Dephosphorylation of 2,3-bisphosphoglycerate by MIPP expands the regulatory capacity of the Rapoport–Luebering glycolytic shunt

Abstract

The Rapoport-Luebering glycolytic bypass comprises evolutionarily conserved reactions that generate and dephosphorylate 2,3-bisphosphoglycerate (2,3-BPG). For >30 years, these reactions have been considered the responsibility of a single enzyme, the 2,3-BPG synthase/2-phosphatase (BPGM). Here, we show that Dictyostelium, birds, and mammals contain an additional 2,3-BPG phosphatase that, unlike BPGM, removes the 3-phosphate. This discovery reveals that the glycolytic pathway can bypass the formation of 3-phosphoglycerate, which is a precursor for serine biosynthesis and an activator of AMP-activated protein kinase. Our 2,3-BPG phosphatase activity is encoded by the previously identified gene for multiple inositol polyphosphate phosphatase (MIPP1), which we now show to have dual substrate specificity. By genetically manipulating Mipp1 expression in Dictyostelium, we demonstrated that this enzyme provides physiologically relevant regulation of cellular 2,3-BPG content. Mammalian erythrocytes possess the highest content of 2,3-BPG, which controls oxygen binding to hemoglobin. We determined that total MIPP1 activity in erythrocytes at 37°C is 0.6 mmol 2,3-BPG hydrolyzed per liter of cells per h, matching previously published estimates of the phosphatase activity of BPGM. MIPP1 is active at 4°C, revealing a clinically significant contribution to 2,3-BPG loss during the storage of erythrocytes for transfusion. Hydrolysis of 2,3-BPG by human MIPP1 is sensitive to physiologic alkalosis; activity decreases 50% when pH rises from 7.0 to 7.4. This phenomenon provides a homeostatic mechanism for elevating 2,3-BPG levels, thereby enhancing oxygen release to tissues. Our data indicate greater biological significance of the Rapoport-Luebering shunt than previously considered.2,3-BPG ͉ erythrocyte ͉ glycolysis T he Rapoport-Luebering glycolytic shunt generates and dephosphorylates 2,3-bisphosphoglycerate (2,3-BPG). These reactions have been considered to be catalyzed by a single 2,3-BPG synthase/2-phosphatase (BPGM) (1, 2). This enzyme has mutase activity that converts the glycolytic intermediate, 1,3-BPG, to 2,3-BPG. BPGM also acts as a phosphatase, converting 2,3-BPG to 3-phosphoglycerate (3-PG), which then reenters the main glycolytic pathway. This has been the textbook perception of this metabolic pathway for Ͼ25 years (3).Metabolic flux through the Rapoport-Luebering shunt carries an energetic cost for the cell because it bypasses the ATPgenerating phosphoglycerate kinase. Nevertheless, in vertebrates and lower eukaryotes, 2,3-BPG fulfills an essential role in glycolysis by priming the phosphoglycerate mutase reaction that converts 3-PG to 2-PG (4). More recently, experiments in Dictyostelium have suggested that 2,3-BPG, through an unknown mechanism, provides a molecular link between the turnover of phosphorylated inositol derivatives and glycolytic flux (5). Nonetheless, the most well studied function for 2,3-BPG is its regulation of whole-body oxygen homeostasis; 2,3-BPG executes this role because it is the main allosteri...

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