Characterization of a vanadate-based transition-state-analog complex of phosphoglucomutase by kinetic and equilibrium binding studies. Mechanistic implications

Abstract

The inhibitor complex produced by the binding of alpha-D-glucose 1-phosphate 6-vanadate to the dephospho form of muscle phosphoglucomutase exhibits an unusually small dissociation constant: about 15 fM for the Mg2+ enzyme at pH 7.4, when calculated in terms of the tetraanion. Such tight binding suggests that the enzyme/vanadate/glucose phosphate complex mimics a state that at least approaches the transition state for (PO3-) transfer in the normal enzymic reaction. This hypothesis also is supported by the observation that replacement of Mg2+, the normal metal ion activator, by Li+, a poor activator, substantially reduces the binding constant for the glucose phosphate/vanadate mixed diester. Other indicators that support this hypothesis are described. One is the derived equilibrium constant for replacement of a PO4(2-) group in bound glucose bisphosphate by VO4(2-): 3 x 10(6) when the replaced group is the phosphate at the (PO3-) transfer site of the Mg2+ enzyme--in contrast to about 10 for the same replacement (of PO4(2-) by VO4(2-)) in an aqueous solution of a phosphate ester. Another is the greatly decreased rate at which Mg2+ dissociates from the glucose phosphate/vanadate complex of the enzyme, relative to the rate at which it dissociates from the corresponding bisphosphate complex (rate ratio less than or equal to 3 x 10(-4)), presumably because Mg2+ binds more tightly to the glucose phosphate/vanadate complex than to the corresponding bisphosphate complex. This apparent increase in Mg2+ binding occurs in spite of what appears to be a reduced charge density at the bound vanadate grouping, relative to the bound phosphate grouping, and in spite of the somewhat weaker binding of Mg2+ by dianionic vanadate than by the phosphate dianion. Although a direct assessment of the binding constant for Mg2+ was not possible, the equilibrium constant for Mg2+/Li+ exchange could be evaluated for the complexes of dephospho enzyme with glucose bisphosphate or glucose 1-phosphate 6-vanadate. The results suggest that the glucose phosphate/vanadate complex of the Mg2+ enzyme mimics a state about halfway between the ground state and the transition state for (PO3-) transfer. This estimate also is in accord with the binding of glucose phosphate/vanadate relative to that expected for transition-state binding of glucose bisphosphate. A possible scenario for the (PO3-) transfer catalyzed by the Mg2+ form of phosphoglucomutase is discussed, on the basis of these observations, together with possible reasons why the bound vanadate group appears to mimic an intermediate state for (PO3-) transfer rather than the ground state for phosphate binding.

Influence of vanadium(V) complexes on the catalytic activity of ribonuclease A. The role of vanadate complexes as transition state analogues to reactions at phosphate

“…This agrees with our analysis above for the studies carried out here with ribonuclease A. A different set of arguments has been advanced for phosphoglucomutase, suggesting that the reaction with vanadate proceeds about 45% of the way to the transition state in that particular system (14).…”

“…Enzyme kinetics studies have also been carried out in conjunction with these NMR studies. The results obtained here have been compared with those reported for phosphoglycerate mutase (12) and for phosphoglucomutase (13,14).…”

Unusual Starch Degradation Pathway via Cyclodextrins in the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus Strain 7324

“…This stimulating effect, which is typical for other PGMs, was attributed to a removal of inhibiting heavy metals by these compounds (16,41,58). The PGM activity was inhibited by vanadate, a structure analog of phosphate, as has been reported for other PGMs (54,55,56).…”

α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

“…A basic question, unanswered by the present results, is why the adduct of vanadate and -glucose displays partial transitionstate analogy instead of simply mimicking the ground-state binding of phosphate and -glucose. Inorganic vanadate resembles phosphate in many respects ; the pK a values of all three ionizations of vanadic acid are similar to those of phosphoric acid [33]. However, the V-O bonds are reported to be approx.…”