A Kinetic Study of the Effect of α-d-Galactose, α-d-Mannose, α-d-Glucosamine, N-Acetyl-α-d-glucosamine, and α-d-Ribose Diphosphate on the Activity of Phosphoglucomutase

Abstract The specificity of phosphoglucomutase for various α-d-sugar diphosphates was studied by examining the relative ability of these compounds to activate the dephosphorylated form of the enzyme prepared from rabbit muscle. The rate of the reaction measured spectrophotometrically at 340 mµ with a coupled assay system containing TPN+ and excess d-glucose 6-phosphate dehydrogenase was linear with time only when the incubation was carried out in the presence of α-d-glucose 1,6-diphosphate. When other sugar diphosphates were used, the rate of disappearance of α-d-glucose 1-phosphate increased with time until a maximum linear rate was attained. The final rate, in each case, was dependent on the concentration of α-d-sugar diphosphate added to the incubation mixture, but it was independent of the order of addition of reactants and the concentration of components of the coupled assay system. The apparent Km values were as follows: α-d-glucose 1,6-diphosphate, 0.1 µm; α-d-mannose 1,6-diphosphate, 3.7 µm; α-d-ribose 1,5-diphosphate, 3.2 µm; α-d-glucosamine 1,6-diphosphate, 11 µm; N-acetyl-α-d-glucosamine 1,6-diphosphate, 1.6 µm; α-d-galactose 1,6-diphosphate, 13 µm. The most effective activator of phosphoglucomutase was α-d-glucose 1,6-diphosphate. The Km values of α-d-galactose 1,6-diphosphate and α-d-glucosamine 1,6-diphosphate, the poorest activators, were about 120-fold greater than that of α-d-glucose 1,6-diphosphate. The maximum velocity varied only 2- to 3-fold when different sugar diphosphates were used to activate the reaction. The following maximum velocities, expressed as micromoles min-1 mg-1, were obtained: α-d-glucose 1,6-diphosphate, 500; α-d-mannose, 1,6-diphosphate, 360; α-d-ribose 1,5-diphosphate, 210; α-d-glucosamine 1,6-diphosphate, 370; N-acetyl-d-glucosamine 1,6-diphosphate, 220; α-d-galactose 1,6-diphosphate, 240. Addition of the appropriate α-d-sugar 1-phosphate or d-sugar 6-phosphate decreased the velocity of the reaction being maintained by either α-d-sugar 1,6-diphosphate or α-d-glucose 1,6-diphosphate. The findings reported are consistent with and support a mechanism in which each of the sugar diphosphates combines with dephosphoenzyme to form a sugar diphosphate-enzyme complex which then further reacts to yield a phosphoenzyme intermediate and free sugar monophosphate.

The specificity of phosphoglucomutase for various CPDsugar diphosphates was studied by examining the relative ability of these compounds to activate the dephosphorylated form of the enzyme prepared from rabbit muscle.
The rate of the reaction measured spectrophotometrically at 340 rnp with a coupled assay system containing TPN+ and excess D-glUcOSe 6-phosphate dehydrogenase was linear with time only when the incubation was carried out in the presence of a-o-glucose 1,6-diphosphate. When other sugar diphosphates were used, the rate of disappearance of ar-D-glucose l-phosphate increased with time until a maximum linear rate was attained.
The final rate, in each case, was dependent on the concentration of a-n-sugar diphosphate added to the incubation mixture, but it was independent of the order of addition of reactants and the concentration of components of the coupled assay system.
The inactive dephosphoenzyme was regenerated by the transfer of a phosphate group from either position of ar-n-glucose 1,6-diphosphate with the concomitant formation of n-glucose g-phosphate or cr-n-glucose l-phosphate (3,4). It is not yet certain whether the form of phosphoenzyme which reacts with ol-n-glucose l-phosphate is the same as that which reacts with n-glucose 6-phosphate, and it is possible that two distinct forms of phosphoenzyme exist.
Phosphorylated and dephosphorylated forms of the enzyme have been isolated by Yankeelov,Horton,and Koshland (5). These workers labeled phosphoglucomutase with a mixture of oL-n-glucose-l-32P and n-glucose-6-32P, and they were able to separate two radioactive peaks containing enzyme activity by chromatography on modified cellulose columns. The active fractions contained 1 mole of phosphate per mole of enzyme. Recently, Harshman, Bocchini, and Najjar (6) isolated a phosphopeptide from phosphoglucomutase which contains 2 serine residues, both of which can be labeled with or-n-glucose-i-32P and n-glucose-6-32P.
Joshi et al. (7) and Joshi and Handler (8) have also isolated two forms of phosphoglucomutase from rabbit, shark, flounder, and human muscle. The different forms in some species appear to be isozymes, since they are similar in molecular weight and catalytic properties, but they differ in charge and amino acid sequence. The amino acid sequence of the peptide containing the active serine was found to be identical in the case of human, rabbit, and flounder phosphoglucomutase (8,9). Ray and Roscelli concluded from their kinetic studies on phosphoglucomutase that the most probable mechanism was one in Issue of August 25, 1970 H. Mulhausen and J. Mendicino 4039 which there is only one form of phosphoenzyme that can react with either of the n-glucose monophosphate esters (4, 10). The results of their studies with s2P-labeled substrates indicated that free ar-n-glucose 1,6-diphosphate was not an obligatory intermediate in the interconversion of sugar monophosphates. They estimated that the rate of turnover of free cr-n-glucose 1,6-diphosphate was only one-twentieth of the rate of interconversion of the n-glucose monophosphates.
