Orientation of 1,3-Bisphosphoglycerate Analogs Bound to Phosphoglycerate Kinase

Abstract

We have previously reported dissociation constants for a range of bisphosphonate analogs of 1,3-bisphospho-D-glyceric acid binding to yeast phosphoglycerate kinase. Data for the unsymmetrical analogs were difficult to interpret because it was not clear in which of the two possible orientations these ligands bound. Here we report a novel NMR method for quantifying orientation preference based on relaxation effects induced by titration with CrADP, which is applied to these ligands. It is shown that all ligands can bind in both orientations but that the driving force for the orientational preference is to put the ␣,␣-difluoromethanephosphonate group in the "basic patch" (nontransferable phosphate) position. The relevance to the design of phosphoglycerate kinase inhibitors is discussed.Phosphoglycerate kinase (PGK) 1 (EC 2.7.2.3) is a glycolytic enzyme that catalyzes the following reaction, where MgADP and MgATP represent the magnesium complexes of ADP and ATP, respectively.

Enzyme-catalyzed synthesis of isosteric phosphono-analogues of sugar nucleotides

“…Phosphonates are structural mimics of phosphates that have found utility in probing a diverse array of biological processes including the development of clinical antiretroviral and osteoporosis drugs. 1,2 The replacement of a labile phosphate linkage with a hydrolytically more stable phosphonate 3 has facilitated the study of a variety of enzymes catalyzing phosphate bond cleavage [4][5][6][7][8] including enzymes where a transient cleavage of the phosphate occurs during catalysis. 9 Phosphonates have also been developed as inhibitors of ester hydrolysis 10,11 or as reagents for proteomic profiling of serine proteases.…”

“…First, the phosphonate 4 has a lower turnover efficiency in comparison to 1, and second, the phosphonate 5 has a higher turnover efficiency, relative to 6. It is interesting that a stereochemical change at C4, distant from the reactive site, can partially be countered by modification of the phosphate moiety (5). Potentially, the change in stereochemistry at C4 may perturb the trajectory of the phosphate, and replacing the anomeric oxygen with a methylene unit may partially realign the nucleophile trajectory as a result of the differences in geometry of the phosphonate.…”

Spin-Lattice Relaxation Time in Drug Discovery and Design

“…During several years the main application of the chemical shift perturbation due to paramagnetic metal ions was used to simplify 1 H NMR spectra and now the range of application is continually growing. Recently, these paramagnetic probes have been used as a tool in the refinement of macromolecular structures [78,[83][84][85][86][87], in the structure determination of the family of intrinsically unstructured proteins (IUPs) [88], medium-resolution structures for proteins in the 40-100 kDa range and for the investigation of intermolecular interactions like protein-protein [79,89]; ligand-protein [88,90,91], DNA-protein [92] peptide-micelle [82,93,94] and peptide-protein [84]. Paramagnetic compounds such as TEMPOL and Gd(III)DTPA-BMA can be used to identify highly accessible regions of proteins [95].…”

Interaction of Human 3-Phosphoglycerate Kinase with Its Two Substrates: Is Substrate Antagonism a Kinetic Advantage?

“…From steady-state and equilibrium studies, the dissociation constant (k off ) for bPG is low, probably b50 s − 1 . 4,23 The paucity of information on the kinetics of bPG binding is due to difficulties in working with this unstable substrate (see, for example, Jakeman et al 26 ). Thus, Negelein found that at pH 7.2 and 38°C, bPG decomposed rapidly (half-life, 27 min).…”