Author ORCID Identifier
Doctor of Philosophy
Martin K. Safo
Bisphosphoglycerate mutase (BPGM) is a part of the erythrocyte glycolysis system. Specifically, it is a central enzyme in the Rapoport-Leubering pathway, a side glycolytic pathway involved in the regulation of the concentration of the natural allosteric effector of hemoglobin (Hb), 2,3-bisphosphoglycerate (2,3-BPG). BPGM catalyses the synthesis and hydrolysis of 2,3-BPG through its synthase and phosphatase activities. The synthase activity is the main role of BPGM, while the phosphatase activity is low and is activated by the physiological effector, 2-phosphoglycolate (2-PG) with the latter mechanism poorly understood.
BPGM activity and 2,3-BPG levels in red blood cells (RBCs) have a significant role in sickle cell disease (SCD) pathology. SCD patients experience a constant state of hypoxia that results in increasing the level of 2,3-BPG as a compensatory mechanism to enhance oxygen delivery to tissues. However, the abnormal increase in 2,3-BPG in RBCs of SCD patients exacerbates the disease’s primary pathophysiology, which is the hypoxia-driven deoxygenated-sickle hemoglobin (HbS) polymerization, that in turn leads to RBCs sickling and consequent numerous downstream multi-organ adverse effects.
Reducing the levels of 2,3-BPG by activating BPGM phosphatase activity using 2-PG has been proposed as a potential therapeutic approach for SCD as 2-PG was found to have an anti-sickling property. Nonetheless, the actual activation mechanism of 2-PG on the phosphatase activity or the binding mode of 2-PG to BPGM is not clear. Moreover, no drug screening studies have been performed to identify small molecules against BPGM for therapeutic purposes.
The objectives of this project are to characterize the steady-state kinetics of BPGM synthase and phosphatase activities, understand the mechanism of phosphatase activation, and elucidate the atomic interaction of BPGM with 2-PG and other effectors such as citrate that can provide valuable insight into their mechanism of actions and provide a framework for developing small molecules with potential SCD therapeutic benefit. In addition, we aim to identify ligands that modulate either BPGM phosphatase and/or synthase activity to reduce 2,3-BPG concentration in RBCs.
First, the steady-state kinetics of BPGM synthase and phosphatase activities were characterized using the previously reported coupled spectrophotometric synthase and phosphatase activities assays. These assays were also optimized for drug screening experiments. Both assays have limitations and proved challenging for drug screening. We also employed the colorimetric malachite green assay to study BPGM phosphatase activity, as well as for compound screening.
Next, we elucidated the mechanism of phosphatase activity activation by 2-PG using kinetic and X-ray crystallography studies. The kinetic study showed the mechanism of 2-PG activation of BPGM to be mixed-type of noncompetitive and competitive, suggesting the binding of 2-PG to the active site and to an allosteric or non-catalytic site of the enzyme. The crystal structures of BPGM with 2-PG in the presence and absence of the substrate 2,3-BPG showed binding of the 2-PG and/or 3-PGA (the reaction product of 2,3-BPG) at the expected active site, and at a novel non-catalytic site at the dimer interface, in agreement with the kinetic analysis. The structural studies of BPGM also showed conformational nonequivalence of the two monomeric active sites: one site in a close catalytic conformation, and the second site in an open conformation, with the residues at the entrance of the active site, including Arg100, Arg116, and Arg117, and the C-terminus region disordered, which we propose to be induced by the dimer interface binding.
In order to gain further insight into the BPGM mechanism of action, we also co-crystallized BPGM with citrate, a known BPGM phosphatase inhibitor. The co-crystal structure of BPGM with citrate showed citrate binding to only one of the dimer active sites and to the dimer interface. The kinetic and crystallographic findings suggest for the first time an allosterism or cooperativity across monomers, in which the binding of a ligand at the dimer interface induces negative cooperativity affecting the affinity of ligand binding at the second active site. In the BPGM•citrtate binary complex, an extreme form of negative cooperativity, where half of the site reactivity is observed, shows that only one active site appears to be functional.
Toward the objective of identifying small molecules modulators of BPGM activity for therapeutic purposes, we identified several compounds that target the active site of BPGM using (1) in-house pharmacophore-based virtual screening and molecular docking; (2) machine learning-based molecular screening in collaboration with the pharmaceutical company Atomwise, and (3) PGM1-004A, a known inhibitor of the homologous enzyme, phosphoglycerate mutase 1 (PGAM1). The compounds were tested for their effect on BPGM synthase and phosphatase activities. Unfortunately, the compounds did not show any modulation except for PGMI-004A, which shows a dose-dependent synthase inhibition with IC50 (50±11 µM). Several attempts were made to co-crystallize BPGM with PGMI-004A but were unsuccessful. The novel allosteric site at the dimer interface was also docked against a library of compounds, which identified several potential binders. The top-scoring compounds will be obtained and tested in the near future.
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