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.
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.
Włodarczyk
Jakub
J
Institute of Experimental Physics, Department of Biophysics, University of Warsaw, 93 Zwirkii Wigury Street, 02-089 Warsaw, Poland.
Stoychev Galitonov
Gerasim
G
Kierdaszuk
Borys
B
D000465:Algorithms; D001665:Binding Sites; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000032:analysis / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005573:Formycins / Q000032:analysis / Q000737:chemistry* / Q000145:classification; D007536:Isomerism; D007700:Kinetics; D046911:Macromolecular Substances / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D011683:Purine-Nucleoside Phosphorylase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D004791:Enzyme Inhibitors; D005573:Formycins; D046911:Macromolecular Substances; C034550:formycin; D011683:Purine-Nucleoside Phosphorylase