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

Steady-state and time-resolved fluorescence spectroscopy, and enzyme kineti cs, 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 fo rms (pk(a) = 7.0), each with characteristic absorption and fluorescence spe ctra, 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 popul ation of the zwitterion varied from 10% to 100%. This demonstrated that, wh ereas the zwitterion is a 3- to 6-fold poorer substrate, if at all, than th e cation for the mammalian enzymes, both ionic species are almost equally g ood substrates for E. coli PNP. The imidazole-ring-opened form of m(7)Guo i s neither a substrate nor an inhibitor of phosphorolysis. Enzyme fluorescen ce quenching, and concomitant changes in absorption and fluorescence spectr a of the two ionic species of m(7)Guo on binding, showed that both forms ar e bound by the enzyme, the affinity of the zwitterion being 3-fold lower th an 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 interact ion of the enzyme with the parent Guo, for which the association constant i s independent of concentration. The weakly fluorescent N(7)-methylguanine ( m(7)Gua), the product of phosphorolysis of M(7)GUo, is a competitive non-su bstrate inhibitor of phosphorolysis (K-i = 8 +/- 2 <mu>M) and exhibits nega tive 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-s tate lifetime, [tau]=2.7 ns, is unchanged in the presence of the ligands, a nd the constants K-SV may therefore be considered as the association consta nts for the enzyme-ligand complexes. In the pH range 9.5-11 there is an ins tantaneous reversible decrease in PNP emission of similar to 15%, correspon ding 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) 200 1 Elsevier Science B.V. All rights reserved.