Abstract
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.
Highlights
The intrinsic tryptophan fluorescence of Schizosac- sites are less selective since they bind guanosine as well as charomycespombe mitochondrialF1is a very sensitive adenosine nucleotides or analogues; they are assumed to be probe to differentiate nucleotide binding to catalytic and noncatalytic sites
F1 titrationwithmantGTP,as followed by theextrinsic fluorescence enhancement (Fig. lA),showed a hyperbolic saturation curve with an apparent dissociation constant of 11.2 p ~ M.ant GDP behavedvery with a K d value of 12.6 p ~Th. e bindingof these analogueswas monitored by quenching of the intrinsic tryptophanfluorescence of F1, which was previously shown t o be related toselective binding of nucleotides or analogues to the catalytic site
A very similar curve was of the nucleotide analogue extrinsic fluorescence at 435nm ( A )on produced by mant GDP with a maximal quenching of 45% and an apparent dissociation constant of 13 p ~ S.ince the enzyme retained all its endogenous nucleotides,the hyperbolic typesaturationcurvesobtainedwith mant derivatives of GTP and GDPsuggest analogue binding to asingle site, as alsoconcludedwiththecorresponding unmodified nucleotides (Divita etal., 1992)
Summary
Mant-nucleotide Binding to S. pombe Fl-The binding of mant-nucleotide analogues could be monitored by both the enhancement of extrinsicanalogue fluorescence andthe quenching of F1 intrinsic tryptophan fluorescence. A very similar curve was of the nucleotide analogue extrinsic fluorescence at 435nm ( A )on produced by mant GDP with a maximal quenching of 45% and an apparent dissociation constant of 13 p ~ S.ince the enzyme retained all its endogenous nucleotides (near 4 mol/ mol),the hyperbolic typesaturationcurvesobtainedwith mant derivatives of GTP and GDPsuggest analogue binding to asingle site, as alsoconcludedwiththecorresponding unmodified nucleotides (Divita etal., 1992). Derivatives, whereas a second emission peak was observed at 435 nm, the effect being higher with mant GDP than with mant ATP Such nucleotide analogues could beused as acceptor probes for fluorescence energy transfer, since their absorption spectra overlap the emission spectrum of F1-tryptophan residues (Fig. 4). This allowed accurate determination of the relative distance between the catalytic binding site, assumed to be mainly located on the @-subunit, and trypto-
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