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

Abstract

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 7Guo), which consists of an equilibrium mixture of cationic and zwitterionic forms (p K 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 7Guo 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 7Guo 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 7Guo 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 7Guo 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 7Gua), the product of phosphorolysis of m 7Guo, is a competitive non-substrate inhibitor of phosphorolysis ( K i=8±2 μM) 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, 〈 τ〉=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 ∼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.