Calf spleen purine nucleoside phosphorylase: complex kinetic mechanism, hydrolysis of 7-methylguanosine, and oligomeric state in solution

Abstract

The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 μg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 μg/ml–0.29 mg/ml). At concentrations below 0.9 μg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-methylguanosine (m⁷Guo) with a catalytic rate constant 1.3×10⁻³ s⁻¹ as compared with the rate of 38 s⁻¹ for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of m⁷Guo, 70 s⁻¹, is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 μM–30 mM phosphate is consistent with only one dissociation constant for phosphate, K_d=220±120 μM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 μM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.