Structural mapping of catalytic site with respect to alpha-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer.
The intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1 is a very sensitive probe to differentiate nucleotide binding to catalytic and noncatalytic sites (Divita, G., Di Pietro, A., Roux, B., and Gautheron, D. C. (1992) Biochemistry 31, 5791-5798), the catalytic site saturation producing quenching of Trp-257 fluorescence (Divita, G., Jault, J.-M., Gautheron, D. C., and Di Pietro, A. (1993) Biochemistry 32, 1017-1024). The present results indicate that two types of fluorescent nucleotide analogues, bearing either 2'(3')N-methylanthraniloyl (mant) or 2',3'-O-(2,4,6-trinitrophenyl) (TNP) group, exhibit high-affinity binding and behave similarly to the corresponding unmodified nucleotides. Selective binding of mant GDP to the catalytic site produces a marked quenching of intrinsic fluorescence which is due to resonance energy transfer between Trp-257 and the mant group. The high efficiency of the transfer allows the determination of a short distance, 10.5 A, indicating the close proximity of catalytic site and alpha-subunit Trp-257. Selective saturation of the noncatalytic site by TNP-ADP produces a marked quenching of the extrinsic fluorescence of mant GDP bound to the catalytic site, which is correlated to an important resonance energy transfer between the two fluorescent groups. A rather short distance of 17.5 A is calculated, indicating vicinity of catalytic and noncatalytic sites.
Divita
G
G
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Goody
R S
RS
Gautheron
D C
DC
Di Pietro
A
A
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases