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Oxidation of the alpha(3)(betaD311C/R333C)(3)gamma subcomplex of the thermophilic Bacillus PS3 F(1)-ATPase indicates that only two beta subunits can exist in the closed conformation simultaneously


Oxidation of the alpha(3)(betaD311C/R333C)(3)gamma subcomplex of the thermophilic Bacillus PS3 F(1)-ATPase indicates that only two beta subunits can exist in the closed conformation simultaneously



Journal of Biological Chemistry 274(44): 31366-31372



ISSN/ISBN: 0021-9258

PMID: 10531337

In the crystal structure of the bovine heart mitochondrial F(1)-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the two liganded beta subunits, one with MgAMP-PNP bound to the catalytic site (beta(T)) and the other with MgADP bound (beta(D)) have closed conformations. The empty beta subunit (beta(E)) has an open conformation. In beta(T) and beta(D), the distance between the carboxylate of beta-Asp(315) and the guanidinium of beta-Arg(337) is 3.0-4.0 A. These side chains are at least 10 A apart in beta(E). The alpha(3)(betaD311C/R333C)(3)gamma subcomplex of TF(1) with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl(2) with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[(14)C]acetamide after prolonged treatment with CuCl(2) in the presence or absence of 1 mM MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a beta subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V(max) of ATP hydrolysis is attained when three catalytic sites of F(1) are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening.

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Accession: 046899662

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