI. The addition of 10 mM inositol 2-phosphate to the enzyme before the iodination reagent considerably diminished the inactivating effect. The results shown in Table 1 represent the percentage inhibition obtained with iodination, as well as with the diazo coupling reaction, nitration, and acetylation. Included
Biochemistry: Layne and Najjar
5012 E 16-
Proc. Natl. Acad. Sci. USA 76 (1979)
40/
E 14-
7612
-30-
E C
0 20-
8
4-
0 ~~
0
20
~
~
~
40
60
80
Time, min
~
~
100 120
4
1
Time, min
FIG. 3. Relative stimulatory activity of arsenate and vanadate.
(Left) One hundred microliters of 2 mM glucose in 80 mM imidazole/2 mM MgCl2 at pH 7.5 was added to 100 ,l containing 0.02 ,gmol of
[32P]phosphoglucomutase in the presence and absence of 1 mM
so-
dium arsenate. The reaction mixture was incubated at 370C for 15, 30, 60, 90, and 120 min. (Right) Same as Left except that 1 mM sodium 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. 0, Glucose alone; A, glucose with arsenate; 0, glucose with vanadate.
also is the protective effect of inositol 2-phosphate. It was not feasible to attempt protection of the enzyme with either of the hexose monophosphate substrates, glucose 1-phosphate or glucose 6-phosphate, because under these conditions there would be an instantaneous transfer of the enzyme phosphate to the substrate at a far more rapid rate than the rate of iodination at 0WC. Acetylation of Phosphoglucomutase with N-Acetylimidazole. Acetylation of 0.02 ,umol of enzyme in 200 mM NTable 1. Inactivation of phosphoglucomutase by derivatizing agents and protective effect of substrate analog inositol 2-phosphate % inactivation Without With inositol inositol 2-phosphate 2-phosphate Derivatizing reaction Diazo coupling (00C, 30 min) 7 0 Diazosulfanilate (3 mM) 16 0 Diazosulfanilate (6 mM) 34 0 Diazosulfanilate (12 mM) Iodination (00C, 10 min) 41 0 Iodine (0.5 mM) 100 15 Iodine (1.0 mM) 100 55 Iodine (2.0 mM) Nitration (250C, 30 min) 41 Tetranitromethane (0.75 mM) 57 Tetranitromethane (1.50 mM) 82 15 Tetranitromethane (3.00 mM) Acetylation (00C, 30 min) 35 N-acetylimidazole (100 mM) 55 23 N-acetylimidazole (200 mM) Derivatizing reagent (100 ul) was added to 0.02 Amol of [32PJphosphoglucomutase in the presence or absence of inositol 2-phosphate in a final volume of 200 pl. The concentrations of inositol 2phosphate used were as follows: 6 mM for the diazo coupling reaction, 10 mM for iodination, 3 mM for nitration, and 5 mM for acetylation. Each concentration of iodine listed in the table contains 4.8 molar excess of KI. A 1000-fold excess of glucose 1-phosphate was added at the end of the incubation period. The reaction was stopped with trichloroacetic acid and assayed as described in Fig. 1.
acetylimidazole was carried out at 0WC. After 30 min only 45% of the enzyme activity remained (Table 1). Inclusion of 5mM inositol 2-phosphate in the acetylation reaction medium afforded good protection of the enzyme to the extent of 58% of the lost activity. Incubation of the derivatized enzyme in 1 M hydroxylamine (pH 7) or 250mM Tris-HCl (pH 9) at 250C for 20 min was found to regenerate 90 or 52% of the inhibited activity, respectively. Nitration of Phosphoglucomutase by Tetranitromethane. Incubation of the enzyme for 30 min at 250C in 0.75-6 mM tetranitromethane, under the conditions described above, resulted 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 out of a total of 20 (5) were nitrated. Inositol 2-phosphate (3mM) afforded considerable protection and reduced inactivation caused by 3 mM tetranitromethane from 83% of the original activity to 15% (Table 1). The derivatizing reagents so far utilized, although highly reactive with the tyrosine residues, nevertheless react to some degree also with cysteine. Consequently, the enzyme was tested for sensitivity to two cysteine reagents-namely, p-hydroxymercuribenzoate and ethyleneimine. Neither reagent caused any loss of activity. This excludes cysteine as a participant in phosphoglucomutase catalysis, as was shown earlier (10). Coupling of Sulfanilic Acid Diazonium Salt to Phosphoglucomutase. Reaction between 0.1 mM enzyme and 3, 6, and 12 mM diazonium salt of sulfanilic acid in 0.1 M bicarbonate buffer at pH 8.8 and 0°C yielded a maximal inactivation of 40% (Table 1). However, no inhibition was observed when 6 mM inositol 2-phosphate was added before the diazonium salt.
