Ribocation transition state capture and rebound in human purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and alpha-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization.