Abstract
Abstract Pyruvate kinase (PK), a crucial enzyme in glycolysis, catalyzes the transfer of phosphate from Phosphoenolpyruvic acid (PEP) to Adenosine diphosphate (ADP) to produce pyruvate, which is further synthesized into acetyl-coA and enters into Tricarboxylic acid cycle (TCA cycle) for high-energy production in normal cells. In cancer cell, an isoform Pyruvate kinase muscle 2 (PKM2) is frequently overexpressed; it is now recognized that PKM2 is a key molecule involved in Warburg effect, or aerobic glycolysis. Even in the presence of sufficient oxygen, the product pyruvate primarily shifts to the production of lactic acid by lactate dehydrogenase. PKM2 is a less efficient isoform of pyruvate kinase which is thought to lead to the accumulation of glycolytic intermediates for rapid cell growth. Structures of PKM2 show conformational change with or without that the allosteric effector fructose 1, 6-biphosphate (FBP), in which the FBP-binding complex stays at an active R-state conformer. H391Y and K422R are natural variants found from Bloom syndrome patients, prone to developing cancer. R399E, an artificial mutant is also reported to lead to the development of cancer. In this study, we aimed to understand the structure-activity relationship of these variants in allosterically modulate enzymatic activity. We have determined H391Y and R399E structure, showing an overall R-state-like conformation. Notably, Tyr 391 could strongly hydrogen-bond with Glu 386 which could prevent alpha-helix translation in opposition to WT with phenylalanine. R399E stays in loose oligomeric conformation, as a result of fewer contacts between Glu 399 with the neighbor subunit (Glu 399 makes 2 H bonds with Glu 418 in the neighbor subunit as opposed to 4 H bonds between Arg 399 and Glu 418 in the WT enzyme). The lower allosteric effect by FBP and serine seen in R399E supports the contribution of oligomeric interactions to allosteric modulation. Our results demonstrate that PKM2 variants H391Y and R399E affects the modulation of allosteric effect and oligomeric interactions, which may further contribute to disease development.