“…These results do not support an O2 protonation mechanism. Deuterium solvent isotope effects (14) and substrate substituent (O2 3 S2) experiment (15) have been presented in support of the mechanism involving O2 protonation. However, such D 2 O effects, as pointed out by Rishavy and Cleland (6), do not prove the existence of a protonation step before the decarboxylation.…”
Section: Discussionmentioning
confidence: 72%
“…Ab initio calculations have shown that O2-or O4-protonation of OMP lowers the activation barrier for decarboxylation by 22-24 kcal͞mol in the gas phase (5). Experimentally determined normal 15 N kinetic isotope effects (KIEs) at N1 suggested no change in the bond order of N1 before decarboxylation (6), but in the following computational study, calculated 15 N KIEs at N1 were rather insensitive to the bond order change (7). Recently, a mechanism involving an enamine protonation at C5 has been proposed as the first step in the decarboxylation reaction (8).…”
“…These results do not support an O2 protonation mechanism. Deuterium solvent isotope effects (14) and substrate substituent (O2 3 S2) experiment (15) have been presented in support of the mechanism involving O2 protonation. However, such D 2 O effects, as pointed out by Rishavy and Cleland (6), do not prove the existence of a protonation step before the decarboxylation.…”
Section: Discussionmentioning
confidence: 72%
“…Ab initio calculations have shown that O2-or O4-protonation of OMP lowers the activation barrier for decarboxylation by 22-24 kcal͞mol in the gas phase (5). Experimentally determined normal 15 N kinetic isotope effects (KIEs) at N1 suggested no change in the bond order of N1 before decarboxylation (6), but in the following computational study, calculated 15 N KIEs at N1 were rather insensitive to the bond order change (7). Recently, a mechanism involving an enamine protonation at C5 has been proposed as the first step in the decarboxylation reaction (8).…”
“…Prevalent among them is proton transfer to the 2-oxygen (the “ylide” mechanism) or to the 4-oxygen (the “carbene” mechanism), proposed by Beak and co-workers, and Lee and Houk, respectively (Scheme , reactions B and C). , Lee and Houk also proposed that the intermediate formed upon 4-protonation and decarboxylation ( 6 ) could be stabilized as reflected in the carbene resonance structure. , More recently, and very importantly, four different crystal structures of ODCase have been solved and reported, by the groups of Ealick and Begley, Short and Wolfenden, Larsen, and Pai and Gao. − Examination of these crystal structures gave rise to a third mechanistic proposal, involving a direct decarboxylation without proton transfer, where catalysis is achieved through ground-state destabilization. The ground-state destabilization hypothesis has led to additional studies probing this mechanistic hypothesis, as well as much debate, including isotope effect studies by Cleland et al, described in more detail later in this paper, that appear consistent with a direct decarboxylation mechanism, where no proton transfer is involved. ,− 1 …”
The mechanism of orotidine 5'-monophosphate decarboxylase was studied computationally by using the decarboxylation of orotic acid analogues as model systems. These calculations indicate that mechanisms involving proton transfer to the 2-oxygen or the 4-oxygen are energetically favorable, as compared to direct decarboxylation without proton transfer, for a series of model compounds where N1 is substituted with respectively H, CH(3), and a tetrahydrofuran moiety. Proton transfer to the 4-oxygen during decarboxylation is found to be energetically more favorable than 2-protonation, which is attributable to both the 4-oxygen site being more basic and an apparent intrinsic preference for the 4-protonation pathway. (15)N isotope effect calculations were also conducted, and compared to experimental (15)N isotope effects previously measured at N1 by Rishavy and Cleland (Biochemistry 2000, 39, 4569-4574). The theoretical isotope effects establish, for the first time, that the experimental (15)N isotope effect is consistent with decarboxylation without protonation, as well as with decarboxylation with protonation, at either O2 or at O4. Furthermore, we propose herein an isotope measurement that could potentially distinguish among mechanisms involving protonation from those that do not involve proton transfer.
“…This interaction, which may involve proton transfer or hydrogen bonding, appears to offer an explanation for the observed isotope effect data. Considering the enormous (11 orders of magnitude) decrease in ODCase activity toward orotidine nucleosides lacking phosphoryl groups 33 and the rescue of activity using phosphite, 34 and the very large (at least 7 orders of magnitude) decrease in ODCase activity toward 2-thioOMP, 35 this proposed feature of the enzyme reaction, which still remains incompletely characterized, seems critical to enzyme activity.…”
Section: Interpretation Of the O2 Isotope Effectmentioning
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
