Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf(spleen purine nucleaside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found K-d = 0.12 +/- 0.02 mu M for Gun and 0. 16:L 0. 01 mu M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were Kd = 0. 15 0. 01 mu M for Gua (pH 9.0) and 0.25 +/- 0.02 mu M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction of catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N(7) position.