Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase.
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 alpha R365 and beta Y354 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 (alpha W365) or intermediate (beta W354) polarity in unoccupied sites. alpha W365 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 beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365. With either alpha W365 or beta W354 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 > or = 27 A.
Weber
J
J
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Wilke-Mounts
S
S
Grell
E
E
Senior
A E
AE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012265:Ribonucleotides / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012265:Ribonucleotides; D014364:Tryptophan; D006180:Proton-Translocating ATPases; D008274:Magnesium