Pre-protonation

John A. Glidden, VP-Finance Darren R. Jamison, Pres., Northern Power Systems Mark E. Murray, Pres., Proton Energy Systems Robert J. Friedland, Sr. VP-Hydrogen Generation Robert B. Nieszczezewski, Controller... 

Stepwise decarboxylation and C6-protonation via transition state stabilization 206 Pre-protonation at C5 209... 

This particular reaction model was chosen because the authors proposed that proton transfer should be concerted with decarboxylation. This model reaction is quite exothermic in the gas phase (— 61.9 kcal mol-1), but in an environment of low dielectric (s = 4), as might be expected in an enzyme active site,38 the AH is a reasonable 17.6 kcal mol-1. This barrier is —25 kcal mol-1 less than the AH calculated by these authors for the uncatalyzed decarboxylation of orotate in a water dielectric, which is almost identical to the magnitude of catalysis observed experimentally.1,6 The authors thus concluded that concerted decarboxylation and proton transfer to the 4-oxygen appears to be a viable catalytic pathway. This particular viewpoint has been challenged by Warshel et al., whose quantum mechanical studies argue against pre-protonation.61... 

Unfortunately, no single mechanism has emerged from these studies as the most likely candidate for the decarboxylation mechanism employed by ODCase. Of the protonation mechanisms, only C6-protonation (mechanism iv, Scheme 2) appears to be consistently discounted,27 59 and the 02 and 04 pre-protonation mechanisms (mechanisms ii and iii, Scheme 2) still appear to be viable possibilities.16,46,47,59 The C5-protonation pathway is also a contender.27... 

Nonetheless, there is still hope that quantum mechanical studies may play a key role in deducing the ODCase mechanism. What these studies have shown is that several mechanisms are energetically viable. They have also provided structural models of transition states and their complexes with active site groups that can be used to design experiments for distinguishing between the several mechanisms that remain in the running. One particularly promising experiment that has already been proposed is the measurement of the 1SN decarboxylation isotope effects for the N3 site of OMP. Phillips and Lee have made the computational prediction that while decarboxylation via 2-protonation and without pre-protonation should have normal isotope effects (1.0027 and 1.0014, respectively), the 4-protonation pathway should display an inverse IE of 0.9949.47 Thus, the combination of computationally predicted and experimentally measured IE values may ultimately lead to elucidation of the enzyme mechanism. 

In the gas phase, calculations predict that decarboxylation via 04 pre-protonation should be preferred over decarboxylation via 02 pre-protonation. While this result has not been tested directly in an experimental setting, the greater proton affinity of 04 over 02 in uracil has been established experimentally in the gas phase. 

Gas-phase quantum mechanical calculations have also revealed the energetic favorability of C5 pre-protonation, which also awaits experimental verification. 

The enzyme mechanism, however, remains elusive. Quantum mechanical models generally disfavor C6-protonation, but 02, 04, and C5-protonation mechanisms remain possibilities. Free energy computations also appear to indicate that C5-protonation is a feasible mechanism, as is direct decarboxylation without preprotonation O-protonation mechanisms have yet to be explored with these methods. Controversy remains, however, as to the roles of ground state destabilization, transition state stabilization, and dynamic effects. Because free energy models do take into account the entire enzyme active site, a comprehensive study of the relative energetics of pre-protonation and concerted protonation-decarboxylation at 02, 04, and C5 should be undertaken with such methods. In addition, quantum mechanical isotope effects are also likely to figure prominently in the ultimate identification of the operative ODCase mechanism. 

The reaction catalyzed by ODCase is ostensibly quite simple—the decarboxylation of orotate ribose monophosphate (OMP) to produce uracil ribose monophosphate (see below). The problem with this reaction is that direct decarboxylation would lead to an anion whose lone pair of electrons is not aligned with any r-system that could lead to stabilization through delocalization. Various proposals have been put forth to overcome this apparent obstacle. These include selective stabilization of the transition state for direct decarboxylation by non-covalent interactions with ODCase, selective destabilization of the reactant by repulsive noncovalent interactions that are reduced or removed during direct decarboxylation, pre-protonation of the reactant on one of its carbonyl oxygens or alkene carbons such that decarboxylation would lead to a stabilized ylide, and concerted protonation-decarboxylation which would avoid the formation of a discrete uracil anion. The validity of these mechanistic proposals is analyzed from various viewpoints in this volume. 

Oxalacetic acid is reduced in four two-electron waves in acidic media the protonated keto form and the protonated enol form, in medium pH-range the monoanion pre-protonated on the carbonyl group and in slightly alkaline solutions the unprotonated dianion. The carbanion-enolate present at pH > 14 is not reducible. 

Diethyl-a-ketoglutarate is reduced up to pH 7 in the pre-protonated form. Hydrolysis prevented studies at pH < 2 and pH > 7. 

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