ABSTRACT
Orotidine-5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) catalyzes the decarboxylation of orotidine-5'-monophosphate (OMP) to uridine-5'-monophosphate (UMP). Despite extensive enzymological, structural, and computational studies, the mechanism of ODCase remains incompletely characterized. Herein, carbon kinetic isotope effects were measured for both the natural abundance substrate and a substrate mixture synthesized for the purpose of carrying out the remote double label isotope effect procedure, with O2 of the substrate as the remote position. The carbon kinetic isotope effect on enzymatic decarboxylation of this substrate mix was measured to be 1.0199 +/- 0.0007, compared to the value of 1.0289 +/- 0.0009 for natural abundance OMP, revealing an (18)O2 isotope effect of 0.991 +/- 0.001. This value equates to an intrinsic isotope effect of approximately 0.983, using a calculated commitment factor derived from previous isotope effect data. The measured (18)O2 isotope effect requires a mechanism with one or more enzymatic processes, including binding and/or chemistry, that contribute to this substantial inverse isotope effect. (18)O2 kinetic isotope effects were calculated for four proposed mechanisms: decarboxylation preceded by proton transfer to 1) O2; 2) O4; and 3) C5; and 4) decarboxylation without a preceding protonation step. A mechanism involving no pre-decarboxylation step does not appear to have any steps with the necessary substantial inverse (18)O2 effect, thus calling into question any mechanism involving simple direct decarboxylation. Protonation at O2, O4, or C5 are all calculated to proceed with inverse (18)O2 effects, and could contribute to the experimentally measured value. Recent crystal structures indicate that O2 of the substrate appears to be involved in an intricate bonding arrangement involving the substrate phosphoryl group, an enzyme Gln side chain, and a bound water molecule; this interaction likely contributes to the observed isotope effect.
