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J. Biol. Chem., Vol. 265, Issue 8, 4340-4347, 03, 1990

Substrate binding-induced alteration of nucleotide binding site properties of chloroplast coupling factor 1

AB Shapiro and RE McCarty
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.

Two adenine nucleotide binding sites of chloroplast coupling factor 1 (CF1) were shown previously to switch their properties after exposure of the enzyme to Mg2(+)-ATP or Ca2(+)-ATP (Shapiro, A. B., and McCarty, R. E. (1988) J. Biol. Chem. 263, 14160-14165). The change in binding properties was monitored by fluorescence resonance energy transfer between Lucifer Yellow vinyl sulfone covalently bound to one alpha subunit and trinitrophenyl-ATP (TNP-ATP) tightly bound to nucleotide binding site 1. When the nucleotide binding properties of sites 1 and 3 switch during catalysis, site 3, which is nearer Lucifer Yellow than site 1, switches its nucleotide binding properties with site 1, allowing TNP-ATP to become tightly bound near Lucifer Yellow. The smaller separation allows energy transfer to occur, resulting in decreased Lucifer Yellow fluorescence. In this paper, we show that adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) bound to CF1 and caused nucleotide binding sites 1 and 3 to switch properties, but was not hydrolyzed. Using AMP-PNP, we also found that relaxation of the properties of the sites to the precatalysis state after removal of substrate occurred in the absence of hydrolysis of the last bound nucleotide. When Mg2+ was omitted during exposure of CF1 to ATP, there was very little hydrolysis or nucleotide site switching. When Mg2+ was added to a very low concentration which was more than stoichiometric with CF1, however, site switching occurred at its maximal level with virtually no increase in ATP hydrolysis. These results support a model in which binding of substrate Mg2(+)-ATP, not hydrolysis, causes the putative catalytic sites to switch properties, in agreement with the alternating site catalytic cooperativity hypothesis (Boyer, P. D. (1989) FASEB J. 3, 2164-2178). TNP-ATP, the fluorescence acceptor, did not cause nucleotide site switching when incubated with CF1 in the presence of EDTA to eliminate free Mg2+. Two possible additional nucleotide binding sites were detected, in addition to the three well characterized sites. At least one of these sites was close to the Lucifer Yellow site, judging by the amount of energy transfer caused by partial occupancy with TNP-ATP.
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