PreprintArticleVersion 1Preserved in Portico This version is not peer-reviewed
Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase
Version 1
: Received: 25 April 2024 / Approved: 26 April 2024 / Online: 28 April 2024 (08:43:50 CEST)
How to cite:
Anashkin, V.A.; Kirillova, E.A.; Orlov, V.N.; Baykov, A.A. Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase. Preprints2024, 2024041753. https://doi.org/10.20944/preprints202404.1753.v1
Anashkin, V.A.; Kirillova, E.A.; Orlov, V.N.; Baykov, A.A. Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase. Preprints 2024, 2024041753. https://doi.org/10.20944/preprints202404.1753.v1
Anashkin, V.A.; Kirillova, E.A.; Orlov, V.N.; Baykov, A.A. Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase. Preprints2024, 2024041753. https://doi.org/10.20944/preprints202404.1753.v1
APA Style
Anashkin, V.A., Kirillova, E.A., Orlov, V.N., & Baykov, A.A. (2024). Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase. Preprints. https://doi.org/10.20944/preprints202404.1753.v1
Chicago/Turabian Style
Anashkin, V.A., Victor N Orlov and Alexander A. Baykov. 2024 "Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase" Preprints. https://doi.org/10.20944/preprints202404.1753.v1
Abstract
Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the “half-of-the-sites” ligand binding stoichiometry. Seven amino acid residues were selected in CBS1 domain based on the available X-ray structure of the regulatory domains and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP activator at low concentrations but inhibitor at high concentrations. Replacements of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding twofold. These findings identify several key protein residues and suggest “two non-interacting pairs of interacting regulatory sites” concept in CBS-PPase regulation.
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
