STRUCTURE OF THE RIBONUCLEASE-CENTER-DOT-URIDINE-VANADATE TRANSITION-STATE ANALOG COMPLEX BY RAMAN DIFFERENCE SPECTROSCOPY - MECHANISTIC IMPLICATIONS

Raman difference spectroscopy is used to assess changes in the interna l bonding of the oxygens of a drop VO2- group when that group is incor porated into a complex also involving RNase A, uridine, and a water mo lecule (the RNase/UVO2/H2O complex). We find that the strengths of the nonbridging V chemical anion O bonds are decreased by 0.055 vu and th eir bond lengths are decreased 0.012 Angstrom, based on the stretching frequency changes of nonbridging V chemical anion O bonds upon format ion of the enzymic RNase/UVO2/H2O complex from the cyclic vanadate die ster in solution. The bond lengths are 1.638 Angstrom for the solution complex and 1.650 Angstrom for the enzymic complex. The values found for the bond lengths are about 0.15 Angstrom shorter than those found previously in crystallographic studies. Assuming the RNase/UVO2/H2O ad duct is a reasonably good transition state analogue, our Raman results suggest that the RNase-catalyzed hydrolysis of uridine 2',3'-cyclic p hosphate proceeds via an S(N)2-like process. The process also may invo lve a small associative character, since the summed bond strength of n onbridging P chemical anion 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 P chemical anion O bonds of the entering a nd leaving groups is correspondingly increased.