Role of {alpha}-subunit VISIT-DG sequence residues Ser-347 and Gly-351 in the catalytic sites of Escherichia coli ATP synthase

• Published in: The Journal of biological chemistry 2009-04, Vol.284 (16), p.10747-10754
• Main Authors: Li, Wenzong, Brudecki, Laura E, Senior, Alan E, Ahmad, Zulfiqar
• Format: Article
• Language: English
• Subjects: 4-Chloro-7-nitrobenzofurazan - metabolism; Amino Acid Sequence; Animals; ATP Synthetase Complexes - antagonists & inhibitors; ATP Synthetase Complexes - chemistry; ATP Synthetase Complexes - genetics; ATP Synthetase Complexes - metabolism; Base Sequence; Catalytic Domain; Dicyclohexylcarbodiimide - metabolism; Dithiothreitol - metabolism; Enzyme Inhibitors - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Glycine - metabolism; Humans; Molecular Conformation; Molecular Sequence Data; Mutation; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Sequence Alignment; Serine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M809209200

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.