Phosphoglucose isomerase: a ketol isomerase with aldol C2-epimerase activity.

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RESUMO

With 13C NMR, phosphoglucose isomerase (PGI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) is shown to produce mannose 6-phosphate (M6P) slowly from a much more rapidly catalyzed equilibrium between glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The identity of M6P and its formation from G6P plus F6P are confirmed by 1H NMR and by the ability of PGI to convert M6P to F6P plus G6P. The possibility of contaminating phosphomannose isomerase (PMI, D-mannose-6-phosphate ketol-isomerase, EC 5.3.1.8) is ruled out by finding no exchange of the C1 proton of G6P or of M6P, whereas exchange occurs with a mixture of PMI and PGI in 2H2O. The pro-R and pro-S protons of F6P become the anomeric protons of M6P and G6P through the actions of PMI and PGI, respectively. Both isomerases exchange the C2 proton of their substrate with the medium; hence, when PGI and PMI are added together to hexose phosphate solutions in 2H2O, both the substrate and anomeric protons are exchange rapidly with deuterons from the medium. The rates of C2-epimerization of G6P and M6P by PGI are shown to be proportional to enzyme concentration and inhibited by 5-phosphoarabinoate, a competitive inhibitor of the previously demonstrated isomerase and anomerase activities of PGI. These data show that the epimerization is enzymatically catalyzed and suggest the involvement of the same active site for all three activities. A primary kinetic isotope effect of 7.5 (H/2H) on the rate constant kcat of the M6P C2-epimerase activity was determined by using a coupled enzymatic assay. A model of the mechanism of PGI is offered, which relates C2-epimerase activity to the isomerase and anomerase activities by allowing the cis-enediol intermediate to rotate about the C2-C3 bond axis followed by protonation at C2 but not at C1 from the si face.

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