More recent studies by Lowry and Passonneau indicate that this ratio may be as high as one-sixtieth (11). Phosphoglucomutase will catalyze the conversion of sugar l-phosphates to sugar diphosphates in the presence of a-n-glucose 1,6&phosphate. Thus, ol-D-mannose l-phosphate was converted to n-mannose 1,6-diphosphate (12), ar-n-ribose l-phosphate and or-D-galactose l-phosphate were converted to the corresponding sugar diphosphates (13), and Dfructose l-phosphate was converted to n-fructose 1, B-diphosphate by this enzyme (11). Various sugar diphosphates can also be used to activate phosphoglucomutase; however, the specificity of the enzyme for different sugar diphosphates has not yet been examined.

EXPERIMENTAL PROCEDURE
Assay of Phosphotransjerase Activity-The spectrophotometric assay for phosphoglucomutase activity was based on the rate of conversion of oc-D-glucose-l-P to D-glucose&P in the presence of excess /?-D-glucose-6-P dehydrogenase and TPN+ (14). The standard reaction mixture was incubated at 25" and contained in a final volume of 1 ml: 25 mM imidazole buffer (pH 7.0), 0.5 mM TPN+, 5 mM MgCl2, 1 mM EDTA, 0.005% bovine serum albumin, 6.4 Mg of /?-n-glucose-6-P dehydrogenase, 8.9 x lo-* PM phosphoglucomutase, 250 pM cu-n-glucose-l-P, and varying amounts of a-n-sugar diphosphate.
Phosphoglucomutase was first incubated for 10 min at 3" in a solution containing 0.05 M imidazole buffer (pH 7.0), 10 rnM MgCl2, 2 mM EDTA, and 0.01% bovine serum albumin just before it was added to the reaction mixture (15). The reaction was initiated by the addition of n-D-glucose-l-P or cl-n-sugar diphosphate, and the increase in absorbance at 340 rnp was measured with a Gilford recording spectrophotometer.
Glucose-6-P dehydrogenase was obtained from Calbiochem, and phosphoglucomutase was purchased from Sigma and it was further purified (5). One unit of activity is defined as the amount of enzyme required to convert 1 pmole of ol-n-glucose-l-P to D-glucose-6-P per min at 25" under the standard assay conditions. Specific activity is expressed as units per mg of protein.
The concentration of protein was determined calorimetrically (16) and spectrophotometrically at 278 rnp with an absorbance index of 7.7 for a 1% solution (17).
Methods and Materials-a-n-Sugar diphosphates were prepared from acetylated sugar monophosphates by condensation with crystalline phosphoric acid as described by Hanna and Mendicino in the preceding communication (18). The analysis of these compounds indicated a ratio of acid-labile phosphate to acid-labile reducing sugar to total phosphate of 1 :1:2, within the experimental error of the methods of assay used. The CY-Dsugar diphosphates and the products formed on acid hydrolysis of these compounds were chromatographically pure in each case. However, a very small amount of contamination by ar-n-glucose-1,6-di-P, not detectable by the calorimetric and chromatographic procedures used, could seriously interfere with the assay of the other a-n-sugar diphosphates.
Therefore, each of the samples was hydrolyzed and assayed spectrophotometrically with Dglucose-6-P dehydrogenase and TPN+ in order to determine whether a trace amount of a-D-glucose 1 ,6-diphosphate was present.
n-Glucose-6-P, cr-D-glucose-l-P, cu-D-ribose-l-P, b-n-ribose-l-P, and n-ribose-5-P were purchased from Sigma and Calbiochem. a-n-Mannose-l-P, oc-D-galactose-l-P, and N-acetyl-ar-D-glucosamine-1-P were synthesized from the corresponding pentaacetates by a modification of the procedure of MacDonald (19). n-Mannose-6-P and n-glucosamine-6-P were prepared by phosphorylation of n-mannose and n-glucosamine with ATP and crystalline yeast hexokinase (20). The D-glucosamine-6-P was N-acetylated with acetic anhydride (21) and purified by ion exchange chromatography.
All of the sugar monophosphate esters were completely freed from small amounts of D-glucose-6-P, cr-n-glucose-1,6-di-P, and any other diphosphate compounds by the following procedure.
Approximately 500 pmoles of the sugar monophosphate were incubated with D-glucose-6-P dehydrogenase and TPNf to oxidize any n-glucose-6-P to 6-Pgluconic acid. The TPNf, TPNH, and 6-P-gluconic acid present in this reaction mixture were retained on the anion exchange column with other diphosphate esters. The incubation mixture was passed through a Dowex I-Cl column (2 x 3 cm) and the column was washed with 200 ml of water.
The sugar monophosphate ester was then eluted with 0.01 N HCl, and the eluant was neutralized with LiOH and concentrated under reduced pressure. The oc-D-ribose-1-P and b-n-ribose-l-P, which were somewhat labile in dilute acid, were eluted from the column with 0.05 M LiCl.
The compounds were applied to washed Whatman No. 3MM paper and LiCl was removed by paper chromatography with a solvent containing ethanol and acetone (2:8).