DISCUSSION The kinetics of the enzyme reaction with increasing pH and the derivatization reactions that lead to inactivation establish the importance of the tyrosine residue in the catalytic activity of the enzyme. The kinetics of inhibition of glucose phosphorylation by inositol 2-phosphate, as shown in Fig. 1, exhibit competitive characteristics. This indicates that inositol 2-phosphate binds at the active site of the enzyme. It also explains the protection afforded the enzyme against inactivation caused by the derivatization of a residue at the active site. The data obtained by the chemical modification of the enzyme and the profile of glucose phosphorylation at elevated pH values indicate the presence of a tyrosine residue at the active site. The compelling evidence for this conclusion includes the inactivation by nitration, iodination, coupling with diazonium salt, and acetylation. All the modifying reagents are known to possess both a high reactivity and a good degree of specificity for the tyrosine residue. Other amino acid residues that might possess some reactivity with these reagents include cysteine, lysine, and histidine. However, the activity of the enzyme is not inhibited by the cysteine derivatizing reagents p-hydroxymercuribenzoate and ethyleneimine. Lysine, on the other hand, is readily eliminated from consideration because tetranitromethane and I2 plus I- do not react with this residue under the conditions used. Furthermore, the facile reactivation of the acetylated enzyme with hydroxylamine or at high pH renders the lysine residue an unlikely candidate because the N-acetyllysine amide bond would not be cleaved under these conditions. By contrast, the O-acetyl ester bond, such as would occur at tyrosine, is easily cleaved by high concentrations of hydroxylamine and hydroxyl ion. The possibility that histidine might be involved should be considered because it is derivatized in the iodination and diazo
Biochemistry: Layne and Najjar coupling reactions. However, other reasons negate its involvement. The pH profile of the transfer reaction indicates a maximum rate at pH 11. This is far removed from the pK of histidine. The acetylation of histidine in proteins by N-acetylimidazole is presumably unlikely because it has not been considered by others (12, 13). The most important fact that singles out tyrosine as the derivatized residue is the nitration reaction. Tetranitromethane has been shown not to nitrate histidine or N-acetylhistidine (8). Fig. 2 depicts the relationship between activity and pH. As noted above, the pattern is reminiscent of a titration curve of a residue with a pKa of about 10. This value corresponds very well to the pKa of 10.07 for the hydroxyl group of free tyrosine.
In the presence of vanadate and arsenate, the phosphate transfer reaction is strongly stimulated. More importantly, the pH optimum of this reaction is shifted to lower values by these anions. Vanadate shifts the pH at which a half-maximal reaction rate is obtained from pH 10 to pH 7.7. On the other hand, the corresponding shift in pH caused by arsenate is only from pH 10 to pH 9.5. The obvious conclusion is that vanadate and arsenate bind to the enzyme in such a way as to promote the transfer reaction. This might occur by assisting in the ionization of the hydroxyl function of the tyrosine at the active site. The manner by which such an ionized residue functions is uncertain. It might abstract a proton, neutralize a positive charge, or participate or assist in a nucleophilic attack on a susceptible bond. Whatever the mechanism, it is certain that the tyrosine residue plays an essential role. The possibility of the involvement of O-phosphoryltyrosine is worthy of consideratior. This report adds yet another enzyme to several that have
Proc. Natl. Acad. Sci. USA 76 (1979)
5013
