Transition State Analysis for Human and Plasmodium falciparum Purine Nucleoside Phosphorylases

Recent studies have shown that Plasmodiumfalciparum is sensitive to a purine salvage block at purine nucleoside phosphorylase (PNP) and that human PNP is a target for T-cell proliferative diseases. Specific tight-binding inhibitiors might be designed on the basis of specific PNP transition state structures. Kinetic isotope effects (KIEs) were measured for arsenolysis of inosine catalyzed by P. falciparum and human purine nucleoside phosphorylases. Intrinsic KIEs from [1′-³H]-, [2′-³H]-, [1′-¹⁴C]-, [9-¹⁵N]-, and [5′- ³H]inosines were 1.184 ± 0.004, 1.031 ± 0.004, 1.002 ± 0.006, 1.029 ± 0.006, and 1.062 ± 0.002 for the human enzyme and 1.116 ± 0.007, 1.036 ± 0.003, 0.996 ± 0.006, 1.019 ± 0.005, and 1.064 ± 0.003 for P. falciparum PNPs, respectively. Analysis of KIEs indicated a highly dissociative D _N*A_N (S_N1) stepwise mechanism with very little leaving group involvement. The near-unity 1′-¹⁴C KIEs for both human and P. falciparum PNP agree with the theoretical value for a 1′-¹⁴C equilibrium isotope effect for oxacarbenium ion formation when computed at the B1LYP/6-31G(d) level of theory. The 9- ¹⁵N KIE for human PNP is also in agreement with theory for equilibrium formation of hypoxanthine and oxacarbenium ion at this level of theory. The 9-¹⁵N KIE for P. falciparum PNP shows a constrained vibrational environment around N9 at the transition state. A relatively small β-secondary 2′-³H KIE for both enzymes indicates a 3′-endo conformation for ribose and relatively weak hyperconjugation at the transition state. The large 5′-³H KIE reveals substantial distortion at the 5′-hydroxymethyl group which causes loosening of the C5′-H5′ bonds during the reaction coordinate.