After development for 20 hours with this solvent, the band containing LiCl was well separated from the sugar phosphate ester. The lithium salt of the monophosphate ester was eluted from the paper with distilled water and the solution was passed through a Dowex 50-H+ column (2 x 2 cm). The phosphate ester, in each case, was converted to the imidazole salt by neutralizing the free acid with imidazole to pH 7.0. Inorganic phosphate and acid-labile phosphate were determined by the method of Fiske and SubbaRow (22), and total phosphate by the procedure of King (23). Reducing sugars were analyzed by the method of Somogyi and Nelson (24,25), and for small quantities the Park and Johnson (26) assay was used. Glucosamine and N-acetyl+glucosamine and their phosphate esters were assayed by a modification of the procedure of Morgan and Elson (21,27).

RESULTS
Influence of cr-D-Glucose-l,6-c&P on Activity of Phosphoglucomutase-Some phosphotransferase activity was observed in the absence of added ol-D-glucose-l, 6-di-P, even when highly purified a-D-glucose-l-P was added. The cr-D-glucose-l-P used in these studies was completely freed of sugar diphosphate by chromatography on Dowex l-Cl columns as described in the previous section. The residual activity of the enzyme in the absence of cr-D-glucose-1,6-di-P was less than 5% of the activity which could be obtained under similar conditions in the presence of the sugar diphosphate.
The velocity was 0.24 nmole per min with 8.9 X 10e4 PM enzyme and 250 pM cr-D-glucose-l-P in the absence of a-D-glucose-l, 6-di-P, and it was 5.34 nmoles per min in the presence of 0.046 PM cr-D-glucose-1,6-di-P.
The velocity in the absence of cr-D-glucose-1,6-d&P was strictly dependent on the concentration of enzyme. Under the standard assay conditions an activity of 0.24 nmole per min was obtained with 8.9 X 10e4 PM enzyme and the velocity increased IO-fold (2.48 nmoles per min) when 8.9 x 10d3 PM enzyme was added. In order to eliminate the possibility that this residual activity was due to contamination of the enzyme preparation with ar-D-glucose 1,6diphosphate, the solution was slowly passed through a Dowex 1-imidazole column to exchange any anions present. After chromatography the same residual activity was observed. The possible presence of tightly bound ac-D-glucose-l, 6-di-P in the enzyme preparation was examined by denaturing 8.9 x 10m6 pmole of enzyme with trichloracetic acid at 3". The resulting suspension was centrifuged and the supernatant was extracted with ether to remove trichloracetic acid. The solution was then neutralized and concentrated under vacuum. The velocity of the reaction did not increase when this sample was added to the standard assay mixture in the absence of ar-D-glucose-l, 6-d&P. Since an extract prepared from 10 times the amount of enzyme normally used in the assay failed to activate the reaction, it is likely that the enzyme preparation is free of ar-D-glucose-1,6di-P. The residual activity could be due to the presence of some active phosphoenzyme in the preparation. However, when the enzyme was treated with D-glucose-6-P or cy-D-glucose-l-P according to the procedure of Najjar and Pullman (3) to remove the phosphorylated form of phosphoglucomutase, the same level of residual activity was found.
The cr-D-glucose-l-P and Dglucose-6-P used to inactivate the enzyme preparation were completely free of a-D-sugar diphosphate esters and, therefore, the persistence of a small amount of residual activity could not be at different concentrations of a-D-glucose-1-P. The reaction mixture was incubated at 25" and contained in 1 ml: 25 mM imidazole-HCl (pH 7.0), 5 mM MgC12, 1 mM EDTA, 0.005% bovine serum albumin, 0.5 mM TPN+, 6.4 pg of glucose-6-P dehydrogenase, 8.9 X W4 PM phosphoglucomutase, and the indicated amounts of cY-D-glucose-l-P and a-n-glucose-1,6-di-P.
The ammonium salts of both the mono-and diphosphate sugars were used in this experiment.
The reaction was initiated by the addition of a-D-glucose-l-P. attributed to a contaminant in these preparations which reactivated some of the enzyme. The final preparation was considered suitable for the present study since at low enzyme concentrations only a very small activity was observed in the absence of added a-D-sugar diphosphates.
The effect of the concentration of a-D-glucose 1-P on the activity of the enzyme was determined over a range of 0.25 mM to 15 mM a-D-glucose-l-P and 1.2 PM to 36 pM &D-glucose-l ,6di-P (Fig. 1). Substrate inhibition was observed, especially at high concentrations of a-D-glucose-l-P.
The apparent K, of or-D-glucose-l ,6-di-P in the presence of 15, 10, and 5 mM a-Dglucose-l-P was 25, 13.7, and 9.1 PM, respectively. There was little or no inhibition at low concentrations of or-D-glucose-l-P. Similar plots of l/V against l/c+D-glucose-l ,6-di-P were obtained under these conditions with 0.25 mM and 0.5 mM a-Dglucose-l-P, as shown in Fig. 1. The apparent K, for (Y-Dglucose-l ,6-di-P at 0.25 mM ar-D-glucose-l-P was calculated to be 0.1 PM. The activity and the dependence on cr-D-glucose-l, 6di-P were strictly proportional to the concentration of enzyme. It should be noted that the inhibition of the enzyme by high concentrations of magnesium ion, caused by the formation of inactive magnesium complexes of cr-D-glucose-l-P and the sugar diphosphates, was also avoided in the present studies by activating the enzyme prior to the assay and by using low concentrations of magnesium ion relative to the amount of phosphate esters present (28, 30). The rate of the phosphoglucomutase reaction in this assay system may also be limited by the fact that D-glucose-6-P dehydrogenase is specific for fl-D-glucose-6-P, whereas or-D-glucose-6-P is formed in the phosphoglucomutase reaction (29). It has been noted that the addition of P-glucose isomerase, a mutarotase for the Q! and fl forms of D-glucose-6-P, accelerates the rate of TPNH formation in the coupled assay by at least 4-fold (11,29), presumably by increasing the spontaneous rate of anomerization of a-D-glucose-6-P to &D-glucose 6-P. However, the addition of P-glucose isomerase to the assay system used in these studies did not accelerate the rate of reduction of TPN+.