already been shown to have a tyrosine at the active sitenamely, carboxypeptidase A (7) and arginine kinase (14). This work was supported by Public Health Service Grant 5R01 AL09116, National Science Foundation Grant PCM76-23008, and the National Foundation March of Dimes Grant 1-556. 1. Layne, P. P. & Najjar, V. A. (1975) J. Biol. Chem. 250, 966972. 2. Layne, P. P. & Najjar, V. A. (1978) Biochim. Biophys. Acta 526, 429-439. 3. Najjar, V. A. (1955) Methods Enzymol. 1, 294-299. 4. Najjar, V. A. & Pullman, M. (1954) Science 119, 631-634. 5. Najjar, V. A. (1962) in The Enzymes, ed. Boyer, P. D. (Academic, New York), Vol. 6, pp. 161-178. 6. Azari, P. R. & Feeney, R. E. (1961) Arch. Biochem. Biophys. 92, 44-52. 7. Simpson, R. T., Riordan, J. F. & Vallee, B. L. (1963) Biochemistry 2,616-622. 8. Sokolovsky, M., Riordan, J. F. & Vallee, B. L. (1966) Biochemistry 5,3582-3589. 9. Riordan, J. F. & Vallee, B. L. (1972) Methods Enzymol. 25, 521-531. 10. Bocchini, V., Alioto, M. R. & Najjar, V. A. (1967) Biochemistry 6,313-322. 11. Layne, P. P. & Najjar, V. A. (1979) Fed. Proc. Fed. Am. Soc. Exp. Biol. 38, in press. 12. Riordan, J. F., Wacher, W. E. C. & Vallee, B. L. (1965) Biochemistry 4, 1758-1765. 13. Means, G. E. & Feeney, R. E. (1971) Chemical Modification of Proteins (Holden-Day, San Francisco), pp. 72-74. 14. Roustan, C., Prudel, L. A., Kassab, R., Fattoum, A. & Thoai, N. V. (1970) Biochim. Biophys. Acta 206, 369-379.
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
The rate of transfer of [32Plphosphate from [32P-labeled phosphoglucomutase (a-D-glucose-1,6-bisphos-
ABSTRACT
phate:a-D-glucose--phosphate phosphotransferase, EC 2.7.5.1) to glucose increases dramatically between pH 8.5 and 10.5 with a half maximal rate at pH 9.8. This suggests the participation of a residue containing an ionizable group with a pK close to
10. The inhibition of enzyme activity obtained with tyrosinederivatizing reactions-iodination, nitration, acetylation, and diazo coupling-is strongly indicative of tyrosine participation. Thiol reagents, p-hydroxymercuribenzoate and ethyleneimine, were without effect. Vanadate and arsenate augmented the transfer reaction 200- and 2.5-fold, respectively, and lowered the pH optimum of the reaction.
The physiological function of phosphoglucomutase (a-D-glucose-1,6-bisphosphate:a-D-glucose-l-phosphate phosphotransferase, EC 2.7.5.1) is to mediate the interconversion of glucose 1-phosphate and glucose 6-phosphate via the intermediate glucose 1,6-bisphosphate. Both phospho- and dephospho-enzyme forms participate in this interconversion. We have recently shown that 32P-labeled phosphoglucomutase ([32P]phosphoglucomutase) is capable of transferring the phosphate group to several nucleophiles (1) including glucose and its analogs (2). The rate of this transfer is several orders of magnitude slower than to glucose monophosphates. This made possible a detailed study of the initial rate of the reaction, the effect of variations in reaction conditions, and the structural requirements of the acceptor molecule. In fact, with this approach, we have made several observations that would have been unlikely otherwise and that are valid and relevant to the catalytic reaction. For example, we have previously been able to define quite accurately several stringent structural requirements of the substrate for reaction, including the essential orientations of the hydroxyl functions of the glucose molecule (2). We have now obtained additional interesting findings, again through the use of the phosphate transfer reaction. In this article, we present evidence for the participation of a tyrosine residue at the active site of phosphoglucomutase. This is based on the behavior of the reaction rate with increasing pH and on the sensitivity of the enzyme to tyrosine derivatizing reagents. No residue other than tyrosine could account fully for the results observed. In addition, vanadate and arsenate, which stimulate the transfer reaction markedly, appear to manifest their activating effect by lowering the pKa of the active site tyrosine. MATERIALS AND METHODS All chemicals were reagent grade and used without further purification. These were obtained as follows: D-glucose, a-D-glucose 1-phosphate, myo-inositol 2-phosphate, NThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
