Structure of the Ribonuclease·Uridine−Vanadate Transition State Analogue Complex by Raman Difference Spectroscopy:  Mechanistic Implications

Raman difference spectroscopy is used to assess changes in the internal bonding of the oxygens of a ⪫VO 2 - group when that group is incorporated into a complex also involving RNase A, uridine, and a water molecule (the RNase/UVO 2 /H 2 O complex). We find that the strengths of the nonbridging V O bonds are decreased by 0.055 vu and their bond lengths are decreased 0.012 Å, based on the stretching frequency changes of nonbridging V O bonds upon formation of the enzymic RNase/UVO 2 /H 2 O complex from the cyclic vanadate diester in solution. The bond lengths are 1.638 Å for the solution complex and 1.650 Å for the enzymic complex. The values found for the bond lengths are about 0.15 Å shorter than those found previously in crystallographic studies. Assuming the RNase/UVO 2 /H 2 O adduct is a reasonably good transition state analogue, our Raman results suggest that the RNase-catalyzed hydrolysis of uridine 2‘,3‘-cyclic phosphate proceeds via an S N 2-like process. The process also may involve a small associative charactor, since the summed bond strength of nonbridging P O bonds is reduced by only 0.11−0.22 vu in the transition state compared with the ground state, which means the summed bond strength of the PO bonds of the entering and leaving groups is correspondingly increased.