The components of the assay system used in the present studies were adjusted so that the rate-limiting factor was the concentration of the sugar diphosphate added.
Activity of Phosphoglucomutase in Presence of ar-D-Mannose-1,6-&-P-The K, of phosphoglucomutase for ar-D-mannose-1,6-di-P has been reported to be about the same as that for cr-D-glucose-l, 6-di-P (31). The effect of ar-D-mannose-l , 6-di-P concentration on the enzyme activity was determined over a range of 0.46 PM to 6.9 PM (Fig. 2). An interesting characteristic of the system, when phosphoglucomutase was activated with any sugar diphosphate other than cr-D-glucose-1,6-diphosphate, was the increase in velocity for approximately 10 min before a steady state was reached.
As seen in Fig. 2, the rate of increase as well as the final velocity attained was dependent on the concentration of ac-D-mannose-1,6-di-P. The velocity observed after the 1st min also increased with increasing concentrations of or-D-mannose-l , 6-di-P.
The same curves were obtained when the reaction was initiated with either ar-D-mannose-l , 6-di-P or or-D-glucose-l-P.
Preliminary incubation of the reaction mixture for periods of up to 20 min before the addition of CY-Dglucose-l-P or a-D-mannose-l , 6-di-P had no effect on the velocity observed in the 1st min. Moreover, this treatment did not affect the rate of change of the velocity or the final velocity attained at each concentration of or-D-mannose-l , 6-di-P. The double reciprocal plot of the data, based on the maximal of or-n-mannose-1,6-di-P concentration on the rate of the phosphoglucomutase reaction.
The reaction was initiated by the addition of 0.25 rmole of the imidazole salt of a-n-glucose-l-P. The rate of the reaction was measured for 15 min; however, the rate was nearly constant after 10 min.
velocities attained at corresponding concentrations of a-D-mannose-l ,6-di-P, is shown in Fig. 3. The apparent K, and maximum velocity for ar-D-mannose-l , 6-di-P, calculated from the data by the method of least squares, was 3.7 x 10B6 M and 360 nmoles per min per mg, respectively (Table I). Thus, under identical conditions the K, of muscle phosphoglucomutase for a-D-mannose-l , 6-di-P was at least 40-fold higher than that of or-D-glucose 1,6-diphosphate. In order to be certain that the activity observed with a-D-mannose-1,6-di-P was not due to contamination of this preparation with cr-D-glucose-l, 6&P, 3 pmoles of a-D-mannose-1,6-di-P were hydrolyzed with 0.1 N HCl at 100" for 10 min and analyzed as described previously. No D-glucose-6-P was detected when the sample was assayed in the presence of TPNf and glucose-6-P dehydrogenase. A small amount of D-glucose-6-P added to the cuvette after the sample was assayed resulted in the reduction of an equivalent amount of TPN+. Thus, a negative test in this assay could not be attributed to an inhibition of D-glucose-6-P dehydrogenase by products formed upon acid hydrolysis of a-D-mannose-l , 6-di-P. As little as 0.02 pmole of D-glucose-6-P could have been detected by this method, and, at the concentrations of cr-D-mannose-l , 6di-P used in the kinetic experiments, smaller amounts of contamination by a+D-glucose-l, 6-di-P would have had no detectable effect on the rate of formation of TPNH in this system.
If oc-D-mannose-1,6-di-P reacted with phosphoglucomutase to form a phosphorylated enzyme, then a-D-mannose-1-P and Dmannose-6-P should also be formed. Furthermore, the addition of these compounds should influence the rate of the reaction by suppressing the formation of phosphoenzyme. was initiated with the addition of 0.25 bmole of the imidazole salt of or-n-glucose-l-P. cr-D-mannose-1-P and D-mannose-6-P on the rate of the phosphoglucomutase reaction in the presence of ar-D-glucose-l, 6-di-P and a-D-mannose-1,6-di-P was examined and the results are summarized in Fig. 4. It may be noted that a maximal velocity was obtained immediately with Lu-D-glucose-1,6-di-P, whereas a slow increase in velocity is observed for 10 min with a-D-mannose-l, 6-di-P. As shown in Table II, the addition of a-D-mannose-l-P to the reaction mixture containing ar-D-glucose-1,6-di-P decreased the velocity by 1.9 nmoles per min in less than 3 min. When D-mannose-6-P was added to the reaction mixture, the velocity decreased by 1.4 nmoles per min. A new lower steady state was attained in each case. The addition of a-D-mannose-1-P to a reaction mixture containing 0.46 PM cr-D-mannose-l ,6di-P decreased the velocity by 1.9 nmoles per min, whereas adding D-mannose-6-P decreased the rate of the reaction by 1.5 nmoles per min. The addition of D-mannose-6-P decreased the velocity by approximately 50% (1.4:2.7 and 1.5:2.7) when a velocity of 2.7 nmoles per mm was being maintained by either  The reaction was initiated by the addition of 0.25 rmole of Lu-n-glucose-l-P.