acetylimidazole, tetranitromethane, sodium nitrite, and phydroxymercuribenzoate (Sigma); glucose 6-phosphate (Boehringer); sodium orthovanadate, (ICN); sodium arsenate and sulfanilic acid, (Fisher); ethyleneimine (K&K); [y32P]ATP (25 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) (New England Nuclear); ACS scintillant (Amersham); bovine serum albumin (Miles). The enzyme was crystallized from rabbit skeletal muscle (3). 32P-Labeled enzyme was prepared as before by allowing exchange of glucose 6-[32P]phosphate and phosphorylated phosphoglucomutase according to the mechanism of the reaction (4, 5). The phosphate transfer reaction was carried out as described (2). The extent of reaction was measured by the production of trichloroacetic acid-soluble radioactive organic phosphate. Chemical Modification of Enzyme with Tyrosine Reagents. Iodination. The method of Azari and Feeney (6) was used. [32P]Phosphoglucomutase (0.02 ,gmol) was incubated in 0.1 M borate buffer at pH 9.5 with various dilutions of a stock iodine/iodide solution of 0.05 M 12 in 0.24 M KI at 0C. The iodine reagent was bleached within 15 s; however, the incubation was continued for 10 mm. Final volume was 200 y1. The extent of inactivation with this reagent, as with all the other derivatizing reagents discussed below, was determined by adding a 1000-fold excess of the substrate glucose 1-phosphate to the reaction. The amount of radioactive phosphate removed served as a measure of the remaining active enzyme. Acetylation. The reaction was carried out in a manner similar to that described by Simpson et al. (7). [32P]Phosphoglucomutase (0.02 .umol) was acetylated by incubation in 200 mM N-acetylimidazole at 0C for 30 min in 40 mM 1,4-piperazinediethanesulfonic acid (Pipes) buffer at pH 7.5 in a final volume of 200 ,p. The extent of inactivation was determined as in the iodination reaction. Deacetylation with subsequent regeneration of enzyme activity was performed in two ways. The inactivated acetylated enzyme was incubated with 1 M hydroxylamine (pH 7) at room temperature for 20 min. Alternatively, deacetylation was effected by incubation of the enzyme in 250 mM Tris-HCl (pH 9.0) at room temperature for 20 min. The extent of regeneration was then determined as before. Nitration. This reaction was carried out according to the method of Sokolovsky et al. (8). [32P]Phosphoglucomutase (0.02 of tetrani,gmol) was incubated with various concentrations tromethane at room temperature for 30 min in 0.05 M Tris-HCI (pH 8) in a final volume of 200 ,g. The desired dilutions were made from a stock solution of 0.84 M tetranitromethane in methanol. The extent of inactivation was then determined. Diazo Coupling. This reaction was carried out according to Riordan and Vallee (9). [32P]Phosphoglucomutase (0.02 ,mol) was incubated with 5 mM diazotized sulfanilic acid in 0.1 M sodium bicarbonate buffer (pH 8.8) at 0°C for 5 and 30 min in a final volume of 200 ,l. The extent of inactivation was again determined by the addition of glucose 1-phosphate. 5010
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Layne and Najjar
5011
Chemical Modification of the Enzyme with Thiol Reagents. 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 rates of phosphate transfer (2). Inositol 2-phosphate is an inhibitor of this reaction with a Ki of 0.58 mM. Double reciprocal plots of initial rates at varying glucose concentrations were made at inositol 2-phosphate concentrations of 0.1 and 0.5 mM. The plot appears to represent a clear case of competitive inhibition (Fig. 1). Effect of pH on Rate of Phosphate Transfer. Fig. 2 shows that a small, gradual increase in the initial reaction rate was obtained with increasing pH from pH 5 to pH 8.5. However, beyond pH 8.5 a rapid increase in rate began with a maximum at pH 10.5 and a half-maximal rate at pH 9.8. Beyond this, the transfer activity decreased. This segment of the pH curve suggests the presence of an ionizable group having a pKa of about 10.0. This would signal the involvement of any one of
6
7
8
pH
9
10
11
