The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase
A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymati...
| Main Authors: | , , , , |
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| Format: | Journal Article |
| Language: | English |
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2016
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| Online Access: | https://hdl.handle.net/10356/82139 http://hdl.handle.net/10220/41122 |
| _version_ | 1811689998010810368 |
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| author | Singh, Dhirendra Sielaff, Hendrik Sundararaman, Lavanya Bhushan, Shashi Grüber, Gerhard |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Singh, Dhirendra Sielaff, Hendrik Sundararaman, Lavanya Bhushan, Shashi Grüber, Gerhard |
| author_sort | Singh, Dhirendra |
| collection | NTU |
| description | A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s− 1 and 30.4 s− 1, respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis. |
| first_indexed | 2024-10-01T05:57:00Z |
| format | Journal Article |
| id | ntu-10356/82139 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:57:00Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | ntu-10356/821392023-02-28T17:00:43Z The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase Singh, Dhirendra Sielaff, Hendrik Sundararaman, Lavanya Bhushan, Shashi Grüber, Gerhard School of Biological Sciences A1AO ATP synthase Subunit F A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s− 1 and 30.4 s− 1, respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis. NMRC (Natl Medical Research Council, S’pore) Accepted version 2016-08-11T09:23:22Z 2019-12-06T14:47:28Z 2016-08-11T09:23:22Z 2019-12-06T14:47:28Z 2016 Journal Article Singh, D., Sielaff, H., Sundararaman, L., Bhushan, S., & Grüber, G. (2016). The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1857(2), 177-187. 0005-2728 https://hdl.handle.net/10356/82139 http://hdl.handle.net/10220/41122 10.1016/j.bbabio.2015.12.003 en Biochimica et Biophysica Acta (BBA) - Bioenergetics © 2016 Elsevier B.V. This is the author created version of a work that has been peer reviewed and accepted for publication by Biochimica et Biophysica Acta (BBA) - Bioenergetics, Elsevier B.V. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1016/j.bbabio.2015.12.003]. 36 p. application/pdf |
| spellingShingle | A1AO ATP synthase Subunit F Singh, Dhirendra Sielaff, Hendrik Sundararaman, Lavanya Bhushan, Shashi Grüber, Gerhard The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase |
| title | The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase |
| title_full | The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase |
| title_fullStr | The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase |
| title_full_unstemmed | The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase |
| title_short | The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase |
| title_sort | stimulating role of subunit f in atpase activity inside the a1 complex of the methanosarcina mazei go1 a1ao atp synthase |
| topic | A1AO ATP synthase Subunit F |
| url | https://hdl.handle.net/10356/82139 http://hdl.handle.net/10220/41122 |
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