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

Abstract

Experimental observations of fluoromagnesate and fluoroaluminate complexes of β-phosphoglucomutase (β-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of β-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of β-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.ground-state analogues | enzyme mechanism T he stabilization of transition states (TS) is central to the high reaction rates achieved by enzymes (1-3). Most studies to date have focused on TSs that include a chemical transformation step, where the reacting species have some partial chemical bonding character. However, enzymes also have to avoid substantial barriers associated with nonchemical transformation steps and hence may be required to stabilize other regions of the complex energy surface corresponding to the reaction coordinate. The concept of near attack conformers (NACs) was introduced as a useful tool to help analyze these different roles, by partitioning chemical transformation and nonchemical transformation steps (4). NACs were defined as species where the reactants have an appropriate geometry for the development of the chemical TS (5, 6). Furthermore, the component of the TS barrier ascribed to the chemical TS was proposed to be relatively constant within systems carrying out similar chemistry, on the basis of a linear relationship between the (calculated) population of NACs and the corresponding reaction rates (5). Hence, in free-energy terms, the NAC model apportions the TS barrier (ΔG † ) between a variable conformational component (ΔG NAC ) and an invariant chemical (covalent) component (ΔG chem ; Fig. 1). For some uncatalyzed reactions, it was proposed that a major component of the TS free-energy barrier is the population of NACs, t...

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

“…19 F NMR resonances for these complexes additionally have provided in situ probes for the electronic and protonic environment of the phosphate moiety in the active site (4-6, 15, 16). Such studies have confirmed a trigonal bipyramidal (tbp) TS associated with inline stereochemistry and general acid-base catalysis, following the rearrangement of near-attack conformers (6). By contrast, step 1, involving phosphorylation of the 6-OH group of βG1P, is not well understood.…”

“…It identifies the structural basis for the strong discrimination by βPGM between two, diastereoisomeric α-fluoromethylenephosphonate analogs of β-D-glucose 1-phosphate. Step 2 has been studied intensively, with analyses focused on structural studies of trifluoromagnesate (MgF 3 − ) and tetrafluoroaluminate (AlF 4 − ) transition state analogs (TSAs) and trifluoroberyllate ground state analogs for G6P complexes (4)(5)(6)15). 19 F NMR resonances for these complexes additionally have provided in situ probes for the electronic and protonic environment of the phosphate moiety in the active site (4-6, 15, 16).…”

J-UNIO protocol used for NMR structure determination of the 206-residue protein NP_346487.1 from Streptococcus pneumoniae TIGR4

“…The NMR structure of the cap domain (residues 17–80) shows an antiparallel four-helix bundle with α1, α2, α3 and α4, which is typical for the subfamily I of phosphohydrolases (Griffin et al 2012; Strange et al 2009). A short helix, α5, at the C-terminal end of the cap domain forms a helix–kink–helix motif with α4.…”

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

“…When combined with NMR computations, they also act as indirect reporters of changes in electronic environment experienced by phosphoryl oxygen atoms in transfer reaction TSs [44,61,62]. 19 F NMR resonances display a high degree of dispersion and are calculable with good precision from QM analysis of electronic distribution [37,51,63], showing resonances strongly affected by neighboring H-bond donors. Reduction in the number of H-bond partners generally results in upfield shift of 19 F resonances, as shown clearly in a comparison of the G6P and the 2-deoxy G6P Fig.…”

“…Thus, fluoroberyllates have been used beneficially to study changes in major conformations of proteins by crystallography, NMR, and EM, while studies on ADPÁBeF 3 -have supported investigations on ATPases that drive various mechanical processes at a molecular level, particularly for myosin [31][32][33][34][35][36]. They have proved especially valuable for the identification of near attack conformations (NACs) in enzyme mechanisms, notably for b-phosphoglucomutase (bPGM) [37]. [38,39].…”