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

Abstract

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 (4,6,20,21). PGI was found to catalyze a second kind of reaction when the interconversions of the a-and ,B-pyranose anomers of G6P (7) and the a-and l3-furanose anomers of F6P (8) by PGI were first demonstrated. These findings have more recently been confirmed by using 31P NMR (9) and 13C NMR (10) spectroscopic techniques. The anomerase activity requires ring cleavage, subsequent torsional rotation about the C1-C2 bond for G6P and C2-C3 bond for F6P, and finally cyclization to the opposite face ofthe carbonyl plane. Studies with epoxide-inactivated PGI (8) suggest that both anomers of each substrate react at the same active site responsible for catalyzing the isomerase reaction. PGI was also shown to mutarotate the anomers of mannose 6-phosphate (M6P) at a rate similar to its effect on G6P (11).Reported here are experiments that demonstrate and characterize yet a third enzymatic reaction catalyzed by PGI: G6P C2-epimerization. This reaction results in the production of MGP from G6P and F6P. A mechanism is proposed that shows the relationship between the isomerase, anomerase, and epimerase activities of PGI. MATERIALS AND METHODSRabbit muscle PGI was obtained from Sigma as a suspension in ammonium sulfate. The suspension was centrifuged and decanted. The pellet was dissolved in a small volume of Hepes buffer and then dialyzed against several changes of buffer (0.01 M Hepes/0.0074 M KOH/0.1 M KC1, pH 8.0) to remove ammonium sulfate. The protein concentration was determined spectrophotometrically by using e280 p.i = 1.32 ml/mg-cm (12). Where necessary, the enzyme was concentrated by using Amicon Centricon 10 centrifugal ultrafiltration devices. PGI activity was typically 580 units/mg when assayed spectrophotometrically in 0.1 M Hepes/0.1 M KC1, pH 8/8 mM F6P/10 units of G6P dehydrogenase (G6PDH)/ 0.5 mM NADP, 25°C.G6P, F6P, and M6P were obtained as their sodium salts from Sigma. G6P was assayed spectrophotometrically (340 nm) with NADP (0.3 mM) and G6PDH (bakers' yeast, Sigma) in a buffer containing 0.1 M triethanolamine, 0.1 M KCl, 0.01 M MgCl2 (pH 7.6). F6P and M6P were assayed in the same cuvette by sequentially adding PGI and then phosphomannose isomerase (PMI, yeast, Sigma), recording the A30 endpoint after each addition.Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; M6P, mannose 6-phosphate; PGI, phosphoglucose isomerase; PMI, phosphomannose isomerase; G6PDH, G6P dehydrogenase; kat, catalytic rate constant.

The Crystal Structure of Mouse Phosphoglucose Isomerase at 1.6Å Resolution and its Complex with Glucose 6-Phosphate Reveals the Catalytic Mechanism of Sugar Ring Opening

“…M6P could be positioned so that the proton to be abstracted from C2 was pointing toward the base catalyst, Glu357, suggesting that, at least, abstraction of this proton is possible in PGI. But, in order for the proton to be donated back to C1, M6P must undergo rotation about the C2-C3 bond 11 to position C1 near Glu357. Rotation in one direction is prevented by a clash between the C2 hydroxyl group and Glu357 (clockwise about the C3-C2 bond, viewed from C3).…”

“…In principle, such rotations when coupled with isomerization could generate mannose 6-phosphate. 11 However, PGI is essentially specific for F6P and G6P, and it will not isomerase mannose 6-phosphate to F6P except at an extremely slow and nonphysiological rate. 1 Interestingly, distantly related members of the PGI superfamily from certain Archaea do show dual phosphoglucose/phosphomannose isomerase (PGI/PMI) activities 12 but how "conventional" PGI prevents the epimerisation of G6P to M6P is unknown.…”

Experimental validation of in silico model‐predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi

“…). This residue is pivotal for the catalytic activities of archaeal PGI/PMIs because it stabilizes the formation of crucial enediol intermediates during their PGI activity (Seeholzer, ; Hansen et al ., ; Swan et al ., ). Hence, these changes in the Dm PMI sequence are likely responsible for its inability to function as PGI.…”

Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5‐phospho‐d‐arabinonhydrazide

“…Secondly, it stabilizes the binding of the 1,2-cis-enediolate HEI. Zinc bidentate coordination by O1 and O2 oxygens atoms of the substrates and HEI also clearly explain why Type I PMI, unlike PGI, does not have anomerase [20][21][22]27] nor C2-epimerase [27] activities: rotation about the C1-C2 and C2-C3 bonds, respectively, cannot occur in this bound state. Hence, the CaPMI-5PAHz structure provides a conclusive experimental evidence to support the early hypothesis of Gracy and Noltmann who proposed that the substrate formed a bidentate ligand to the zinc cofactor, one from the carbonyl oxygen, and the second from the adjacent hydroxyl group [1].…”