Interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with the cationic and zwitterionic forms of the fluorescent substrate N(7)-methylguanosine

Steady-state and time-resolved fluorescence spectroscopy, and enzyme kinetics, were applied to study the reaction of purine nucleoside phosphorylase (PNP) from Escherichia coli with its substrate N(7)-methylguanosine (m(7)Guo), which consists of an equilibrium mixture of cationic and zwitterionic forms (pk(a) = 7.0), each with characteristic absorption and fluorescence spectra, over the pH range 6-9, where absorption and intrinsic fluorescence of the enzyme are virtually unchanged. The pH-dependence of kinetic constants for phosphorolysis of m(7)Guo were studied under condition where the population of the zwitterion varied from 10% to 100%. This demonstrated that, whereas the zwitterion is a 3- to 6-fold poorer substrate, if at all, than the cation for the mammalian enzymes, both ionic species are almost equally good substrates for E. coli PNP. The imidazole-ring-opened form of m(7)Guo is neither a substrate nor an inhibitor of phosphorolysis. Enzyme fluorescence quenching, and concomitant changes in absorption and fluorescence spectra of the two ionic species of m(7)Guo on binding, showed that both forms are bound by the enzyme, the affinity of the zwitterion being 3-fold lower than that of the cation. Binding of m(7)Guo is bimodal, i.e., an increase in ligand concentration leads to a decrease in the association constant of the enzyme-ligand complex, typical for negative cooperativity of enzyme-ligand binding, with a Hill constant <1. This is in striking contrast to interaction of the enzyme with the parent Guo, for which the association constant is independent of concentration. The weakly fluorescent N(7)-methylguanine (m(7)Gua), the product of phosphorolysis of M(7)GUo, is a competitive non-substrate inhibitor of phosphorolysis (K-i = 8 +/- 2 M) and exhibits negative cooperativity on binding to the enzyme at pH 6.9. Quenching of enzyme emission by the ligands is a static process, inasmuch as the mean excited-state lifetime, [tau]=2.7 ns, is unchanged in the presence of the ligands, and the constants K-SV may therefore be considered as the association constants for the enzyme-ligand complexes. In the pH range 9.5-11 there is an instantaneous reversible decrease in PNP emission of similar to 15%, corresponding to one of the six tyrosine residues per subunit readily accessible to solvent, and OH- ions. Relevance of the overall results to the mechanism of phosphorolysis, and binding of substrates/inhibitors is discussed. (C) 2001 Elsevier Science B.V. All rights reserved.