Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation

Luis F. Schachner; Benjamin Des Soye; Soo Ro; Grace E. Kenney; Ashley N. Ives; Taojunfeng Su; Young Ah Goo; Michael C. Jewett; Amy C. Rosenzweig; Neil L. Kelleher

doi:10.1021/acschembio.2c00324

Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation

Luis F. Schachner, Benjamin Des Soye, Soo Ro, Grace E. Kenney, Ashley N. Ives, Taojunfeng Su, Young Ah Goo, Michael C. Jewett, Amy C. Rosenzweig, Neil L. Kelleher^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

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.

Original language	English (US)
Pages (from-to)	2769-2780
Number of pages	12
Journal	ACS chemical biology
Volume	17
Issue number	10
DOIs	https://doi.org/10.1021/acschembio.2c00324
State	Published - Oct 21 2022

Funding

This work was supported by the National Institute of General Medical Sciences P41 GM108569 for the National Resource for Translational and Developmental Proteomics at Northwestern University and NIH grants S10OD025194 and RF1AG063903 (Kelleher lab). L.F.S. is a Gilliam Fellow of the Howard Hughes Medical Institute. Research in this publication is also supported by Thermo Fisher Scientific and a fellowship associated with the Chemistry of Life Processes Predoctoral Training Grant T32GM105538 at Northwestern University. We acknowledge R. Mishra and CMIDD for molecular dynamics simulation work. M.C.J. gratefully acknowledges the Army Research Office Grants W911NF-16-1-0372 and W911NF-18-1-0200, the David and Lucile Packard Foundation and the Camille Dreyfus Teacher–Scholar Program. KD-SepRS-EFSep-5x tRNASep (B40 OTS) was a gift from J. Rinehart (Addgene plasmid # 52054; http://n2t.net/addgene:52054 ; RRID:Addgene_52054).

ASJC Scopus subject areas

Biochemistry
Molecular Medicine

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10.1021/acschembio.2c00324

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@article{4eac6078a59c42afa1f512fa2521ffdf,

title = "Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation",

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.",

author = "Schachner, {Luis F.} and Soye, {Benjamin Des} and Soo Ro and Kenney, {Grace E.} and Ives, {Ashley N.} and Taojunfeng Su and Goo, {Young Ah} and Jewett, {Michael C.} and Rosenzweig, {Amy C.} and Kelleher, {Neil L.}",

note = "Funding Information: This work was supported by the National Institute of General Medical Sciences P41 GM108569 for the National Resource for Translational and Developmental Proteomics at Northwestern University and NIH grants S10OD025194 and RF1AG063903 (Kelleher lab). L.F.S. is a Gilliam Fellow of the Howard Hughes Medical Institute. Research in this publication is also supported by Thermo Fisher Scientific and a fellowship associated with the Chemistry of Life Processes Predoctoral Training Grant T32GM105538 at Northwestern University. We acknowledge R. Mishra and CMIDD for molecular dynamics simulation work. M.C.J. gratefully acknowledges the Army Research Office Grants W911NF-16-1-0372 and W911NF-18-1-0200, the David and Lucile Packard Foundation and the Camille Dreyfus Teacher–Scholar Program. KD-SepRS-EFSep-5x tRNASep (B40 OTS) was a gift from J. Rinehart (Addgene plasmid # 52054; http://n2t.net/addgene:52054 ; RRID:Addgene_52054). Publisher Copyright: {\textcopyright} 2022 American Chemical Society. All rights reserved.",

year = "2022",

month = oct,

day = "21",

doi = "10.1021/acschembio.2c00324",

language = "English (US)",

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T1 - Revving an Engine of Human Metabolism

T2 - Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation

AU - Schachner, Luis F.

AU - Soye, Benjamin Des

AU - Ro, Soo

AU - Kenney, Grace E.

AU - Ives, Ashley N.

AU - Su, Taojunfeng

AU - Goo, Young Ah

AU - Jewett, Michael C.

AU - Rosenzweig, Amy C.

AU - Kelleher, Neil L.

N1 - Funding Information: This work was supported by the National Institute of General Medical Sciences P41 GM108569 for the National Resource for Translational and Developmental Proteomics at Northwestern University and NIH grants S10OD025194 and RF1AG063903 (Kelleher lab). L.F.S. is a Gilliam Fellow of the Howard Hughes Medical Institute. Research in this publication is also supported by Thermo Fisher Scientific and a fellowship associated with the Chemistry of Life Processes Predoctoral Training Grant T32GM105538 at Northwestern University. We acknowledge R. Mishra and CMIDD for molecular dynamics simulation work. M.C.J. gratefully acknowledges the Army Research Office Grants W911NF-16-1-0372 and W911NF-18-1-0200, the David and Lucile Packard Foundation and the Camille Dreyfus Teacher–Scholar Program. KD-SepRS-EFSep-5x tRNASep (B40 OTS) was a gift from J. Rinehart (Addgene plasmid # 52054; http://n2t.net/addgene:52054 ; RRID:Addgene_52054). Publisher Copyright: © 2022 American Chemical Society. All rights reserved.

PY - 2022/10/21

Y1 - 2022/10/21

N2 - 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.

AB - 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.

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Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation

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