After a maximal velocity was attained, the sugar monophosphate was added and the rate of the reaction was followed until a new steady rate was reached. This lower rate was usually attained in about 3 min. All of the sugar phosphate esters were the WD anomers unless otherwise indicated. When shown in the table, 0.39 PM cu.n-glucose-1,6-di-P, 0.46 pM ol-n-mannose-1,6-di-P, 3.18 pM a-n-ribose-l,5-di-P and 1 PM N-acetyl-cu-n-glucosamine-1,6-di-P were added. Vol. 245,No. 16 FIG. 4. The influence of a-n-mannose-1-P and D-mannose-6-P on the phosphoglucomutase reaction in the presence of cu-n-glueose-1,6-di-P, cu-n-mannose-1,6-di-P, and a-n-glucose-l-P. The reaction mixture contained in 1 ml: 25 mM imidazole-HCI (pH 7.0), 5 mM MgCh, 1 mM EDTA, 0.005yo bovine serum albumin, 0.5 mM TPNf, 6.4 rg of glucose-6-P dehydrogenase, 8.9 X 10-4 PM phosphoglucomutase, and 0.039 PM imidazole salt of a-n-glucose-1,6-di-P or 0.46 pM a-n-mannose-1,6-di-P. The reaction was initiated by the addition of 0.25 pmole of a-n-glucose-l-P. At the time indicated in the figure, 1.74 pmoles of the imidazole salt of ol-n-mannose-1-P or 1.46 pmoles of the imidazole salt of n-mannose-6-P were added and the rate of the reaction was followed for an additional 10 min. The plots of l/P against l/sugar diphosphate were calculated from the maximal velocities reached at each concentration of or-n-sugar diphosphate.
0.46 PM a-n-mannose-l ,6-di-P or 0.39 FM cY-n-glucose-l, 6-di-P. The addition of a-n-mannose-1-P decreased the velocity by approximately 70% (1.9:2.7) under the same conditions. Since 20% more cr-n-mannose-1-P was added (with a 20% difference in the final rate of the reaction), these results would suggest that the same intermediates are formed from both sugar diphosphates, and that the sugar monophosphates influence the steady state concentrations of these intermediates to the same extent. It appears that about 12 times (0.46 :0.039) as much ar-n-mannose-1,6-di-P is required to maintain the same concentration of the rate-limiting intermediate as cu-o-glucose 1,6-diphosphate. On the basis of evidence presently available, the reaction of dephosphoenzyme with a-n-sugar 1 ,6-diphosphate before the addition of or-n-glucose-l-P would result in the formation of sugar-l-P, sugar-6-P, and various derivatives of the enzyme represented as E .sugar 1 , 6-diphosphate and EP (Reactions 1 to 3). 2E + 2 sugar-1,6-di-P F? 2E.sugar-1,6-di-P (1) E-sugar-1,6-di-P = EP + sugar-l-P (2) E.sugar-1,6-di-P g EP + sugar-6-P 2E + 2 sugar-1,6-di-P e 2[EP] + sugar-l-P + sugar-6-P (Sum l-3) The over-all equilibrium between the reactants would be dependent on the concentration of the components shown as Sum l-3.
The initial rate observed in the experiments described in Fig. 2 may depend on the amount of EP formed before WDglucose-l-P is added. After the addition of cr-n-glucose-l-P the D-glucose-6-P formed in Reactions 4 and 5 would be removed by the action of glucose-6-P dehydrogenase.

(glucose 1-P)
This relationship indicates that the rate of conversion of Cr-Dglucose-l-P to n-glucose-6-P will be adversely influenced by in-creasing concentrations of sugar-l-P and sugar-6-P.
The rate of the reaction should increase with increasing concentrations of a-n-glucose-l-P and ar-n-sugar-l ,6-di-P. In other experiments, not shown, it was shown that increasing concentrations of a-!-~glucose-l-P and a-n-glucose-l, 6-di-P or cY-n-mannose-l , 6-di-P increased the velocity of the reaction in the presence of the sugar monophosphates.
The possibility of forming a-D-ghCOSe-1,6-di-P by the reactions shown below in Equations 6 and 7 should also be considered. It is unlikely that there is very much of this conversion occurring under these conditions, since glucose-6-P dehydrogenase rapidly removes n-glucose-6-P formed in Reaction 5 and thereby competes very favorably with Reaction 7 for any glucose-l-P-E. P formed in Reaction 4. Ray and Roscelli (4) and Lowry and Passonneau (11) have estimated that the ratio of the rate of dissociation of or-n-glucose-l, 6-di-P from the enzyme (Reaction 7) to the rate of formation of n-glucose-6-P (Reaction 5) is probably less than one-sixtieth.
Preliminary incubation of the enzyme with ar-n-glucose-l-P or ol-n-ribose-1 ,5-di-P did not alter the initial velocity observed or the final velocity attained in this system. The same curves were obtained when the reaction was initiated with either Lu-n-glucose-l-P or cr-n-ribose-1 ,5di-P. However, both the initial rate and final velocity increased with increasing concentrations of cr-n-ribose-1 ,5-di-P.
