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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 tetrani- tromethane 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 pre- viously 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 as- sayed as in Fig. 1. A, Glucose alone; 0, glucose with arsenate; 0, glu- cose with vanadate. 4000 three functional groups: the hydroxyl of tyrosine, the E-amino 3500 of lysine, or the thiol of cysteine.

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