Significance of αThr-349 in the catalytic sites of Escherichia coli ATP synthase.
This paper describes the role of α-subunit VISIT-DG sequence residue αThr-349 in the catalytic sites of Escherichia coli F1Fo ATP synthase. X-ray structures show the highly conserved αThr-349 in the proximity (2.68 Å) of the conserved phosphate binding residue βR182 in the phosphate binding subdomain. αT349A, -D, -Q, and -R mutations caused 90-100-fold losses of oxidative phosphorylation and reduced ATPase activity of F1Fo in membranes. Double mutation αT349R/βR182A was able to partially compensate for the absence of known phosphate binding residue βR182. Azide, fluoroaluminate, and fluoroscandium caused insignificant inhibition of αT349A, -D, and -Q mutants, slight inhibition of the αT349R mutant, partial inhibition of the αT349R/βR182A double mutant, and complete inhibition of the wild type. Whereas NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) inhibited wild-type ATPase and its αT349A, -D, -R, and -Q mutants essentially completely, βR182A ATPase and double mutant αT349A/βR182A were inhibited partially. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in the wild type, αT349R, and double mutant αT349R/βR182A but not in αT349A, αT349D, or αT349Q. The results demonstrate that αThr-349 is a supplementary residue involved in phosphate binding and transition state stabilization in ATP synthase catalytic sites through its interaction with βR182.
Department of Biochemistry, Kirksville College of Osteopathic Medicine, A. T. Still University of Health Sciences , Kirksville, Missouri 63501, United States.
Zulfiqar
Z
Ahmad
Mumeenat
M
Winjobi
M Anaul
MA
Kabir
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000535:Aluminum / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology*; D005461:Fluorine / Q000494:pharmacology; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D009578:Nitrobenzenes / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*
C511330:7-chloro-4-nitrobenz-2-oxa-1,3-diazole; D001386:Azides; D004791:Enzyme Inhibitors; D009578:Nitrobenzenes; D010080:Oxazoles; C053629:fluoroaluminum; D005461:Fluorine; D013912:Threonine; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; D000535:Aluminum; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases; D004229:Dithiothreitol