| Autor: |
Schachner LF; Department of Chemistry, the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States., Soye BD; Department of Chemistry, the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States.; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States., Ro S; Department Molecular and Biological Sciences, Northwestern University, Evanston, Illinois 60208, United States., Kenney GE; Department Molecular and Biological Sciences, Northwestern University, Evanston, Illinois 60208, United States.; Department of Chemistry, Harvard University, Cambridge, Massachusetts 02140, United States., Ives AN; Department of Chemistry, the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States., Su T; Department of Chemistry, the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States., Goo YA; Department of Chemistry, the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States., Jewett MC; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States., Rosenzweig AC; Department Molecular and Biological Sciences, Northwestern University, Evanston, Illinois 60208, United States., Kelleher NL; Department of Chemistry, the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States.; Department Molecular and Biological Sciences, Northwestern University, Evanston, Illinois 60208, United States. |
| Abstrakt: |
Triosephosphate isomerase (TPI) performs the 5th step in glycolysis, operates near the limit of diffusion, and is involved in "moonlighting" functions. Its dimer was found singly phosphorylated at Ser20 (pSer20) in human cells, with this post-translational modification (PTM) showing context-dependent stoichiometry and loss under oxidative stress. We generated synthetic pSer20 proteoforms using cell-free protein synthesis that showed enhanced TPI activity by 4-fold relative to unmodified TPI. Molecular dynamics simulations show that the phosphorylation enables a channel to form that shuttles substrate into the active site. Refolding, kinetic, and crystallographic analyses of point mutants including S20E/G/Q indicate that hetero-dimerization and subunit asymmetry are key features of TPI. Moreover, characterization of an endogenous human TPI tetramer also implicates tetramerization in enzymatic regulation. S20 is highly conserved across eukaryotic TPI, yet most prokaryotes contain E/D at this site, suggesting that phosphorylation of human TPI evolved a new switch to optionally boost an already fast enzyme. Overall, complete characterization of TPI shows how endogenous proteoform discovery can prioritize functional versus bystander PTMs. |
