α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction

Abstract

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-Dglucose 1-phosphate (βG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-D-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-D-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼30°between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.phosphonate analogs | phosphoryl transfer mechanism | 19 F NMR | X-ray crystallography | water-mediated substrate recognition E fficient enzyme catalysis of the manipulation of phosphates is one of the great achievements of evolution (1). Enzymes that operate on phosphate monoesters and anhydrides transfer the phosphoryl moiety, PO 3 − , with rate accelerations approaching 10 21 for monoesters, placing them among the most proficient of all enzymes (1). Phosphomutases, including α-phosphoglucomutase (αPGM) (2, 3) and β-phosphoglucomutase (βPGM) (4-6), phosphoglycerate mutase (7), α-phosphomannomutase (αPMM/PGM) (8), and N-acetylglucosamine-phosphate mutase (9), merit special attention because these enzymes have to be effective in donating a phosphoryl group to either of two hydroxyl groups that have intrinsically different reactivity. Only when both half-reactions of a phosphomutase are accessible to mechanistic analysis can the problem of how an enzyme accommodates two distinct chemistries within a single active site be resolved. Hexose 1-phosphate mutases, including enzymes central to glycolysis and other metabolic pathways, are well characterized (10, 11). They are generally activated by phosphorylation to form a covalent phosphoenzyme, which then donates its PO 3 − group to either of its substrates to deliver a common, transient, hexose 1,6-bisphosphate intermediate species. However, structural studies on phosphomutases are complicated by the rapid and often imbalanced equilibrium position between the substrates, and kinetic studies are problematic because of competitive, parallel pathways of enzyme activation and substrate inhibition (12, ...

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

“…The observation of MgF 3 -metal fluoride complexes for other phospho-transfer enzymes including beta-phosphoglucomutase (Jin et al 2014) and phosphoglycerate kinase ) led us to consider whether this mode of inhibition may explain the previously observed inhibition of PEPs by fluoride (Cooper and Kornberg 1965). is the concentration of the inhibitor and K i is the inhibition constant.…”

“…The use of metal fluoride complexes has led to recent advances in elucidating the mechanisms of enzymes which form phospho-4 Asp and phospho-Arg intermediates, including β-phosphoglucomutase (Jin et al 2014;Baxter et al 2006;Kagawa et al 2004;Lee and Yang 2006;Graham et al 2002).…”

“…Phosphoryl transfer transition state analogues for β-phosphoglucomutase, in complex with metal fluorides, were observed by 19 F NMR and X-ray crystallography through the synthesis of fluorine containing phosphonate-based chemical probes (Jin et al 2014).…”

Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes

“…14a, b) [44,50,51,59,60]. In the context of TSAs and GSAs, the chemical shifts of 19 F resonances provide a key measure of interactions between MF x moieties and their protein hosts, and report the electronic environment of the fluorine nuclei.…”

“…In those cases, presaturation of the free fluoride resonance at -120 ppm can be applied to eliminate the unbound metal fluoride signals complexes of bPGM (-18.1 ppm) [53]. In general, the resonance of the fluorine coordinated to a catalytic magnesium is always the most upfield, because of depletion of H-bond coordination [44,45,50,51,53,60,62]. Proton distribution near fluorine nuclei can be further assessed through the quantitation of 19 F-1 H NOEs using perdeuterated enzyme in protonated buffer to suppress the 1 H-1 H spin diffusion [49,51], while resonance assignment of exchangeable 1 H nuclei in the protein enables unambiguous assignment of individual 19 F NMR resonances.…”