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Comparative Study
. 2004 Aug;33(5):377-85.
doi: 10.1007/s00249-003-0369-9. Epub 2003 Dec 4.

Identification of the tautomeric form of formycin A in its complex with Escherichia coli purine nucleoside phosphorylase based on the effect of enzyme-ligand binding on fluorescence and phosphorescence

Affiliations
Comparative Study

Identification of the tautomeric form of formycin A in its complex with Escherichia coli purine nucleoside phosphorylase based on the effect of enzyme-ligand binding on fluorescence and phosphorescence

Jakub Włodarczyk et al. Eur Biophys J. 2004 Aug.

Abstract

Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, formycin A (FA), a close structural analogue of adenosine (natural substrate), in the absence and presence of phosphate (P(i), substrate). Formation of enzyme-FA complexes led to marked quenching of enzyme tyrosine intrinsic fluorescence and phosphorescence, with concomitant increases in fluorescence and phosphorescence of FA. Fluorescence resonance energy transfer from the protein Tyr160 residue to the FA base moiety was identified as a major mechanism of protein fluorescence quenching, increased by addition of P(i). The effects of enzyme-FA interactions on the nucleoside excitation and emission spectra for fluorescence and phosphorescence revealed shifts in the tautomeric equilibrium of the bound FA, i.e. from the N(1)-H tautomer (predominant in solution) to the N(2)-H form, enhanced by the presence of P(i). The latter was confirmed by enzyme-ligand dissociation constant ( K(d)) values of 5.9+/-0.4 and 2.1+/-0.3 microM in the absence and presence of P(i), respectively. Addition of glycerol (80%, v/v) led to a lower enzyme affinity ( K(d) approximately 70 microM), without changes in binding stoichiometry. Enzyme-FA complex formation led to a higher increase of the fluorescence than the phosphorescence band of the ligand, consistent with the fact that the N(2)-H tautomer is characterized by a weaker phosphorescence than the N(1)-H tautomeric form. These results show, for the first time, the application of phosphorescence spectroscopy to the identification of the tautomeric form of the inhibitor bound by the enzyme.

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References

    1. Biochim Biophys Acta. 1996 May 21;1290(1):9-17 - PubMed
    1. Biochemistry. 1997 Sep 30;36(39):11749-56 - PubMed
    1. Methods Enzymol. 1997;278:221-57 - PubMed
    1. Biochem J. 2002 Jan 1;361(Pt 1):1-25 - PubMed
    1. J Biol Chem. 1990 Feb 25;265(6):3066-9 - PubMed

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