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 (m(7)Guo), and orthophosphate (P(i), natural cosubstrate) and its thiophosphate analogue (SP(i)), 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 (pK(a) 9.2 and 8.8); m(7)Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pK(a) 7.0); and P(i) and SP(i) exhibit equilibria between monoanionic and dianionic forms (pK(a) 6.7 and 5.4, respectively). The phosphorolysis of m(7)Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SP(i), independently of pH. With P(i), the human enzyme shows Michaelis kinetics only at pH approximately 5. However, in the pH range 5-8 for the bacterial enzyme, and 6-8 for the human enzyme, enzyme kinetics with P(i) 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 P(i), characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K (m1) and K (m2)) and apparent maximal velocities (V (max1) and V (max2)). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K (m1) < K (m2)) and enzyme kinetics (V (max1)/K (m1) > V (max2)/K (m2)). Comparison of the pH-dependence of the substrate properties of P(i) versus SP(i) points to both monoanionic and dianionic forms of P(i) as substrates, with a marked preference for the dianionic species in the pH range 5-8, where the population of the P(i) 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 n (H) approximately 1 to n (H) approximately 0.7 for Guo with the human enzyme, and to n (H) approximately 0.7 and 0.5 for m(7)Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SP(i) in relation to its structure.
Modrak-Wójcik
Anna
A
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Warsaw, Poland.
Kirilenko
Aneta
A
Shugar
David
D
Kierdaszuk
Borys
B
D001667:Binding, Competitive; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism*; D013379:Substrate Specificity
C116616:8-methylguanosine; D010710:Phosphates; D006151:Guanosine; D011683:Purine-Nucleoside Phosphorylase; C035638:thiophosphoric acid