Inhibition of phosphoglucomutase by vanadate

Phosphoglucomutase is inhibited by a complex formed from alpha-D-glucose 1-phosphate (Glc-1-P) and inorganic vanadate (Vi). Both the inhibition at steady state and the rate of approach to steady state are dependent on the concentrations of both Glc-1-P and Vi. Inhibition is competitive versus alpha-D-glucose 1,6-bisphosphate (Glc-P2) and is ascribed to binding of the 6-vanadate ester of Glc-1-P (V-6-Glc-1-P) to the dephospho form of phosphoglucomutase (E). The inhibition constant for V-6-Glc-1-P at pH 7.4 was determined from steady-state kinetic measurements to be 2 x 10(-12) M. The first-order rate constant for approach to steady state increases hyperbolically with inhibitor concentration. The results are consistent with rapid equilibrium binding of V-6-Glc-1-P to E, with dissociation constant 1 x 10(-9) M, followed by rate-limiting conversion of the E.V-6-Glc-1-P complex to another species, E*.V-6-Glc-1-P, with first-order rate constant 4 x 10(-2)s-1. The rate constant determined for the reverse reaction, conversion of E*.V-6-Glc-1-P to E.V-6-Glc-1-P, is 2.5 x 10(-4)s-1. Formation of E*.V-6-Glc-1-P can also occur via binding of glucose 6-vanadate to the phospho form of phosphoglucomutase (E-P) followed by phosphoryl transfer and rearrangement of the enzyme-product complex.     

The mechanism of the phosphoglucomutase reaction. Studies on rabbit muscle phosphoglucomutase with flux techniques

1. The kinetics of phosphoglucomutases from different sources are discussed and it is concluded that on the available evidence there are in all cases three possible mechanisms for the reaction. These are an indirect transfer of phosphate involving the phosphoenzyme (mechanism 1), a direct transfer of phosphate (mechanism 2), and an intermolecular transfer of phosphate from glucose 1,6-diphosphate to the substrate (mechanism 3). Conventional net flux measurements are shown not to differentiate between these mechanisms. 2. Flux equations are developed and it is shown that there are three flux ratios that characterize and distinguish between the mechanisms. 3. To examine these flux ratios induced-transport tests are described with ¹⁴C- and ³²P-labelled substrates. The fluxes determined with ¹⁴C- and ³²P-labelled substrates are also compared at chemical equilibrium. 4. With rabbit muscle phosphoglucomutase the results of these tests were completely consistent with mechanism 1 and unequivocally excluded any substantial part of the reaction proceeding by mechanism 2 or mechanism 3. Evidence was also obtained for an isomerization of the phosphoenzyme with an apparent rate constant about 4·5×10⁷sec.⁻¹. Taking into account the activity coefficients of the substrates the true rate constant appears to be about one-sixth of this value. 5. Isotope effects and non-ideal behaviour of the solutions are discussed and the activity coefficients of the substrates are shown to be equal by measurement of the depression of freezing point. It is concluded that these factors do not influence the tests significantly. 6. Alternative mechanisms are considered and it is concluded that the tests show that the glucose residue is transferred directly, that the phosphate is transferred indirectly with one intermediate phosphate, and that there is an isomerization of the free phosphoenzyme without reference to any other details of the reaction. Further, no assumptions are required about the constancy of rate constants. 7. The relative merits of induced transport and product inhibition for detecting isomerization of the enzyme are discussed. It is concluded that the induced-transport test is more sensitive and that its interpretation is less equivocal. 8. The application of the tests to other enzyme systems is briefly considered.     

The inhibition of phosphoglucomutase by beryllium

1. The inhibition of phosphoglucomutase by beryllium has been examined. 2. Inhibition by beryllium does not occur unless a complex-forming agent such as cysteine or imidazole is present. It is therefore similar to activation by magnesium. 3. The inhibition is progressive and the rate follows first-order kinetics, which may be defined by a bimolecular rate constant. 4. In the presence of magnesium the rate of inhibition is less. By using a fixed time for inhibition competition between beryllium and magnesium may be demonstrated. After inhibition has taken place, the addition of magnesium does not reverse it.     

