Ground-State Destabilization in Orotate Phosphoribosyltransferases by Binding Isotope Effects

Intrinsic kinetic isotope effects (KIEs) are commonly used to provide bond structural information for enzymatic transition states (TSs). Competing isotopic label methods provide KIEs on the kinetic parameter of kcat/Km (1, 2). But the kcat/Km KIEs include all kinetically important steps between reactants free in solution and the first irreversible step of the reaction (Figure 1) (3). By measuring equilibrium binding isotope effects (BIEs), bond vibrational changes upon formation of the Michaelis complex are obtained and KIEs from the chemical step can be resolved.(4). Reactions involving N-ribosidic bond-loss commonly form ribocation transition states where the anomeric carbon undergoes rehybridization from sp3 in the reactant to sp2 at the transition state. α-Secondary [1′-3H] BIEs are a sensitive way to measure bonding changes in reactants upon binding to catalytic sites and therefore report on the degree of ground state destabilization for the enzyme-substrate complexes. Enzyme-based ground state destabilization is established for several enzymes and been summarized by Anderson (5), although earlier reports have questioned the ability of enzymes to distort substrates (6). Figure 1 Reactions catalyzed by PfOPRT and HsOPRT and the relationship between BIE, kcat/Km KIE and kcat KIE. The substrate pair of orotidine–pyrophosphate (PPi) is shown. A similar transition state is formed when PPi is replaced with phosphonoacetic acid. ... Recent BIE studies with purine nucleoside phosphorylase (PNP) reported a [5′-3H] BIE remote from the site of chemical bond breaking and gave a BIE of 1.5%. For TS analogues of PNP, normal [5′-3H] BIEs of 13 to 29% were reported for human PNP, indicating that TS analogue interactions differ considerably from those in Michaelis complexes or at the TS (7). BIEs with PNP were explained by local C5′-H5′ bond distortion and are not necessarily linked to ground-state destabilization. Ground-state destabilization can be established by BIEs of atoms involved in the chemical reaction by showing that the distortion leads toward the transition state. Orotate phosphoribosyltransferases from Plasmodium falciparum (PfOPRT) and human sources (HsOPRT) catalyze the reversible pyrophosphorolysis of orotidine and orotidine 5′-monophosphate (OMP). Transition state analyses from isotope effects and quantum chemical calculations have established ribocation TS structures with fully dissociated dianionic orotates (Figure 1) (8, 9). Large normal α-secondary [1′-3H] kcat/Km intrinsic KIEs of 19 to 26% were measured for reactions catalyzed by PfOPRT and HsOPRT. These isotope effects are primarily the result of out-of-plane bending mode changes as C1′ geometry is distorted from sp3 in the reactant to sp2 at the SN1 TS. Secondary [1′-3H] kcat/Km KIEs from in vacuo calculations provide a two-state model for the conversion of unbound reactants to the transition state. In reality, kcat/Km KIEs also include changes to the [1′-3H] bond vibrational changes in the Michaelis complex. Here we use BIE to resolve these effects. The X-ray crystal structure of HsOPRT in complex with OMP (PDB code: 2WNS) proposes multiple catalytic site contacts to OMP. Structural models predict an extended hydrogen-bond network surrounding the pyrimidine group to enforce leaving group activation. Leaving group forces have been confirmed in OPRT by isotope-edited Fourier transform infrared (FTIR) spectroscopy of the pyrimidine group (10). Enzyme-bound OMP differs in geometry from the in vacuo energy minima, most obviously for the N-ribosyl torsion angle between the orotate base and ribose ring. The [1′-3H] bond vibrations are sensitive to the N-ribosyl torsion angle (11). When the enzyme stabilizes a conformation away from the energetic minimum of unbound substrate, the degree of sp3 hybridization at C1′ may be altered to cause a BIE (12). Here, [1′-3H] BIEs of OMP and orotidine were measured in binary and ternary complexes of PfOPRT and HsOPRT. Large normal BIEs with [1′-3H]OMP (alone or with sulfate, a competitive inhibitor with pyrophosphate) demonstrate ground state destabilization toward the TS. Lack of [1′-3H]orotidine BIEs indicate that without the 5′-phosphate, substrate undergoes no significant distortion in the Michaelis complexes. This is the first work to report a [1′-3H] BIE for any N-ribosyltransferase. In combination with experimental KIEs, the [1′-3H] BIEs reveal the 5′-phosphate to assist is a two-step C1′-distortion for OPRT-catalyzed OMP pyrophosphorolysis.