Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site- directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues αR365 and βY354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (αW365) or intermediate (βW354) polarity in unoccupied sites. αW365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy. Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis. Fluorescence from βW354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue β354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue α365. With either αW365 or βW354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non- catalytic and catalytic sites is ≥27 Å.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Apr 15 1994|