title: Functional importance of αIle-346 and αIle-348 in the catalytic sites of Escherichia coli ATP synthase.
abstract: We studied the functional role of highly conserved VISIT-DG sequence residues αIle-346 and αIle-348 in the catalytic sites of Escherichia coli F1Fo ATP synthase. αIle-346 is in close proximity, 2.98 and 3.63 Å, to the two known phosphate binding residues αR376 and βR182; αIle-348 is situated within 3.66 Å from βR182. Single or double mutants of both αI346 and αI348 resulted in a variable loss of oxidative phosphorylation and ATPase activity. Azide, fluoroaluminate, and fluoroscandium caused insignificant to significant inhibition of mutants. Whereas the wild-type enzyme was completely inhibited by NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole), a variable extent of inhibition was observed for αI346 and αI348 mutants. MgPi protection against NBD-Cl induced inhibition of wild-type, αI346, and αI348 demonstrated that, although strongly conserved, αI346 and αI348 have no direct role in phosphate binding. Insertion of Arginine in the form of αI346R/βR182A, αI346R/αR376A, or αI348R/βR182A was able to compensate for the absence of known phosphate-binding Arginine residues βR182 and αR376. Results also suggest that αIle-346 and αIle-348 seem to have functional importance in upholding the phosphate-binding subdomain and transition state stabilization in the catalytic sites of E. coli ATP synthase.
authors.affiliation: Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, 63501, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, 63501, USA.
authors.forename: Hiba
authors.initials: H
authors.lastname: Syed
authors.affiliation: Department of Anatomy, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, 63501, USA; Faculty of Medicine, Cairo University, Cairo, Egypt.
authors.forename: Sherif S
authors.initials: SS
authors.lastname: Hassan
authors.affiliation: Department of Microbiology & Immunology, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, 63501, USA.
authors.forename: Vineet K
authors.initials: VK
authors.lastname: Singh
authors.affiliation: Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, 63501, USA. Electronic address: zahmad@atsu.edu.
authors.forename: Zulfiqar
authors.initials: Z
authors.lastname: Ahmad
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D010710:Phosphates; D006180:Proton-Translocating ATPases
keywords: ATPase inhibition; Escherichia coli ATP synthase; F(1)F(o) ATP synthase; Oxidative phosphorylation; Pi binding residues