When the concentration of the sugar diphosphate was varied from 1.59 PM to 7.95 pM the maximum velocity increased from 5.1 nmoles per min to 9.2 nmoles per min. The apparent K, and maximum velocity, calculated from plots of l/velocity against l/S, were 3.16 x 10m6 M and 210 pmoies min-l mg-l, respectively (Table  I). The K, for cr-n-ribose-1 ,5-di-P was approximately 35 times greater than the K, for a-n-glucose-l, 6-di-P under these conditions.
In order to establish that the extent of activation of the dephosphoenzyme by or-n-ribose-1,5-di-P was related to the amount of n-ribose monophosphates formed in the reaction, experiments were carried out to examine the effects of a-~ribose-1-P and n-ribose-5-P on the velocity of the reaction in the presence of sugar diphosphates.
The results of experiments designed to compare the effects of n-ribose monophosphate esters on the activity in the presence of ar-n-glucose 1,6-diphosphate are summarized in Table II. The addition of 1.1 ,emoles of cY-n-ribose-1-P decreased the observed velocity by 0.9 nmole per min in less than 3 min, where the addition of 3.1 pmoles of Dribose-5-P decreased the velocity by 1.4 nmoles per min. The n-ribose monophosphates also decreased the velocity of the reaction when the enzyme was activated with or-n-ribose-1 ,5di-P.
The addition of 1.1 pmoles of ac-n-ribose-1-P decreased the velocity by 2.5 nmoles per min, and under the same conditions 3.1 pmoles of n-ribose-5-P decreased the velocity by 2.9 nmoles per min (Table II).
In other experiments, in which the concentration of the n-ribose monophosphates were varied between 1 and 5 pmoles, it was found that or-n-ribose-1-P was consistently about 2 to 3 times as effective as n-ribose-5-P in decreasing the velocity of the phosphoglucomutase reaction. The addition of 1.1 pmoles of b-n-ribose-l-P, which is inactive as a substrate in the phosphoglucomutase reaction, was completely ineffective in altering the velocity of the reaction in the presence of either cY-n-glucose-l, 6-di-P or cY-n-ribose-l , 5-di-P.
Since almost the same relative effects were observed with ar-n-glucose-1,6-di-P and a-n-ribose-1 ,5-di-P, some common enzyme intermediates are probably formed from both of the sugar diphosphate esters. Moreover, the capability of the monophosphate sugars to decrease the level of these intermediates appears to be related to the nature of the sugar, and perhaps the position of the phosphate group. The two terminally phosphorylated derivatives of n-mannose were equally effective, whereas those of n-ribose were different.
The maximum velocities for the interconversion of a-n-mannose-l-P (V,,, = 19.4 pmoles mg-l min-l) and n-mannose-6-P (V,,, > 0.2) or cr-n-ribose-1-P (V,, = 2.9) and n-ribose-5-P (Vmx = 0.24) are very low compared to that of cY-n-glucose-l-P (Vnmx = 328) (11). The K, values for cr-n-mannose-1-P (245 PM), n-mannose-6-P (500 PM), a-n-ribose-1-P (900 j&M), and Dribose-5-P (400 PM) are correspondingly high compared to that of a-D-ghCOSe-1-P (8 PM) (11). It is, therefore, rather unlikely that any extensive equilibration of the two sugar monophosphate esters occurs under the conditions used in these experiments. However, the data obtained from preliminary incubation studies strongly indicate that complete and rapid equilibration of the sugar monophosphates with the corresponding a-n-sugar diphosphate does occur under these conditions. From this evidence and the fact that at equal concentrations ar-n-ribose-1-P was more effective than n-ribose-5-P in removing phosphoenzyme, it might be tentatively suggested that after the new steady state is reached 2 to 3 times more P-enzyme-cr-n-ribose-1-P is formed than P-enzyme-n-ribose-5-P as shown in the following general equations: EP + ol-D-sugar-l-P = a-n-sugar I-P+EP (24 EP + n-sugar-5-P G n-sugar 5-P.EP (34 cr-n-Sugar-1-P.EP ti E.sugar-1,5-di-P @b) n-Sugar-5-P.EP $ E.sugar-1,5-di-P (3b) The compounds interconverted in these reactions represent the appropriate n-ribose phosphate intermediates of Equations 2 and 3. Activation of Phosphoglucomutase with N-Acetyl-or-o-Glucosamine-l ,6-&P-In Fig. 3 are shown the change in velocity with concentration and the Lineweaver-Burk plot of data obtained when phosphoglucomutase was activated with N-acetyl-cr-nglucosamine-1 ,6-di-P.
The velocity increased for about 10 min at all concentrations of N-acetyl-a-n-glucosamine-1 ,6-di-P tested. The final maximum rate attained was dependent on the amount of sugar diphosphate added. Essentially identical curves were obtained when the enzyme was first incubated for various times with either sugar diphosphate or cr-n-glucose-l-P, thus supporting the previous observation that rapid and complete equilibration of enzyme and sugar diphosphate occurs in this system. These results, as well as the data obtained with the other sugar diphosphates, are consistent with the mechanism shown in Equations 1 through 7. Calculations based on data obtained from the double reciprocal plots of l/velocity with respect to l/substrate concentration in Fig. 3 showed a K, and maximum velocity of 1.6 X 10m6 M and 220 pmoles mihl mg-I, respectively.
