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Enzyme Architecture: Optimization of Transition State Stabilization from a Cation–Phosphodianion Pair
[Image: see text] The side chain cation of R269 lies at the surface of l-glycerol 3-phosphate dehydrogenase (GPDH) and forms an ion pair to the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), which is buried at the nonpolar protein interior. The R269A mutation of GPDH results in a 110...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2015
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416717/ https://www.ncbi.nlm.nih.gov/pubmed/25884759 http://dx.doi.org/10.1021/jacs.5b02202 |
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author | Reyes, Archie C. Koudelka, Astrid P. Amyes, Tina L. Richard, John P. |
author_facet | Reyes, Archie C. Koudelka, Astrid P. Amyes, Tina L. Richard, John P. |
author_sort | Reyes, Archie C. |
collection | PubMed |
description | [Image: see text] The side chain cation of R269 lies at the surface of l-glycerol 3-phosphate dehydrogenase (GPDH) and forms an ion pair to the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), which is buried at the nonpolar protein interior. The R269A mutation of GPDH results in a 110-fold increase in K(m) (2.8 kcal/mol effect) and a 41 000-fold decrease in k(cat) (6.3 kcal/mol effect), which corresponds to a 9.1 kcal/mol destabilization of the transition state for GPDH-catalyzed reduction of DHAP by NADH. There is a 6.7 kcal/mol stabilization of the transition state for the R269A mutant GPDH-catalyzed reaction by 1.0 M guanidinium ion, and the transition state for the reaction of the substrate pieces is stabilized by an additional 2.4 kcal/mol by their covalent attachment at wildtype GPDH. These results provide strong support for the proposal that GPDH invests the 11 kcal/mol intrinsic phosphodianion binding energy of DHAP in trapping the substrate at a nonpolar active site, where strong electrostatic interactions are favored, and obtains a 9 kcal/mol return from stabilizing interactions between the side chain cation and transition state trianion. We propose a wide propagation for the catalytic motif examined in this work, which enables strong transition state stabilization from enzyme–phosphodianion pairs. |
format | Online Article Text |
id | pubmed-4416717 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-44167172016-04-17 Enzyme Architecture: Optimization of Transition State Stabilization from a Cation–Phosphodianion Pair Reyes, Archie C. Koudelka, Astrid P. Amyes, Tina L. Richard, John P. J Am Chem Soc [Image: see text] The side chain cation of R269 lies at the surface of l-glycerol 3-phosphate dehydrogenase (GPDH) and forms an ion pair to the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), which is buried at the nonpolar protein interior. The R269A mutation of GPDH results in a 110-fold increase in K(m) (2.8 kcal/mol effect) and a 41 000-fold decrease in k(cat) (6.3 kcal/mol effect), which corresponds to a 9.1 kcal/mol destabilization of the transition state for GPDH-catalyzed reduction of DHAP by NADH. There is a 6.7 kcal/mol stabilization of the transition state for the R269A mutant GPDH-catalyzed reaction by 1.0 M guanidinium ion, and the transition state for the reaction of the substrate pieces is stabilized by an additional 2.4 kcal/mol by their covalent attachment at wildtype GPDH. These results provide strong support for the proposal that GPDH invests the 11 kcal/mol intrinsic phosphodianion binding energy of DHAP in trapping the substrate at a nonpolar active site, where strong electrostatic interactions are favored, and obtains a 9 kcal/mol return from stabilizing interactions between the side chain cation and transition state trianion. We propose a wide propagation for the catalytic motif examined in this work, which enables strong transition state stabilization from enzyme–phosphodianion pairs. American Chemical Society 2015-04-17 2015-04-29 /pmc/articles/PMC4416717/ /pubmed/25884759 http://dx.doi.org/10.1021/jacs.5b02202 Text en Copyright © 2015 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Reyes, Archie C. Koudelka, Astrid P. Amyes, Tina L. Richard, John P. Enzyme Architecture: Optimization of Transition State Stabilization from a Cation–Phosphodianion Pair |
title | Enzyme
Architecture: Optimization of Transition State
Stabilization from a Cation–Phosphodianion Pair |
title_full | Enzyme
Architecture: Optimization of Transition State
Stabilization from a Cation–Phosphodianion Pair |
title_fullStr | Enzyme
Architecture: Optimization of Transition State
Stabilization from a Cation–Phosphodianion Pair |
title_full_unstemmed | Enzyme
Architecture: Optimization of Transition State
Stabilization from a Cation–Phosphodianion Pair |
title_short | Enzyme
Architecture: Optimization of Transition State
Stabilization from a Cation–Phosphodianion Pair |
title_sort | enzyme
architecture: optimization of transition state
stabilization from a cation–phosphodianion pair |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416717/ https://www.ncbi.nlm.nih.gov/pubmed/25884759 http://dx.doi.org/10.1021/jacs.5b02202 |
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