BINDING OF PHOSPHATE AND SULFATE ANIONS BY PURINE NUCLEOSIDE PHOSPHORYLASE FROM ESCHERICHIA-COLI - LIGAND-DEPENDENT QUENCHING OF ENZYME INTRINSIC FLUORESCENCE

Steady-state and time-resolved emission spectroscopy was applied to a study of the binary and ternary complexes of pure E, coli purine nucle oside phosphorylase (PNP) with phosphate(P-i; a substrate) and a close non-substrate analogue (sulfate; SA). The quenching of enzyme fluores cence by P-i was bimodal, best described by two modified Stern-Volmer equations fitted independently for ''low'' (below 0.5 mM P-i) and ''hi gh'' (above 0.5 mM P,) ligand concentrations. At P-i>0.5 mM, binding i s characterized by a fortyfold higher dissociation constant (K-d2=1.12 +/-0.10 mM), i.e. by a lower affinity for phosphate, with a sevenfold lower quenching constant and 1.6-fold higher accessibility. By contras t, the binding of SA, and the resultant fluorescence quenching, was un imodal, with K-d=1.36+/-0.07 mM, comparable to the K-d for P-i at ''hi gh'' P-i, with a total binding capacity of one sulfate or phosphate gr oup per enzyme subunit. SA proved to be a competitive inhibitor of pho sphorolysis with K-i=1.2+/-0.2 mM vs. P-i, hence similar to its K-d. S A at a concentration of 5 mM did not affect the P-i affinity at P-i<0. 5 mM, but led to a reduced affinity and twofold higher P-i binding cap acities at P-i>0.5 mM. The resultant fluorescence quenching by P-i dec reased at 5 mM SA, with lower Stern-Volmer constant (K-SV) and fractio nal accessibility (f(a)) values. Increasing concentrations of P-i redu ced the enzyme affinity for SA, characterized by a higher K-d. The Hil l model showed negative cooperative binding of P-i in the absence and presence of 5 mM SA with Hill coefficients h=0.60+/-0.01 and h=0.83+/- 0.07, respectively. SA exhibited non-cooperative binding in the absenc e of P-i (h=1.08+/-0.01) and negative cooperative binding in the prese nce of P-i (h<1). PNP fluorescence decays were best fitted to a sum of two exponentials, with an average lifetime of 2.40+/-0.14 ns, unchang ed on interaction with quenching ligands, and pointing to static quenc hing. The overall results are relevant to the properties of PNP from v arious sources, in particular to the design of potent bisubstrate anal ogue inhibitors.