Anionic Charge Is Prioritized over Geometry in Aluminum and Magnesium Fluoride Transition State Analogs of Phosphoryl Transfer Enzymes

Abstract

Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride.

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

“…Moreover, our experimental results demonstrate that the enzyme overrides the anion-anion repulsion between the fluoroberyllate moiety and the oxygen nucleophile, plus its attendant protein side chain (D10), to such an extent that an almost fully closed conformation (NAC II) is readily populated. This behavior supports the hypothesis that charge balance between the enzyme active site and the substrate is central to accelerated progression along the reaction coordinate (17,24). Finally, this work establishes that, for phosphoryl transfer, the route leading to the formation of a productive, on-enzyme NAC can have other minima (e.g., NAC I) that reduce repulsion between reacting groups, which might otherwise result in obligatory high-energy species on the reaction pathway.…”

“…Phosphoisomerism is achieved through the release of either glucose 6-phosphate (G6P) or β-glucose 1-phosphate (βG1P) from the resulting phosphoenzyme complex, according to Scheme 1. 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).…”

Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF3- rather than by phosphoranes

“…as were used in the solution structure determinations of the PGM-MgF 3 -G6P-TSA and PGM-AlF 4 -G6P-TSA complexes (8,10). Together, these measurements provide a picture of the relationship between the charge distribution of the phosphoryl group transfer mimic and the protein.…”

Shared traits on the reaction coordinates of ribonuclease and an RNA enzyme

“…demonstrate pH sensitivity [7,8], limited solubility [4,8], and may result in partial atomic occupancy (PDB codes 2P7E [9], 1RUV [10] and 1M7G [11]). The relevance of vanadiumoxide binding in the ribonuclease (RNase) A active site [10,12] has been challenged, since amino acids poised for transition-state stabilization did not support roles ascribed by biochemical and kinetic analyses [13].…”