TRYPTOPHAN FLUORESCENCE PROVIDES A DIRECT PROBE OF NUCLEOTIDE-BINDINGIN THE NONCATALYTIC SITES OF ESCHERICHIA-COLI F(1)-ATPASE

Tryptophan fluorescence was investigated as a tool to study the noncat alytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-di rected mutagenesis, affinity labeling, and lin-benzo-ATP binding studi es had shown that residues alphaR365 and betaY354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to try ptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alphaW365) or intermediate (betaW354) polarity in unoccupied sites. AlphaW365 fluorescence was completely quenched by b inding of ATP or ADP, providing a direct, specific probe of noncatalyt ic site nucleotide occupancy. Using this signal, we measured binding p arameters for ATP and ADP, showed that nucleotide binding was magnesiu m-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and EDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditi ons; the results indicated that occupancy of noncatalytic sites was no t required for catalysis. Fluorescence from betaW354 was quenched comp letely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, re sidue beta354 is not as closely juxtaposed to the adenine ring of boun d ATP and ADP as is residue alpha365. With either alphaW365 or betaW35 4 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluo rescence resonance energy transfer was detected, indicating that the d istance between noncatalytic and catalytic sites is greater-than-or-eq ual-to 27 angstrom.