Mutagenesis of residue betaArg-246 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F1-ATPase.
Residues responsible for phosphate binding in F(1)F(0)-ATP synthase catalytic sites are of significant interest because phosphate binding is believed linked to proton gradient-driven subunit rotation. From x-ray structures, a phosphate-binding subdomain is evident in catalytic sites, with conserved betaArg-246 in a suitable position to bind phosphate. Mutations betaR246Q, betaR246K, and betaR246A in Escherichia coli were found to impair oxidative phosphorylation and to reduce ATPase activity of purified F(1) by 100-fold. In contrast to wild type, ATPase of mutants was not inhibited by MgADP-fluoroaluminate or MgADP-fluoroscandium, showing the Arg side chain is required for wild-type transition state formation. Whereas 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by approximately 50%, although reaction still occurred at residue betaTyr-297, proximal to betaArg-246 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in betaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type but not in mutants. The results show that phosphate can bind in the betaE catalytic site of E. coli F(1) and that betaArg-246 is an important phosphate-binding residue.
Ahmad
Zulfiqar
Z
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
Senior
Alan E
AE
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D001386:Azides / Q000494:pharmacology; D025243:Bacterial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D017434:Protein Structure, Tertiary
D001386:Azides; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D025243:Bacterial Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan