Protonation pathway

Hofacker, I., Schulten, K. Oxygen and proton pathways in cytochrome-c oxidase. Proteins Str. Funct. Genet. 29 (1998) 100-107... 

Agmon N (2005) Proton pathways in green fluorescence protein. Biophys J 88 2452-2461... 

Table V Rate Constants for the Solvent and Protonation Pathways in Complex Dissociation (30) ...

When the initial hydride CpRuH(CO)(PH3), It, is placed near H3O+, a proton transfer occurs without the energy barrier to form the dihydrogen complex [CpRu(H2)(CO)(PH3)]+, in good agreement with the data of Orlova and Scheiner [42]. Dihydrogen-bonded intermediates appear on the protonation pathway if the hydride It interacts with the weaker acids TFA and PFTB. However, the... 

Theoretical studies have been reported for the neutral29 and alkaline30,31 hydrolysis of formamide. A theoretical study of the acid hydrolysis of iV-formylaziridine concluded that both N- and O-protonated pathways compete.32 In an historical overview of tetrahedral intermediates in the reactions of carboxylic acid derivatives with nucleophiles, several citations of amide reactions are included.33... 

This model is based on subunit stoichiometry, predicted secondary structure and subunit-subunit interactions. Fi sector (a, /3, y, S, and e subunits) has catalytic sites and Fo sector (a, b and c subunits) forms a proton pathway. 

Genetic analysis and in vitro reconsitution studies suggested that all three Fo subunits (a, b, c) were required for a functional proton pathway.30,31 A single subunit or a combination of two could not form the H+ pathway when incorporated into liposomes. However, these results do not necessarily suggest that amino acid residues from the three subunits actually form a proton pathway. 

Close interactions of the membrane spanning domains of the three subunits have been suggested by a genetic method the effect of 6Gly-9- Asp mutation in the membrane domain of the b subunit was suppressed by a second mutation in the a subunit (aPro-240—Ala, Leu)81 or in the c subunit (cAla-62—Ser).82 The three nonsense mutants (nTrp-111—end aTrp-231—end aGln-252—end) had 50-70% of the membrane ATPase activity, but could not form active proton pathways.83 Thus at least past of the Fi binding site could be formed without a region between aTrp-111 and uHis-271 (carboxyl terminus) of the a subunit. 

A membrane-embedded hairpin structure for the c subunit was established from genetic studies.31 The role of cAsp-61 in the second membrane helix of the c subunit has been studied in detail the cAsp-61— Asn or Gly mutants showed no proton conduction or ATP synthesis.99 Binding of DCCD to this residue blocked proton conduction. These results are consistent with the notion that cAsp-61 is part of the proton pathway. The carboxyl residue at position 61 was able to be transferred to the corresponding position of the first helix.100 Vacuolar-type ATPase has a similar proteolipid subunit possibly evolved from the same ancestral protein as the c subunit.25 The vacuolar proteolipid has glutamate in the middle of the fourth membrane-spanning helix, corresponding to the position of c Asp-61 of the c subunit. The glutamate may play an important role in proton conduction similar to the c subunit.101 ... 

The membrane domain of the b subunit may not play any role in proton-translocation. Thus we may conclude that the a and c subunits form a proton pathway, and residues such as aArg-210, aGlu-219, aHis-245 and cAsp-61 function as the pathway. Further studies will give more detailed understanding of proton translocation. 

The most difficult and interesting question about H+-ATPase is how chemical reaction (ATP synthesis/ hydrolysis) is coupled with vectorial H+ conduction. The mechanism for the stoichiometric coupling between chemical reaction and vectorial transport of ions is a universal question for ion-motive ATPases. The Fo portion is a passive proton pathway but becomes a regulated pathway after the binding of Fi. Mutant analyses suggest that the y subunit has regulatory role(s) for proton conduction. 

The barriers just described were calculated with 1-methylorotic acid (11) as a reference point to model the uncatalyzed reaction in solution. However, the computed free-energy barriers for decarboxylation of zwitterions 4b and 6b are 8.4 and 7.6 kcal mol-1, respectively. This difference of 0.8 kcal mol-1 is significantly smaller than the 6 kcal mol-1 difference calculated by Lee and Houk for the 2-protonation and 4-protonation pathways. This discrepancy arises from an internal hydrogen bond (12) between the Nl-H and the carboxylate that artificially stabilizes the 02-protonated zwitterion 4a, and renders its corresponding decarboxylation barrier too high. When the Nl-H is replaced by a methyl, the hydrogen bond is removed, and the ylide and carbene mechanisms become closer in energy nonetheless, 4-protonation is still favored. 

These three main studies of the gas phase behavior of orotate derivatives show that the 4-protonation pathway is always favored over the 2-protonation pathway. When the barriers are calculated relative to a common reference point of orotic acid, as was done in the Singleton-Beak-Lee study, the 4-protonation pathway is favored by a considerable amount, due mostly to the higher basicity of the 4-oxygen over the 2-oxygen in orotate. Still, the 4-protonation pathway also seems to be favored intrinsically, as evidenced by the consistently lower barriers computed for decarboxylation of the 4-protonated zwitterion 6, regardless of the Nl-R group. 

Computed properties of orotate derivatives other than the energetics of decarboxylation have also been published. The computed gas phase proton affinities of the 2- and 4-oxygens of orotate and of C6-deprotonated uracil have been reported by Lee and Houk to be 263 and 274 kcal mol-1, respectively, for orotate 02 and 04, and 285 and 302 kcal mol-1 for deprotonated uracil 02 and 04.16 The authors noted that the greater proton affinity of the 4-oxygen is relevant to the favorability of the 4-protonation pathway. Similar observations were made by Singleton, Beak and Lee and Phillips and Lee.46,47 Kollman and coworkers recently found that the most basic site of orotate appears to be C5, which is calculated to be 7 kcal mol-1 more basic than 04 at MP2/6-31 + G7/HF/6-31 + G. 27 This translates to a very low energy barrier for decarboxylation of the C5 protonated intermediate 10 kcal mol-1 at MP2/6-31 +G7/HF/6-31 + G, and 5 kcal mol-1 at MP2/cc-pVDZ. SCRF sol-... 

As a further step, Phillips and Lee also calculated the 1SN decarboxylation isotope effects for the N3 site. For decarboxylation without proton transfer, and for decarboxylation via 2-protonation, the isotope effect is found to be normal (1.0014 and 1.0027, respectively). The 4-protonation pathway, however, has an inverse IE of 0.9949. Therefore, the authors propose that isotope effects at N3 may be useful for distinguishing between these mechanisms. 

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. 

A structure-function study of a proton pathway in the y-class carbonic anhydrase from Methanosarcina thermophila was conducted in the work of Tripp and Ferry (2000). Four enzyme glutamate residues were characterized by site-directed mutagenesis. It was shown that Glu 84 and an active site residue, Glu 89, are important for CO2 hydration activity, while external loop residues, Glu 88 and Glu 89 are less important. Glu 84 can be substituted for other ionizable residues with similar pKa values and, therefore, participates in the enzyme catalysis not as a chemical reagent but as a proton shuttle. 

Adelroth, P., Paddock, M. L., Tehrani, A., Beatty, J. T., Feher, G., and Okamura, M. Y. (2001) Identification of the proton pathway in bacterial reaction centers decrease of proton transfer rate by mutation of surface histidines at H126 and H128 and chemical rescue by imidazole identifies the initial proton donors, Biochemistry 40 14538 -14546. 

Tesseyre and coworkers21 used the CNDO/2 method with optimized geometries to elucidate the protonation pathway for vinylamine and concluded that protonation occurred only by attack on the nitrogen atom they could not envisage the attack on the carbon atom even by considering the solvation effect via Jano s model39. Consequently, they stated that conversion of the enammonium into the iminium form could not take place via an intramolecular process. 

In conclusion, theoretical calculations suggest that, in the equilibrium non-planar geometry vinylamine features a single protonation pathway on the nitrogen the situation can be changed only if the molecular geometry is adequately distorted, presumably by another molecule. 

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