Transition State Analogue Structures of Human Phosphoglycerate Kinase Establish the Importance of Charge Balance in Catalysis

Abstract

Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF(3)(-)) and tetrafluoroaluminate (AlF(4)(-)) complexes of PGK have been determined using X-ray crystallography and (19)F-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the alpha-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF(3)(0)) complex. The AlF(3)(0) moiety retains the octahedral geometry observed within AlF(4)(-) TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF(3)(0) complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF(3)(-).

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

“…Studies on hPGK have validated this concept by demonstrating that hPGK prioritizes anionic charge over geometry in selection of MFx for TSA complex formation. [60] Based on the geometry of MFx complexes for a wide range of PTx enzymes, it was demonstrated that charge balance is maintained within a sphere of up to 15 Å around the transferring phosphorus even when that borders on bulk water (Fig. 19B).…”

“…19B). [60] A classic example is that of cAPK where charge balance is only achieved by the incursion of the substrate peptide with three +ve charges into a 13.5 Å sphere (Fig. 19A).…”

Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches

“…A single resonance was observed for the AlF 4 − complex ( δ average =−143.6 ppm, Figure S2) due to fast exchange of the four fluorines between the three binding sites for the oxygens of PO 3 − 1b,1d,1g. Compared with WT, the small upfield chemical shift change (1.6 ppm) and marginal fall in average solvent‐induced isotope shift (SIIS) value (0.2 ppm) for the Arg85′Ala complex indicate that loss of the arginine finger leads to a reduction in the overall tightness of coordination of AlF 4 − (Figure S2).…”

Near attack conformers dominate β-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate

“…Work to date has focused on probing the reaction mechanism of β-PGM through the introduction of metal fluoride transition-state analogs (TSAs) and computational analysis based on the derived structural data (15)(16)(17)(18)(19)(20)(21). In particular, trifluoromagnesate (MgF 3 − ) has been established as a very close steric and electronic mimic of the planar PO 3 − group (16,20,(22)(23)(24) that represents the energy maximum as phosphorus moves through the plane of the three PO 3 − oxygen atoms (15,21). In addition to providing atomic resolution structures of β-PGM in a near TS conformation, the introduction of metal fluorides allows 19 F NMR to provide measures of the electronic and protonic environment at the catalytic center (20).…”