Ground-State Destabilization in Orotate Phosphoribosyltransferases by Binding Isotope Effects
2011-05-31T00:00:00Z (GMT) by
Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large α-secondary 1′-3H kcat/Km kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787–8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition-state effects from the kcat/Km KIEs. The BIEs for interactions of [1′-3H]orotidine 5′-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp3 bond hybridization of C1′ toward the sp2 geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1′-3H]OMP kcat/Km KIEs are approximately 1.20, half of the intrinsic kcat/Km KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1′-3H]orotidine BIEs. Thus, OPRTs are unable to initiate ground-state destabilization of orotidine by altered C1′ hybridization because of the missing 5′-phosphate. However the kcat/Km KIEs for [1′-3H]orotidine are also approximately 1.20. The C1′ distortion for OMP happens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1′ distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.