Abstract
Molecular details for the timing and role of proton transfer in phosphoryl transfer reactions are poorly understood. Here, we have combined QM models, experimental NMR measurements, and X-ray structures to establish that the transition of an archetypal phosphoryl transfer enzyme, βPGM, from a very closed near-attack conformation to a fully closed transition state analogue (TSA) conformation triggers both partial proton transfer from the general acid–base residue to the leaving group oxygen and partial dissociation of the transferring phosphoryl group from the leaving group oxygen. Proton transfer continues but is not completed throughout the reaction path of the phosphoryl transfer with the enzyme in the TSA conformation. Moreover, using interacting quantum atoms (IQA) and relative energy gradient (REG) analysis approaches, we observed that the change in the position of the proton and the corresponding increased electrostatic repulsion between the proton and the phosphorus atom provide a stimulus for phosphoryl transfer in tandem with a reduction in the negative charge density on the leaving group oxygen atom. The agreement between solution-phase 19F NMR measurements and equivalent QM models of βPGMWT and βPGMD10N TSA complexes confirms the protonation state of G6P in the two variants, validating the employed QM models. Furthermore, QM model predictions of an AlF4 distortion in response to the proton position are confirmed using high resolution X-ray crystal structures, not only providing additional validation to the QM models but also further establishing metal fluorides as highly sensitive experimental predictors of active-site charge density distributions.
Highlights
Enzyme-catalyzed transfer of phosphoryl groups is a central process in almost all biological processes in all kingdoms of life.[1]
To guide the fixed boundary positions of this QMWT PO3 model, NMR-derived order parameters (S2 values) were determined for the backbone amides in the βPGMWT−AlF4−glucose 6-phosphate (G6P) complex in solution under conditions reported for its backbone resonance assignment (BioMagResBank (BMRB) 15467).[10]
Residue and the promotion of phosphoryl transfer. Both these processes are assisted by the transition of the enzyme from the near attack complexes (NACs) III conformation to the NAC II or transition state analogue (TSA) conformation via a 13° relative rotation of the cap and core domains, which is in line with the βPGM−βG16BP complex (PDB 5OK1) preferring to adopt the NAC III conformation.[18]
Summary
Enzyme-catalyzed transfer of phosphoryl groups is a central process in almost all biological processes in all kingdoms of life.[1]. Which the phosphorus atom of the 1-phosphate group of βG16BP is in van der Waals contact with the nucleophilic carboxylate oxygen of D8; the enzyme has not achieved full domain closure This observation demonstrated that by disfavoring proton transfer from the GAB residue to the bridging oxygen of βG16BP (the leaving group oxygen in this scenario), the phosphate prefers to remain bonded to the sugar and the enzyme does not adopt the conformation that supports the chemical TS. We have combined QM models, X-ray structures, and NMR measurements to establish that the transition of the βPGM−βG16BP complex from the NAC III to the NAC II or TSA conformation delivers full domain closure, which triggers partial proton transfer from the GAB residue to the leaving group oxygen. This provides additional validation to the QM models and further establishes metal fluorides as highly sensitive predictors of active-site charge density distributions
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