FIG. 2. Effect of pH on rate of phosphate transfer reaction. [32P]Phosphoglucomutase (0.02 Amol) in 5 mM Tris-HCl/2 mM MgCl2 at pH 7.5 was added to a previously prepared solution composed of 55 Al of water, 100 gl of 20 mM glucose, and 20 Al of 1 M buffer at the appropriate pH. Final volume was 200 ,ul. The reaction mixture was incubated at 37°C for various time periods. Samples were
measured after 10 s for vanadate and 60 s for arsenate and control. All values are adjusted to cpm transferred. Where appropriate, 2 JAI of 100 mM sodium arsenate or sodium vanadate was included. The buffers used were 4-morpholineethanesulfonic acid (Mes) for pH 6 and 7, Tris-HCl for pH 8 and 9, and sodium carbonate for pH 10 and 11. Because vanadate caused a considerable rate increase, it was necessary to decrease the final glucose concentration from 10 mM as in the control (glucose alone) to 1 mM for accurate measurement of 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 3500 3000
4O . M
25000
E E 2000 _/
0o 1500 .C
-° 1000-
0
0.01
0.02 1/Glucose, mM-1
0.03
FIG. 1. Double reciprocal plot of reaction of glucose with phosphoglucomutase in presence of inositol 2-phosphate. Glucose (100 gl 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 l:12Pjphosphoglucomutase previously dialyzed against 5 mM Tris-HCl, pH 7.5. Final volume was 200 ,l. Where appropriate, 5 ,ul of 20 mM or 4 mM inositol 2-phosphate was added to yield a final concentration of 0.5 and 0.1 mM, respectively. Fifty microliters of 6% bovine serum albumin was added followed immediately by 100 ul of 25% trichloroacetic acid. After centrifugation 200 yl of supernatant was assayed for radioactivity. Controls containing only buffer yield negligible ~roduction of inorganic phosphate (12% per hr). 0, Glucose alone; 0, glucose with 0.1 mm inositol 2-phosphate; A, glucose with 0.5 mM inositol 2-phosphate.
three functional groups: the hydroxyl of tyrosine, the E-amino of lysine, or the thiol of cysteine. It must be noted here that under the conditions of our experiments spontaneous dephosphorylation at elevated pH values in the absence of glucose was negligible. The figure also shows that with 1 mM arsenate or 1 mM vanadate a shift occurred in the activity curve toward more acidic pH values. The effect of vanadate was much more dramatic than that of arsenate. Vanadate caused a shift of the half-maximal rate from pH 9.8 to 7.7, whereas the shift with arsenate was only slight with a half-maximal rate obtained at pH 9.5. In this connection, it is of interest that both anions are also activators 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. Other anions such as tungstate, molybdate, and columbate were ineffective. Inactivation of Phosphoglucomutase by lodination. Table 1 shows that inactivation of this enzyme under the conditions detailed above was very rapid, approximately 15 s, over a range of concentrations (0.25-1 mM 12 and, correspondingly, 1.2-4.8 mM KI). Enzyme concentration was kept at 0.1 mM throughout. Complete inactivation of the enzyme was obtained at approximately 1 mM 12 in KI. The addition of 10 mM inositol 2-phosphate to the enzyme before the iodination reagent considerably diminished the inactivating effect. The results shown in Table 1 represent the percentage inhibition obtained with iodination, as well as with the diazo coupling reaction, nitration, and acetylation. Included
Biochemistry: Layne and Najjar
5012 E 16-
Proc. Natl. Acad. Sci. USA 76 (1979)
40/
E 14-
7612
-30-
E C
0 20-
8
4-
0 ~~
0
20
~
~
~
40
60
80
Time, min
~
~
100 120
4
1
Time, min
FIG. 3. Relative stimulatory activity of arsenate and vanadate.
(Left) One hundred microliters of 2 mM glucose in 80 mM imidazole/2 mM MgCl2 at pH 7.5 was added to 100 ,l containing 0.02 ,gmol of
[32P]phosphoglucomutase in the presence and absence of 1 mM
so-
dium arsenate. The reaction mixture was incubated at 370C for 15, 30, 60, 90, and 120 min. (Right) Same as Left except that 1 mM sodium 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. 0, Glucose alone; A, glucose with arsenate; 0, glucose with vanadate.
