The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Go1 A1Ao ATP synthase

A(1)A(o) ATP synthases couple ion-transport of the A(o) sector and ATP synthesis/hydrolysis of the A(3)B(3)-headpiece via their stalk subunits D and F. Here, we produced and purified stable A(3)B(3)D- and A(3)B(3)DF-complexes of the Methanosarcina mazei Go1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei GM A-ATP synthase subunit F is an ATPase activating subunit The maximum ATP hydrolysis rates (V-max) of A(3)B(3)D and A(3)B(3)DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a V-max 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 A(3)B(3)D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunits epsilon of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis. (C) 2015 Elsevier B.V. All rights reserved.