title: Role of {alpha}-subunit VISIT-DG sequence residues Ser-347 and Gly-351 in the catalytic sites of Escherichia coli ATP synthase.
abstract: This paper describes the role of alpha-subunit VISIT-DG sequence residues alphaSer-347 and alphaGly-351 in catalytic sites of Escherichia coli F(1)F(o) ATP synthase. X-ray structures show the very highly conserved alpha-subunit VISIT-DG sequence in close proximity to the conserved phosphate-binding residues alphaArg-376, betaArg-182, betaLys-155, and betaArg-246 in the phosphate-binding subdomain. Mutations alphaS347Q and alphaG351Q caused loss of oxidative phosphorylation and reduced ATPase activity of F(1)F(o) in membranes by 100- and 150-fold, respectively, whereas alphaS347A mutation showed only a 13-fold loss of activity and also retained some oxidative phosphorylation activity. The ATPase of alphaS347Q mutant was not inhibited, and the alphaS347A mutant was slightly inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium, in contrast to wild type and alphaG351Q mutant. Whereas 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) inhibited wild type and alphaG351Q mutant ATPase essentially completely, ATPase in alphaS347A or alphaS347Q mutant was inhibited maximally by approximately 80-90%, although reaction still occurred at residue betaTyr-297, proximal to the alpha-subunit VISIT-DG sequence, near the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts inbetaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type and alphaG351Q mutant but not in alphaS347Q or alphaS347A mutant. The results demonstrate that alphaSer-347 is an additional residue involved in phosphate-binding and transition state stabilization in ATP synthase catalytic sites. In contrast, alphaGly-351, although strongly conserved and clearly important for function, appears not to play a direct role.
authors.affiliation: Department of Biological Sciences, East Tennessee State University, Johnson City, Tennessee 37614, USA.
authors.forename: Wenzong
authors.initials: W
authors.lastname: Li
authors.forename: Laura E
authors.initials: LE
authors.lastname: Brudecki
authors.forename: Alan E
authors.initials: AE
authors.lastname: Senior
authors.forename: Zulfiqar
authors.initials: Z
authors.lastname: Ahmad
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D025181:ATP Synthetase Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D006801:Humans; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D012694:Serine; D004024:Dicyclohexylcarbodiimide; D025181:ATP Synthetase Complexes; D009327:4-Chloro-7-nitrobenzofurazan; D004229:Dithiothreitol; D005998:Glycine