also is the protective effect of inositol 2-phosphate. It was not feasible to attempt protection of the enzyme with either of the hexose monophosphate substrates, glucose 1-phosphate or glucose 6-phosphate, because under these conditions there would be an instantaneous transfer of the enzyme phosphate to the substrate at a far more rapid rate than the rate of iodination at 0WC. Acetylation of Phosphoglucomutase with N-Acetylimidazole. Acetylation of 0.02 ,umol of enzyme in 200 mM NTable 1. Inactivation of phosphoglucomutase by derivatizing agents and protective effect of substrate analog inositol 2-phosphate % inactivation Without With inositol inositol 2-phosphate 2-phosphate Derivatizing reaction Diazo coupling (00C, 30 min) 7 0 Diazosulfanilate (3 mM) 16 0 Diazosulfanilate (6 mM) 34 0 Diazosulfanilate (12 mM) Iodination (00C, 10 min) 41 0 Iodine (0.5 mM) 100 15 Iodine (1.0 mM) 100 55 Iodine (2.0 mM) Nitration (250C, 30 min) 41 Tetranitromethane (0.75 mM) 57 Tetranitromethane (1.50 mM) 82 15 Tetranitromethane (3.00 mM) Acetylation (00C, 30 min) 35 N-acetylimidazole (100 mM) 55 23 N-acetylimidazole (200 mM) Derivatizing reagent (100 ul) was added to 0.02 Amol of [32PJphosphoglucomutase in the presence or absence of inositol 2-phosphate in a final volume of 200 pl. The concentrations of inositol 2phosphate used were as follows: 6 mM for the diazo coupling reaction, 10 mM for iodination, 3 mM for nitration, and 5 mM for acetylation. Each concentration of iodine listed in the table contains 4.8 molar excess of KI. A 1000-fold excess of glucose 1-phosphate was added at the end of the incubation period. The reaction was stopped with trichloroacetic acid and assayed as described in Fig. 1.
acetylimidazole was carried out at 0WC. After 30 min only 45% of the enzyme activity remained (Table 1). Inclusion of 5mM inositol 2-phosphate in the acetylation reaction medium afforded good protection of the enzyme to the extent of 58% of the lost activity. Incubation of the derivatized enzyme in 1 M hydroxylamine (pH 7) or 250mM Tris-HCl (pH 9) at 250C for 20 min was found to regenerate 90 or 52% of the inhibited activity, respectively. Nitration of Phosphoglucomutase by Tetranitromethane. Incubation of the enzyme for 30 min at 250C in 0.75-6 mM tetranitromethane, under the conditions described above, resulted 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 out of a total of 20 (5) were nitrated. Inositol 2-phosphate (3mM) afforded considerable protection and reduced inactivation caused by 3 mM tetranitromethane from 83% of the original activity to 15% (Table 1). The derivatizing reagents so far utilized, although highly reactive with the tyrosine residues, nevertheless react to some degree also with cysteine. Consequently, the enzyme was tested for sensitivity to two cysteine reagents-namely, p-hydroxymercuribenzoate and ethyleneimine. Neither reagent caused any loss of activity. This excludes cysteine as a participant in phosphoglucomutase catalysis, as was shown earlier (10). Coupling of Sulfanilic Acid Diazonium Salt to Phosphoglucomutase. Reaction between 0.1 mM enzyme and 3, 6, and 12 mM diazonium salt of sulfanilic acid in 0.1 M bicarbonate buffer at pH 8.8 and 0°C yielded a maximal inactivation of 40% (Table 1). However, no inhibition was observed when 6 mM inositol 2-phosphate was added before the diazonium salt.
