TRANSITION-STATE STRUCTURE OF SALMONELLA-TYPHIMURIUM OROTATE PHOSPHORIBOSYLTRANSFERASE

Orotate phosphoribosyltransferase (OPRTase) catalyzes the magnesium-de pendent conversion of alpha-D-phosphoribosylpyrophosphate (PRPP) and o rotate to orotidine 5'-monophosphate (OMP) and pyrophosphate. We have determined kinetic isotope effects on the reaction of OMP with pyropho sphate and with the pyrophosphate analog phosphonoacetic acid. In the latter case, full expression of the kinetic isotope effects allowed us to calculate the structure of the transition state for the pyrophosph orylytic reaction. The transition state resembles a classical oxocarbo nium ion. Using the recently reported three-dimensional structures of the OPRTase-OMP (Scapin et al., 1994) and the OPRTase-PRPP complexes ( Scapin et al., 1995a), we have modeled the calculated transition state structure into the active site of OPRTase. We propose a detailed chem ical mechanism which is consistent with these results.