Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme.

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

Purine nucleoside phosphorylase activity decline is linked to the decay of the trimeric form of the enzyme.

Homotrimeric mammalian purine nucleoside phosphorylase (PNP) plays a key role in the nucleoside and nucleotide metabolic salvage pathway. Each monomer in the active PNP trimer is composed of a central ß-sheet flanked by several α-helices. We investigated the stability of calf PNP using analytical ultracentrifugation, differential scanning calorimetry, circular dichroism, and UV absorption spectroscopy. The results demonstrate that the activity decline (due to protein aging after isolation from cells) of wild type PNP and its two mutants with point mutations in the region of monomer-monomer interface, is accompanied by a decrease of the population of the trimeric enzyme and an increase of the population of its aggregated forms. The data do not indicate a significant population of either folded or unfolded PNP monomers. The enzyme with specific activity lower than the maximal shows a decrease of the helical structure, which can make it prone to aggregation. The presence of phosphate stabilizes the enzyme but leads to a more pronounced aggregation above the melting temperature. These results suggest that the biological role of packing of the PNP monomers into a trimeric structure is to provide the stability of the enzyme since the monomers are not stable in solution.

Trimeric purine nucleoside phosphorylase: exploring postulated one-third-of-the-sites binding in the transition state.

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.