The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase

• Main Authors: Singh, Dhirendra, Sielaff, Hendrik, Sundararaman, Lavanya, Bhushan, Shashi, Grüber, Gerhard
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2016
• Subjects: A1AO ATP synthase; Subunit F
• Online Access: https://hdl.handle.net/10356/82139; http://hdl.handle.net/10220/41122
• Institution: Nanyang Technological University

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.