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Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase

[Image: see text] The side chains of Y208 and S211 from loop 7 of triosephosphate isomerase (TIM) form hydrogen bonds to backbone amides and carbonyls from loop 6 to stabilize the caged enzyme–substrate complex. The effect of seven mutations [Y208T, Y208S, Y208A, Y208F, S211G, S211A, Y208T/S211G] on...

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Autores principales: Zhai, Xiang, Amyes, Tina L., Richard, John P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2015
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694050/
https://www.ncbi.nlm.nih.gov/pubmed/26570983
http://dx.doi.org/10.1021/jacs.5b09328
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author Zhai, Xiang
Amyes, Tina L.
Richard, John P.
author_facet Zhai, Xiang
Amyes, Tina L.
Richard, John P.
author_sort Zhai, Xiang
collection PubMed
description [Image: see text] The side chains of Y208 and S211 from loop 7 of triosephosphate isomerase (TIM) form hydrogen bonds to backbone amides and carbonyls from loop 6 to stabilize the caged enzyme–substrate complex. The effect of seven mutations [Y208T, Y208S, Y208A, Y208F, S211G, S211A, Y208T/S211G] on the kinetic parameters for TIM catalyzed reactions of the whole substrates dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate [(k(cat)/K(m))(GAP) and (k(cat)/K(m))(DHAP)] and of the substrate pieces glycolaldehyde and phosphite dianion (k(cat)/K(HPi)K(GA)) are reported. The linear logarithmic correlation between these kinetic parameters, with slope of 1.04 ± 0.03, shows that most mutations of TIM result in an identical change in the activation barriers for the catalyzed reactions of whole substrate and substrate pieces, so that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The second linear logarithmic correlation [slope = 0.53 ± 0.16] between k(cat) for isomerization of GAP and K(d)(⧧) for phosphite dianion binding to the transition state for wildtype and many mutant TIM-catalyzed reactions of substrate pieces shows that ca. 50% of the wildtype TIM dianion binding energy, eliminated by these mutations, is expressed at the wildtype Michaelis complex, and ca. 50% is only expressed at the wildtype transition state. Negative deviations from this correlation are observed when the mutation results in a decrease in enzyme reactivity at the catalytic site. The main effect of Y208T, Y208S, and Y208A mutations is to cause a reduction in the total intrinsic dianion binding energy, but the effect of Y208F extends to the catalytic site.
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spelling pubmed-46940502016-11-16 Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase Zhai, Xiang Amyes, Tina L. Richard, John P. J Am Chem Soc [Image: see text] The side chains of Y208 and S211 from loop 7 of triosephosphate isomerase (TIM) form hydrogen bonds to backbone amides and carbonyls from loop 6 to stabilize the caged enzyme–substrate complex. The effect of seven mutations [Y208T, Y208S, Y208A, Y208F, S211G, S211A, Y208T/S211G] on the kinetic parameters for TIM catalyzed reactions of the whole substrates dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate [(k(cat)/K(m))(GAP) and (k(cat)/K(m))(DHAP)] and of the substrate pieces glycolaldehyde and phosphite dianion (k(cat)/K(HPi)K(GA)) are reported. The linear logarithmic correlation between these kinetic parameters, with slope of 1.04 ± 0.03, shows that most mutations of TIM result in an identical change in the activation barriers for the catalyzed reactions of whole substrate and substrate pieces, so that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The second linear logarithmic correlation [slope = 0.53 ± 0.16] between k(cat) for isomerization of GAP and K(d)(⧧) for phosphite dianion binding to the transition state for wildtype and many mutant TIM-catalyzed reactions of substrate pieces shows that ca. 50% of the wildtype TIM dianion binding energy, eliminated by these mutations, is expressed at the wildtype Michaelis complex, and ca. 50% is only expressed at the wildtype transition state. Negative deviations from this correlation are observed when the mutation results in a decrease in enzyme reactivity at the catalytic site. The main effect of Y208T, Y208S, and Y208A mutations is to cause a reduction in the total intrinsic dianion binding energy, but the effect of Y208F extends to the catalytic site. American Chemical Society 2015-11-16 2015-12-09 /pmc/articles/PMC4694050/ /pubmed/26570983 http://dx.doi.org/10.1021/jacs.5b09328 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 Zhai, Xiang
Amyes, Tina L.
Richard, John P.
Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase
title Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase
title_full Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase
title_fullStr Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase
title_full_unstemmed Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase
title_short Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase
title_sort role of loop-clamping side chains in catalysis by triosephosphate isomerase
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694050/
https://www.ncbi.nlm.nih.gov/pubmed/26570983
http://dx.doi.org/10.1021/jacs.5b09328
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