Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation
Citations Over TimeTop 12% of 2022 papers
Abstract
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.
Related Papers
- → Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced inEscherichia coli(2013)8 cited
- → Molecular dynamics simulations of active site mutants of triosephosphate isomerase(1990)17 cited
- → Multiple forms in the subunit structure of concanavalin A(1971)56 cited
- → A nonenzyme-coupled assay for triosephosphate isomerase based upon circular dichroism of glyceraldehyde-3-phosphate(1974)6 cited
- → Triosephosphate isomerase from young and old Turbatrix aceti(1976)39 cited