Thermodynamics and mechanism of the PO3 transfer process in the phosphoglucomutase reaction.
The equilibria among the central complexes in the phosphoglucomutase system were evaluated by (a) using an excess of enzyme plus Mg2+ to prepare mixtures with glucose phosphates in which essentially no free glucose phosphates were present; (b) inactivating the enzyme in such mixtures by means of a procedure that prevents substantial interconversion of the central complexes; and (c) assaying the quenched mixture for glucose 1-P, glucose 1-6-P2, and glucose-6-P. The fractional amounts of Ep-Mg-Glc-1-P, ED-Mg-Glc-P2, and Ep-Mg-Glc-6-P present at pH 7.5 and 24 degrees C were 0.13, 0.54, and 0.33. (Ep and ED are the phospho and dephospho forms of the enzyme, respectively). From these fractions and the equilibrium isotope exchange constants for the three sugar phosphates, true dissociation constants can be calculated for each of the above complexes: 8.5 muM, 19 nM, and 57 muM, respectively. Relative to the rate of PO3 transfer to water, a 3 x 10(10)-fold rate increase is produced by binding glucose-1-P to the Mg2+-enzyme (Ray, jr., W.J., Long, J.W., and Owens, J.D. (1976), Biochemistry, the following paper in this issue). This "substrate-induced rate effect" is equivalent to a difference of some 14 kcal in Gibbs activation energies for transfer to chemically similar hydroxyl groups, and most of this energy difference ultimately must be rationalized in terms of binding interactions involving the phosphoglucosyl moiety. Three different mechanisms for using substrate binding energy to reduce the activation energy of the subsequent catalytic step are examined as possible explanations for the substrate-induced rate effect. These mechanisms emphasize (a) enthalpic destabilization and (b) (entropic) immobilization of reactant groups during formation of the enzyme-substrate complex, and (c) increased binding interactions of nonreactant groups during the subsequent approach to the transition state. As a test for enthalpic destabilization of the enzymic phosphate group, values of deltaG degrees' for the hydrolytic cleavage of this group in Ep and Ep-Glc-1-P are calculated from equilibria measured at pH 7.5 and 30 degrees C: about -1 and +1.4 kcal/mol, respectively. To test for destabilization of the acceptor hydroxyl group in the enzyme-substrate complex, deltaG degrees' for equilibrium, Ep-Glc-P in equilibrium ED-Glc-P2, is compared with that for the corresponding process involving the nonrigid acceptor, 1,4-butanediol monophosphate: about -0.9 and -1.9 kcal, respectively. These results are not consistent with a large enthalpic destabilization of the reactant groups in the Ep-Glc-1-P complex. To test for entropic immobilization of reactant groups, glucose-6-phosphate is considered as a bidentate ligand, and the chelate effect on the binding and subsequent enzymic transfer reaction that arises from covalently linked the sugar ring and the PO3 group is evaluated. Reference reactions involving xylose as a PO3 acceptor both in the presence and absence of bond (inorganic) phosphite are used...
D001665:Binding Sites; D005958:Glucosephosphates; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D010710:Phosphates; D010733:Phosphoglucomutase / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics
D005958:Glucosephosphates; D010710:Phosphates; D010733:Phosphoglucomutase; D008274:Magnesium