title: The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.
abstract: 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.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Dhirendra
authors.initials: D
authors.lastname: Singh
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Sielaff
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Lavanya
authors.initials: L
authors.lastname: Sundararaman
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Shashi
authors.initials: S
authors.lastname: Bhushan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore. Electronic address: ggrueber@ntu.edu.sg.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D025181:ATP Synthetase Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D017020:Methanosarcina / Q000737:chemistry* / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology; D017384:Sequence Deletion; D013045:Species Specificity
chemical_list: D019843:Archaeal Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D025181:ATP Synthetase Complexes
keywords: A(1)A(O) ATP synthase; ATP synthase; Archaea; Bioenergetics; Methanosarcina mazei Gö1; Subunit F