Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (E. coli) and mammalian (human) origin

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m7Guo), and orthophosphate (Pi, natural cosubstrate) and its thiophosphate analogue (SPi), found to be a weak cosubstrate, have been studied in the pH range 5–8. In this pH range Guo and Ino exist predominantly in the neutral forms (pKa 9.2 and 8.8); m7Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pKa 7.0); and Pi and SPi exhibit equilibria between monoanionic and dianionic forms (pKa 6.7 and 5.4, respectively). The phosphorolysis of m7Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SPi, independently of pH. With Pi, the human enzyme shows Michaelis kinetics only at pH ∼5. However, in the pH range 5–8 for the bacterial enzyme, and 6–8 for the human enzyme, enzyme kinetics with Pi are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by Pi, characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, Km1 and Km2) and apparent maximal velocities (Vmax1 and Vmax2). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (Km1 < Km2) and enzyme kinetics (Vmax1/Km1 > Vmax2/Km2). Comparison of the pH-dependence of the substrate properties of Pi versus SPi points to both monoanionic and dianionic forms of Pi as substrates, with a marked preference for the dianionic species in the pH range 5–8, where the population of the Pi dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from nH ∼1 to nH ∼0.7 for Guo with the human enzyme, and to nH ∼0.7 and 0.5 for m7Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SPi in relation to its structure.