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

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.

Fluorescence Decay Heterogeneity Model Based on Electron Transfer Processes in an Enzyme-Ligand Complex

“…Moreover, crystal data and previous investigations (see Ref. [32] and literature cited therein) indicate that both the base moiety of FA and the phenol ring of Tyr160 are involved in π−π interactions with the aromatic residue of Phe159. The latter is located between FA and Tyr160, almost perpendicularly to the base moiety of FA from one side and to the phenol ring of Tyr160 on the other side.…”

“…In addition, the kinetics of fluorescence decay is affected by FRET, i.e. the resonance interaction by weak (Förster) coupling between transition dipoles of emission and absorption of protein tyrosine residues (Tyr160) and the N(2)-H form of FA bound by the enzyme [32]. Moreover, crystal data and previous investigations (see Ref.…”

A new approach to interpretation of heterogeneity of fluorescence decay in complex biological systems

“…Moreover, crystal data and previous luminescence studies (see Ref. [18], and literature cited therein) indicate that both the base moiety of FA and the phenol ring of Tyr160 are involved in π−π interactions with the aromatic residue of Phe159. The latter is located between FA and Tyr160, almost perpendicularly to the base moiety of FA from one side and to the phenol ring of Tyr160 on the other side.…”

“…Addition of E. coli PNP-I to the aqueous solution of FA lead to selective binding of the N(2)-H tautomeric form, and to a shift of the tautomeric equilibrium in favor of the N(2)-H species. 17,18 3.2 Effect of enzyme-ligand interaction on tyrosine fluorescence decay of the enzyme E. coli PNP-I and its ternary complex with formycin A (FA, inhibitor) and phosphate (P i , natural co-substrate) in aqueous solutions were chosen as good examples of highly complex fluorescence intensity decays resulting from excitation of tyrosine residues in the enzyme, the N(1)-H and N(2)-H tautomeric forms of FA (Scheme 1), free in solution (see Ref. [16], and literature cited therein), and the latter bound selectively in the active site of the enzyme.…”

“…In addition, the kinetics of fluorescence decay is affected by fluorescence resonance energy transfer (FRET), i.e. the resonance interaction by week (Förster) coupling between transition dipoles of emission and absorption of protein tyrosine residues (Tyr160) and the N(2)-H form of FA bound by the enzyme 18 . Moreover, crystal data and previous luminescence studies (see Ref.…”