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Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate
[Image: see text] l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic m...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238798/ https://www.ncbi.nlm.nih.gov/pubmed/25330457 http://dx.doi.org/10.1021/bi501113u |
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author | Sirithanakorn, Chaiyos Adina-Zada, Abdussalam Wallace, John C. Jitrapakdee, Sarawut Attwood, Paul V. |
author_facet | Sirithanakorn, Chaiyos Adina-Zada, Abdussalam Wallace, John C. Jitrapakdee, Sarawut Attwood, Paul V. |
author_sort | Sirithanakorn, Chaiyos |
collection | PubMed |
description | [Image: see text] l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that l-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. l-Aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of l-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of l-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. l-Aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, l-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and l-aspartate) are spatially distinct but connected by a network of allosteric interactions. |
format | Online Article Text |
id | pubmed-4238798 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-42387982015-10-20 Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate Sirithanakorn, Chaiyos Adina-Zada, Abdussalam Wallace, John C. Jitrapakdee, Sarawut Attwood, Paul V. Biochemistry [Image: see text] l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that l-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. l-Aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of l-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of l-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. l-Aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, l-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and l-aspartate) are spatially distinct but connected by a network of allosteric interactions. American Chemical Society 2014-10-20 2014-11-18 /pmc/articles/PMC4238798/ /pubmed/25330457 http://dx.doi.org/10.1021/bi501113u Text en Copyright © 2014 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 | Sirithanakorn, Chaiyos Adina-Zada, Abdussalam Wallace, John C. Jitrapakdee, Sarawut Attwood, Paul V. Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate |
title | Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate |
title_full | Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate |
title_fullStr | Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate |
title_full_unstemmed | Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate |
title_short | Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate |
title_sort | mechanisms of inhibition of rhizobium etli pyruvate carboxylase by l-aspartate |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238798/ https://www.ncbi.nlm.nih.gov/pubmed/25330457 http://dx.doi.org/10.1021/bi501113u |
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