The reaction was initiated with the addition of 0.25 pmole of a-D-glUCOSe-1-P.
At times the indicated 1.42 and 2.84 pmoles of N-acetyl-a-o-glucosamine-I-P or 1 pmole of N-acetylglucosamine-6-P was added. 6. Inhibition of the phosphoglucomutase reaction by a-n-galactose-1,6-di-P.
The reaction was initiated with the addition of 0.25 rmole of a-D-glucose-l-P.
As seen in Table II, when N-acetyl-a-n-glucosamine-1-P was added to a reaction mixture containing or-n-glucose-l ,6-di-P the velocity decreased by 1.7 nmoles per min. The velocity decreased by 2.3 nmoles per min when N-acetyl-n-glucosamine-6-P was added.
In this case, the decrease was greater when the B-phosphate derivative was added. The ratio of the differences in the velocities was 1.8 (2.3:1.7 x 1.42) after correction for the concentrations of the sugar monophosphates used. When 1.42 pmoles of N-acetyl-a-n-glucosamine-1-P were added to a reaction mixture containing 1 pM N-acetyl-cr-n-glucosamine-1 ,6-di-P, the velocity decreased by 1.1 nmoles per min, whereas the addition of 2.84 pmoles of the l-phosphate sugar caused a decrease of 2.2 nmoles per min (Fig. 5). Similar results were obtained with all of the sugar monophosphates tested, indicating that the extent of decrease was directly proportional to the amount of sugar monophosphate added. The addition of 1 pmole of N-acetyl-n-glucosamine-6-P to the same reaction mixture depressed the velocity by 3.7 nmoles per min, as shown in Table II. It is noteworthy that, in contrast to the results obtained with the n-ribose monophosphate esters, N-acetyl-n-glucosamine-6-P was at least 3 times more active than N-acetyl-a-n-glucosamine-1-P in decreasing the velocity of the phosphoglucomutase reaction. The data obtained in these experiments again suggest that unequal amounts of the intermediate forms of P .enzyme.Nacetyl-oc-n-glucosamine-1-P and P enzyme .N-acetyl-n-glucosamine-6-P may be formed, and that the free forms of these monophosphates are far from equilibrium even after the new steady state is reached.
Effect of a+o-Glucosamine-i ,6-di-P and oc-o-Galacbse-l , B-di-P on Activity of Phosphoglucomutase-Phosphoglucomutase was activated by a-n-glucosamine-1 , 6.di-P. The rate of conversion of ol-n-glucose-l-P to n-glucose-6-P was measured with levels of the sugar diphosphate ranging from 0.9 PM to 3.6 pM. The characteristic increase in velocity with time was also obtained with this compound.
The initial velocity and final rate of the reaction were unaffected by preliminary incubation and the order of addition of the reaction components, but increased with increasing concentrations of the sugar diphosphate.
The K, and maximum velocity calculated from the data shown in Fig. 3 were 1.1 X low5 PM and 370 pmoles mine1 mggl, respectively.
The apparent K, for a+n-glucosamine-1,6-di-P was 7 times larger than that for N-acetyl-cu-n-glucosamine-1 ,6-di-P. The C-4 isomer, cr-n-galactose-1,6-di-P, was a very poor activator of the phosphoglucomutase reaction. At low concentrations, the Ii, and maximum velocity calculated from the data shown in Fig. 6 were 1.3 x lop5 M and 240 pmoles mine* mg-I, respectively.
Not only was or-n-galactose-1 ,6-di-P a poor activator of phosphoglucomutase relative to oL-n-glucose-l, 6-di-P and the other sugar diphosphates, but at very high concentrations it was also a competitive inhibitor.
The fact that the velocity increases with time to a maximum, as shown in Fig. 6, strongly indicates that some phosphoenzyme is being formed from this sugar diphosphate. DISCUSSION The kinetic constants for the sugar diphosphates tested as activators of the phosphoglucomutase reaction are summarized in Table I. With cw-n-glucose-l-P as the substrate, cr-n-glucose-1,6-di-P was the best activator. The K, of N-acetyl-ac-n-glucosamine-1 ,6-di-P was only about 18 times higher, whereas the K, of n-glucosamine-1 ,6-di-P was almost 120 times greater than that of or-n-glucose-l ,6-di-P.
The K, of the pentofuranosy diphosphate sugar was 35 times higher, while the K, of a-Dmannose-1,6-di-P was 40 times higher than that of oc-n-glucose-1,6-di-P.
Comparison of the results obtained with cr-n-galactose-1 ,6-di-P and cr-n-mannose-l ,6-di-P indicates that the configuration of the hydroxyl group at C-2 is not as important for the reactivity of the sugar diphosphate as the configuration of the hydroxyl group at C-4. Substitution of an electron withdrawing group, such as an amino group, at C-2 raises the K, of the sugar diphosphate dramatically, and N-acetylation of the amino group greatly decreases the K,. The variations in the maximum velocities obtained with each of the sugar diphosphates was not as great as the difference in the K, values. The largest decrease was seen with cr-n-ribose-1 ,5-di-P (210 pmoles min-l mg+ compared to 500 pmoles min-1 "g-1 for cr-D-glucose-1,6-di-P) . The effects of the various sugar diphosphates and sugar monophosphates on the rate of conversion of cr-D-glucose-l-P to D-glucose-6-P by phosphoglucomutase may be summarized in the following series of interactions: or-n-sugar diphosphate -I-enzyme 11 P.E.a-D-Sugar-l-P : E.sugar diphosphate ti P.E.o-sugar-6-P 11 11 cY-D-Glucose-l-P D-Sugar-B-P + E.P + L--D-+ E.P + Dsugar-l-P glucose-6-P 11 11 a-D-Glucose-l-P.E.Pe E.glucose-1,6-di-P ti D-glucose-6-P.E.P 11 ~-o-Glucose-l ,6-di-P + enzyme The observations made in the present study may be explained in terms of this scheme and the relative rates of the reactions involved as they influence the rate of formation of D-glucose-6-P. The formation of enzyme. sugar diphosphate or enzyme. phosphate complexes by equilibration of enzyme and sugar diphosphate is a relatively rapid reaction, and it is probable that the same enzyme. phosphate complex is formed from all of the sugar diphosphates.