DISCUSSION The kinetics of the enzyme reaction with increasing pH and the derivatization reactions that lead to inactivation establish the importance of the tyrosine residue in the catalytic activity of the enzyme. The kinetics of inhibition of glucose phosphorylation by inositol 2-phosphate, as shown in Fig. 1, exhibit competitive characteristics. This indicates that inositol 2-phosphate binds at the active site of the enzyme. It also explains the protection afforded the enzyme against inactivation caused by the derivatization of a residue at the active site. The data obtained by the chemical modification of the enzyme and the profile of glucose phosphorylation at elevated pH values indicate the presence of a tyrosine residue at the active site. The compelling evidence for this conclusion includes the inactivation by nitration, iodination, coupling with diazonium salt, and acetylation. All the modifying reagents are known to possess both a high reactivity and a good degree of specificity for the tyrosine residue. Other amino acid residues that might possess some reactivity with these reagents include cysteine, lysine, and histidine. However, the activity of the enzyme is not inhibited by the cysteine derivatizing reagents p-hydroxymercuribenzoate and ethyleneimine. Lysine, on the other hand, is readily eliminated from consideration because tetranitromethane and I2 plus I- do not react with this residue under the conditions used. Furthermore, the facile reactivation of the acetylated enzyme with hydroxylamine or at high pH renders the lysine residue an unlikely candidate because the N-acetyllysine amide bond would not be cleaved under these conditions. By contrast, the O-acetyl ester bond, such as would occur at tyrosine, is easily cleaved by high concentrations of hydroxylamine and hydroxyl ion. The possibility that histidine might be involved should be considered because it is derivatized in the iodination and diazo
Biochemistry: Layne and Najjar coupling reactions. However, other reasons negate its involvement. The pH profile of the transfer reaction indicates a maximum rate at pH 11. This is far removed from the pK of histidine. The acetylation of histidine in proteins by N-acetylimidazole is presumably unlikely because it has not been considered by others (12, 13). The most important fact that singles out tyrosine as the derivatized residue is the nitration reaction. Tetranitromethane has been shown not to nitrate histidine or N-acetylhistidine (8). Fig. 2 depicts the relationship between activity and pH. As noted above, the pattern is reminiscent of a titration curve of a residue with a pKa of about 10. This value corresponds very well to the pKa of 10.07 for the hydroxyl group of free tyrosine.
In the presence of vanadate and arsenate, the phosphate transfer reaction is strongly stimulated. More importantly, the pH optimum of this reaction is shifted to lower values by these anions. Vanadate shifts the pH at which a half-maximal reaction rate is obtained from pH 10 to pH 7.7. On the other hand, the corresponding shift in pH caused by arsenate is only from pH 10 to pH 9.5. The obvious conclusion is that vanadate and arsenate bind to the enzyme in such a way as to promote the transfer reaction. This might occur by assisting in the ionization of the hydroxyl function of the tyrosine at the active site. The manner by which such an ionized residue functions is uncertain. It might abstract a proton, neutralize a positive charge, or participate or assist in a nucleophilic attack on a susceptible bond. Whatever the mechanism, it is certain that the tyrosine residue plays an essential role. The possibility of the involvement of O-phosphoryltyrosine is worthy of consideratior. This report adds yet another enzyme to several that have
Proc. Natl. Acad. Sci. USA 76 (1979)
5013
already been shown to have a tyrosine at the active sitenamely, carboxypeptidase A (7) and arginine kinase (14). This work was supported by Public Health Service Grant 5R01 AL09116, National Science Foundation Grant PCM76-23008, and the National Foundation March of Dimes Grant 1-556. 1. Layne, P. P. & Najjar, V. A. (1975) J. Biol. Chem. 250, 966972. 2. Layne, P. P. & Najjar, V. A. (1978) Biochim. Biophys. Acta 526, 429-439. 3. Najjar, V. A. (1955) Methods Enzymol. 1, 294-299. 4. Najjar, V. A. & Pullman, M. (1954) Science 119, 631-634. 5. Najjar, V. A. (1962) in The Enzymes, ed. Boyer, P. D. (Academic, New York), Vol. 6, pp. 161-178. 6. Azari, P. R. & Feeney, R. E. (1961) Arch. Biochem. Biophys. 92, 44-52. 7. Simpson, R. T., Riordan, J. F. & Vallee, B. L. (1963) Biochemistry 2,616-622. 8. Sokolovsky, M., Riordan, J. F. & Vallee, B. L. (1966) Biochemistry 5,3582-3589. 9. Riordan, J. F. & Vallee, B. L. (1972) Methods Enzymol. 25, 521-531. 10. Bocchini, V., Alioto, M. R. & Najjar, V. A. (1967) Biochemistry 6,313-322. 11. Layne, P. P. & Najjar, V. A. (1979) Fed. Proc. Fed. Am. Soc. Exp. Biol. 38, in press. 12. Riordan, J. F., Wacher, W. E. C. & Vallee, B. L. (1965) Biochemistry 4, 1758-1765. 13. Means, G. E. & Feeney, R. E. (1971) Chemical Modification of Proteins (Holden-Day, San Francisco), pp. 72-74. 14. Roustan, C., Prudel, L. A., Kassab, R., Fattoum, A. & Thoai, N. V. (1970) Biochim. Biophys. Acta 206, 369-379.
