Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E. coli: ligand-dependent quenching of enzyme intrinsic fluorescence

Abstract

Steady-state and time-resolved emission spectroscopy was applied to a study of the binary and ternary complexes of pure E. coli purine nucleoside phosphorylase (PNP) with phosphate (P i; a substrate) and a close non-substrate analogue (sulfate; SA). The quenching of enzyme fluorescence by P i was bimodal, best described by two modified Stern-Volmer equations fitted independently for “low” (below 0.5 mM P i) and “high” (above 0.5 mM P i) ligand concentrations. At P i > 0.5 mM, binding is 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 contrast, the binding of SA, and the resultant fluorescence quenching, was unimodal, with K d = 1.36 ± 0.07 mM, comparable to the K d for P i at “high” P i, with a total binding capacity of one sulfate or phosphate group per enzyme subunit. SA proved to be a competitive inhibitor of phosphorolysis with K i = 1.2 ± 0.2 mM vs. P i, hence similar to its K d. SA 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 capacities at P i > 0.5 mM. The resultant fluorescence quenching by P i decreased at 5 mM SA, with lower Stern-Volmer constant ( K SV) and fractional accessibility ( f a) values. Increasing concentrations of P i reduced the enzyme affinity for SA, characterized by a higher K d. The Hill 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 absence of P i ( h = 1.08 ± 0.01) and negative cooperative binding in the presence 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, unchanged on interaction with quenching ligands, and pointing to static quenching. The overall results are relevant to the properties of PNP from various sources, in particular to the design of potent bisubstrate analogue inhibitors.