Pyrophosphate activation in hypoxanthine-guanine phosphoribosyltransferase with transition state analogue

Isotope-edited difference Raman and FTIR studies complemented by ab initio calculations have been applied to the transition state analogue complex of HGPRT -ImmHP -MgPP; to determine the ionic states of the 5'-phosphate moiety of ImmHP and of PP¡ These measurements characterize electrostatic interactions within the enzyme active site as deduced from frequency shifts of the phosphate groups. The bound 5'-phosphate moiety of ImmHP is dianionic, and this phosphate group exists in two different conformations within the protein complex. In one conformation, a hydrogen bond between the 5'-phosphate of ImmHP and the OH group of Tyrl04 in the catalytic loop appears to be stronger. With the stronger H-bond, the OH of Tyrl04 approaches one of the PO bonds from the bridging oxygen side to cause distortion of the PO₃ moiety, as indicated by a lowered symmetric PO stretch frequency. The asymmetric stretch frequencies are similar in both phosphate conformations. Bound PP_i in this complex is fully ionized to P₂O₇^4-. Bond frequency changes for bound PP_i indicate coordination to Mg²⁺ ions but show no indication of significant PO bond polarization. Extrapolation of these results to reaction coordinate motion for HGPRT suggests that bond formation between Cl' of the nucleotide ribose and the oxygen of PP _iis accomplished by migration of the ribocation toward immobilized pyrophosphate.