The structure determination of rabbit phosphoglucomutase

Tetragonal crystals of rabbit phosphoglucomutase have been grown from solutions containing ammonium sulphate, polyethylene glycol solution and enzyme. There are two molecules, each of relative molecular mass 64 000 per asymmetric unit. A rotation function suggests that these are related by a twofold axis. X-ray diffraction data for five heavy-atom derivatives and native crystals have been collected by using oscillation photography. A tentative and partial solution of the KAu(CN)2 sites has been obtained. The enzyme in the native crystals is phosphorylated, but the phosphate can be removed without harm to the crystals. Similarly the essential Mg2+ ion can be removed or replaced by Zn2+. The enzyme is active in the native crystals.     

Fructose 2,6-bisphosphate inhibition of phosphoglucomutase

Fructose 2,6-bisphosphate inhibits phosphoglucomutase noncompetitively with respect to the cofactor glucose 1,6-bisphosphate. Previous studies from our laboratory had shown that phosphoglucomutase was activated by fructose 2,6-bisphosphate in the absence of added glucose 1,6-bisphosphate. The fructose 2,6-bisphosphate activation previously reported was due to the presence of glucose 1,6-bisphosphate in the commercial preparation of fructose 2,6-bisphosphate.     

Polymorphism of phosphoglucomutase in cattle leucocytes

By means of starch gel electrophoresis the polymorphism of the phospoglucomutase isozymes PGMi has been investigated in cattle leucocytes. The phenotypes and allele frequencies of PGMi are demonstrated in a series of 106 complete and 221 incomplete families of 'Deutsche Schwarzbunte'. The segregation of the 327 daughters' phenotypes agree with the formal genetic model of 2 codominant autosomal alleles. The observed heterozygous PGMi type is more frequent in mating types and progenies as expected under equilibrium.     

Inhibition of phosphoglucomutase by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate inhibits phosphoglucomutase. The inhibition is mixed with respect to glucose 1,6-bisphosphate and non-competitive with respect to glucose 1-phosphate. In contrast with fructose 1,6-bisphosphate and glycerate 1,3-bisphosphate, which also possess inhibitory effect, fructose 2,6-bisphosphate does not phosphorylate phosphoglucomutase. Fructose 2,6-bisphosphate preparations contain contaminants which can explain artefactual results previously reported.     

The mechanism of phosphoglucomutase from Micrococcus lysodeikticus

The mechanism of the phosphoglucomutase from Micrococcus lysodeikticus was investigated. Induced-transport tests at low substrate concentrations (0.15mm) showed co-transport of the (32)P label but no induced transport of the (14)C label, which is in quantitative agreement with a phosphoenzyme mechanism with a rapid isomerization of the phosphoenzyme. The results excluded an intramolecular transfer of phosphate and could only have been compatible with a sequential mechanism if the K(m) for glucose 1-phosphate had been over 20 times smaller than the measured value. The results of induced-transport tests at intermediate concentrations (1mm) with both labels agreed quantitatively with a phosphoenzyme mechanism, and induced-transport tests with (14)C-labelled substrates at high concentrations (26mm) indicated that the rate constants for isomerization of the phosphoenzyme must be greater than about 3x10(6)s(-1). Consistent with these findings is the fact that (14)C label exchanged between the substrates twice as rapidly as the (32)P label at chemical equilibrium. Further, since the (14)C label exchanged between the substrates about ten times more rapidly than between the substrates and glucose 1,6-diphosphate, glucose 1,6-diphosphate is not an obligatory intermediate in the interconversion of the substrates. It is concluded that, contrary to previous evidence, the mechanism of the enzyme from M. lysodeikticus is essentially that of the rabbit muscle enzyme. To account for the rapid isomerization of the phosphoenzyme in both cases a mechanism is proposed in which there is no formal isomerization of the phosphoenzyme.     

Immunological studies on erythrocyte phosphoglucomutase isozymes

Human erythrocytes (phenotype PGM1 a1 or PGM1 a3) contain two sets of phosphoglucomutase isozymes, produced by the expression of the PGM1 and and PGM2 loci. The two sets are constituted each by two forms, of which that called "secondary" is thought to derive from the post-translational modification of that called "primary". Cross-reactivities of these isozymes were studied by means of monospecific rabbit antibodies against purified human red cell PGM1 and PGM2 "primary" isozymes. The results show that the PGM1 and PGM2 forms are not immunologically related and provide further proof of the post-synthetic origin of "secondary" isozymes and of the multifunctionality of PGM2 phosphoglucomutases.