Thus, in no case did preliminary incubation of the enzyme and sugar diphosphate increase the initial rate of the reaction after the addition of ar-D-glucose-l-P.
However, both the initial and final velocities increased with increasing concentrations of a-D-sugar diphosphate.
The large variation in the initial and final rates observed with the five activators tested indicates that the sugar component of the sugar diphosphate as well as the steric orientation of the phosphate groups may be responsible for the differences observed in the K, of these compounds.
The results further indicate that when the enzyme is activated by a sugar diphosphate, other then c+D-glucose 1,6-diphosphate, the increase in the velocity of the reaction may be limited by the slow rate of accumulation of a phosphate.enzyme.D-glucose phosphate, enzyme. a-D-glucose 1,6-diphosphate, or some other obligatory enzyme intermediate between a-D-glucose-l-P and D-glucose6-P, which was not present before the addition of (r-Dglucose-l-P.
The final concentration and rate of accumulation of this intermediate were dependent on the amount of sugar diphosphate added. A maximum level was attained in about 10 min in most cases. In marked contrast, a maximal rate was obtained almost immediately when the enzyme was activated with or-D-glucose 1,6-diphosphate.
The formation of the ratelimiting intermediate must have been very rapid, under these circumstances.
The difference in the activation by other sugar diphosphates and oc-D-glucose 1,6-diphosphate appears to involve the obligatory formation of an enzyme-phosphate intermediate, and, after the addition of ol-D-glucose-l-P, a phosphate. enzyme. cw-D-glucose-l-P complex with the sugar diphosphates. These intermediates may be subsequently converted to the same compound formed directly from a-D-glucose 1,6-diphosphate and free enzyme. Thus, it would seem that the conversion of phosphate. enzyme. cr-D-glucose-l-P to enzyme. a-D-glucose 1,6diphosphate or phosphate .enzyme .D-glucose-6-P is the relatively slow step in the over-all conversion.
The reaction of sugar monophosphates with enzyme. phosphate to form enzyme .oc-D-sugar 1,6-diphosphate, a reversal of the activation process, is at least 3 times as fast as the formation of the rate-limiting intermediate after the addition of or-D-glucose-1-P (less than 3 min compared to 10 min). This result may be explained if the rate of conversion of cr-D-glucose-l-P to D-glucose-6-P becomes dependent on the concentration of another intermediate, enzyme . phosphate, when relatively large amounts of sugar monophosphate are added. High concentrations of the sugar monophosphates, about 1 MM, were required to remove sufficient enzyme. phosphate to make this complex rate-limiting.
Low concentrations, up to 300 PM, had little or no effect on the velocity, although they would be expected to remove some enzyme f phosphate.
In this manner the decrease in the velocity of the reaction and the attainment of a new lower steady state occurs in less than 3 min, even though the concentration of the original rate-limiting enzyme complexes (phosphate. enzyme +Dglucose monophosphates or enzyme. cr-D-glucose diphosphate) remains high because of their slow rate of synthesis and breakdown.
The rate of interconversion of sugar monophosphates appears to be much slower than the rate at which they react with phosphate. enzyme to form phosphate. enzyme + sugar monophosphate, enzyme-a-D-sugar diphosphate, or enzyme and free a-D-sugar diphosphate.
The two enzyme intermediates, not common to both approaches which would not be expected to equilibrate rapidly under these conditions, are the products of the reaction of phosphate-enzyme with each of the sugar monophosphates. The results obtained in the present study show that D-mannose-6-P and ol-D-mannose-1-P were equally effective in depressing the velocity of the reaction. D-Ribose-5-P was less active than a-D-ribose-I-P, and N-acetyl-D-glucosamine-6-P was more active than N-acetyl-ol-D-glucosamine-1-P.
The reaction of phosphate.enzyme and sugar monophosphate appears to be the step which is dependent on the structure of the sugar. The effective concentrations of the sugar monophosphates containing a free aldehyde group might be lower than the amount that was added, since only the OL anomers would be expected to react with phosphoglucomutase.
This factor would further alter the results obtained with each sugar monophosphate.
In agreement with the results of other studies (4, 10, II), free a-D-sugar 1,6-diphosphates do not seem to be obligatory intermediates in the interconversion of the corresponding sugar monophosphates.
However, the mechanism does appear to involve the formation of both enzyme-E-D-sugar diphosphate and enzyme phosphate intermediates. Furthermore, the steady state level of these intermediates maintained during the reaction was dependent on the concentration of both Or-D-sugar diphosphate and cr-D-glucose-l-P.