title: The (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 has altered ATPase activity after cross-linking alpha and beta subunits at noncatalytic site interfaces.
abstract: In crystal structures of bovine MF(1), the side chains of alpha F(357) and beta R(372) are near the adenines of nucleotides bound to noncatalytic sites. To determine if during catalysis these side chains must pass through the different arrangements in which they are present in crystal structures, the catalytic properties of the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the TF(1)-ATPase were characterized before and after cross-linking the introduced cysteines with CuCl(2). The unmodified mutant enzyme hydrolyzes MgATP at 50% the rate exhibited by wild type. Detailed comparison of the catalytic properties of the double mutant enzyme before and after cross-linking with those of the wild-type subcomplex revealed the following. Before cross-linking, the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex has less tendency than wild type to release inhibitory MgADP entrapped in a catalytic site during turnover when MgATP binds to noncatalytic sites. Following cross-linking, ATPase activity is reduced 5-fold, and inhibitory MgADP entrapped in a catalytic site during turnover does not release under conditions wherein binding of ATP to noncatalytic sites of the wild-type enzyme promotes release of MgADP from the affected catalytic site. When assayed in the presence of lauryldimethylamine oxide, which prevents turnover-dependent entrapment of inhibitory MgADP in a catalytic site, ATPase activity of the cross-linked form is 47% that of the unmodified mutant enzyme. These results suggest that, during catalysis, the side chains of alpha F(357) and beta R(372) do not pass through the extremely different relative positions in which they exist at the three noncatalytic site interfaces in crystal structures.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0601, USA.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Bandyopadhyay
authors.forename: Huimiao
authors.initials: H
authors.lastname: Ren
authors.forename: Ching S
authors.initials: CS
authors.lastname: Wang
authors.forename: William S
authors.initials: WS
authors.lastname: Allison
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000378:metabolism; D004789:Enzyme Activation; D009154:Mutation; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D021122:Protein Subunits; D000244:Adenosine Diphosphate; D006180:Proton-Translocating ATPases