Constrained Bonding Environment in the Michaelis Complex of Trypanosoma cruzi Uridine Phosphorylase

The transition state for the Trypanosoma cruzi uridine phosphorylase (TcUP) reaction has an expanded S_N² character. We used binding isotope effects (BIE's) to probe uridine distortion in the complex with TcUP and sulfate to mimic the Michaelis complex. Inverse 1'-³H and 5'- ³H BIE's indicate a constrained bonding environment of these groups in the complex. Quantum chemical modeling identified a uridine conformer whose calculated BIE's match the experimental values. This conformer differs in sugar pucker and uracil orientation from the unbound conformer and the transition-state structure. These results support ground-state stabilization in the Michaelis complex.