Stereospecific isotope effects indicate the geometry of hydrolytic depurination transition states

Numerous N-glycohydrolases are known to proceed through transition states with considerable oxocarbenium ion character. For hydrolases accepting 2'deoxynucleoside substrates, the 2'-prochiral hydrogens allow two separate Osecondary kinetic isotope effects (KIEs) to be determined. Using an empirical relationship for 13-secondary KIE as a function of the leaving group dihedral angle ( 0 ), these KIEs allow two equations in two unknowns to be solved for 0. Given the constrained nature of the planar CI-O4 double bond in the deoxyribosyl oxocarbenium ion, knowledge of 0 enables the sugar ring conformation in such transition states to be predicted. Both pro-R and pro-S [2-³H]-2- deoxyinosine were synthesized and used as geometric probes of the transition states of two ribonucleoside hydrolases, a deoxyribonucleoside hydrolase/transferase and acid-catalyzed solvolysis. When combined with computer modeling studies, these results indicate that acid-catalyzed hydrolysis proceeds through a shallow trough on the potential energy surface, allowing conversion of reactants into products through an ensemble of energetically similar activated complex structures at normal ambient temperatures. In contrast, hydrolysis KIEs for all three enzymes are consistent with transition states that are structurally restrained to maximize hyperconjugative stabilization of positive charge on the anomeric carbon by the sugar hydrogens. Supported by research grant GM41916 from the NIH.