We used mutants of RNase Tl and the R-P isomer of a thiosubstituted su
bstrate to determine stereospecific thioeffects on catalysis. The anal
ysis reveals subtle structural and functional changes in the intermole
cular transition state interactions. Tyr 38 contributes to catalysis t
hrough a hydrogen bond with the pro-R-P oxygen. Y38F RNase Tl prefers
the R-P thiosubstituted analog over the natural phosphodiester substra
te that is favored by the wild type enzyme.