Decarboxylation uncatalyzed

The mechanism for the uncatalyzed decarboxylation of P-ketoacids had previously been established by Bredt and by Pedersen (Bredt, 1927 Pedersen, 1929 1936 Westheimer and Jones, 1941). The acid loses C02 to form the enol of the product, which subsequently ketonizes. The idea behind Pedersen s mechanism for aniline catalysis is that nitrogen is more basic than oxygen, and so could be protonated more readily the protonated imine would provide a better electron sink than the ketone. Although Pedersen offered little or no experimental support for his hypothesis, it provided a basis in physical organic chemistry for the mechanism of the corresponding enzymic process. 

Hamilton marked the carbonyl group of acetoacetic acid with ieO, and then carried out the enzymic decarboxylation (Hamilton and Westheimer, 1959). The product of the decarboxylation, acetone, contained none of the label. This result is demanded by the ketimine mechanism, whereas the mechanism of uncatalyzed decarboxylation would have required that the label appear intact in the product. Of course, in order to make these statements we had to carry out an elaborate set of control experiments, since 180 is washed out of both acetone and acetoacetic acid by buffers and even more... 

Although metal ions do not catalyze the decarboxylation of monocarboxylic acids in solution, a variety of metal ions catalyze the decarboxylation of oxaloacetic acid anion, leading to the formation of pyruvic acid (27). The metal ions involved were cupric, zinc, magnesium, aluminum, ferric, ferrous, manganous, and cadmium, approximately 10-2 to 10-3 M (27). Of these, the aluminum, ferric, ferrous, and cupric ions were the most efficient sodium, potassium, and silver ions were inactive. This process involves the decarboxylation of a / -keto acid, which undergoes a relatively facile uncatalyzed decarboxylation. However, not every decarboxylation of a / -keto acid is catalyzed by metal ions—only those... 

Write a stepwise mechanism for the enzyme-induced decarboxylation, clearly indicating the nature of the bonding between the substrate and enzyme. Show how your mechanism can accommodate hydrogen cyanide inhibition and the results of the borohydride reactions. Utilize the results of the discussion of the ease of decarboxylation of various acids in Section 18-4 to deduce possible structural requirements for the active site so that decarboxylation of the enzyme-substrate complex can occur more readily than the uncatalyzed decarboxylation. 

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... 

So far, three computational studies of isotope effects related to the ODCase mechanism have been published Singleton, Beak and Lee used 13C isotope effects to elucidate the mechanism by which the uncatalyzed decarboxylation of orotic acid takes place.46 Phillips and Lee calculated 15N isotope effects and compared them to known experimental values to show that oxygen-protonation mechanisms are viable for the enzyme-catalyzed process.47 Kollman and coworkers focused on the 15N isotope effect associated with C5-protonation.27 Each study is described further below. 

Carbon-13 isotope effects and the uncatalyzed decarboxylation of orotic acid... 

Results. The goal of this study was to elucidate the pathway by which the uncatalyzed decarboxylation of 1,3-dimethyl orotic acid in sulfolane proceeds. As described earlier, the authors expected this decarboxylation to proceed via 4-protonation, which is the energetically favored pathway according to their calculations. 

Acetic acid and other organic acids are thermodynamically unstable at sedimentary conditions and will eventually decarboxylate to CO2 and alkanes (24). Experimental studies of acetic acid decarboxylation show that the rate is extremely sensitive to temperature and the types of catalytic surfaces available (Table II 25-261. Extrapolated rate constants for acetic acid decarboxylation at 100 C differ by more than 14 orders of magnitude between experiments conducted in stainless steel and catalytically less active titanium (Table II 26). Inherent (uncatalyzed) decarboxylation rates are similar for acetic acid and acetate (26). However, in catalytic environments their rates of decarboxylation differ markedly (25-261. and therefore a pronounced pH effect on total decarboxylation rate is observed. 

Acetoacetate will also readily undergo uncatalyzed decarboxylation to form acetone and CO2. 

Fig. 8. Hammett pa correlations for the cycloheptaamylose-catalyzed ( ) and the uncatalyzed (O) decarboxylation of phenylcyanoacetic acid anions at pH 9.24. For the catalyzed reaction, the Hammett reaction constant p = 2.72. For the uncatalyzed reaction, p = 2.44.

The catalytic power of enzymes is awesome (Table 2.1). A most spectacular example is that of the decarboxylation of orotic acid. It spontaneously decarboxy-lates with tm of 78 million years at room temperature in neutral aqueous solution. Orotidine 5 -phosphate decarboxylase enhances the rate of decarboxylation enzyme-bound substrate by 1017 fold. The classical challenge is to explain the magnitude of the rate enhancements in Table 2.1. We will not ask why enzymatic reactions are so fast but instead examine why the uncatalyzed reactions are so slow, and how they can be speeded up. 

In biochemical decarboxylation reactions where the reactant contains a 3-keto group, the e-amino group of a lysyl side chain of the protein backbone can form an iminium derivative with the substrate.82 Upon loss of carbon dioxide, the delocalized, weakly basic product will not react faster than carbon dioxide can separate. Benner83 showed that the stereochemical consequence of decarboxylation of acetoacetate by acetoacetate decarboxylase involves protonation of the product from either face, consistent with a passive, uncatalyzed step, which is consistent with the view we have presented. 

Orotidine S -monophosphate decarboxylase (ODCase) is a key enzyme in the biosynthesis of nucleic acids, effecting the decarboxylation of orotidine 5 -monophosphate (OMP, 1) to form uridine S -monophosphate (UMP, 2, Scheme l).1,2 The conversion of OMP to UMP is biomechanistically intriguing, because the decarboxylation appears to result, uniquely, in a carbanion (3, mechanism i, Scheme 2) that cannot delocalize into a it orbital.3,4 The uncatalyzed reaction in solution is therefore extremely unfavorable, with a AG of... 

Lee and Houk next examined the decarboxylation of the 2-protonated orotate 4a, which was first proposed by Beak and Siegel in 1976 to be an intermediate for the catalyzed reaction.15 The A// for decarboxylation of 4a is found to be only 21.6 kcal mol-1, which is 22 kcal mol-1 lower than for the uncatalyzed reaction. 

Lee and Houk also proposed a modification of the Beak ylide mechanism, suggesting that protonation on the 4-oxygen to yield the stabilized carbene 7 might be a favorable reaction. This carbene mechanism (decarboxylation of 6a, mechanism iii, Scheme 2) is found to have a A// of 15.5 kcal mol-1, which is 28 kcal mol-1 more favorable than the uncatalyzed reaction. The conclusion from these studies was therefore that both 2- and 4-oxygen protonation would lower the barrier of the decarboxylation, with 4-oxygen protonation (the carbene mechanism) being 6 kcal mol-1 more favorable than 2-oxygen protonation (the ylide mechanism). 

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. 

The authors also compared their values to a previously measured 13C isotope effect of 1.043 0.003 for the carboxylate carbon in the E. coli ODCase-catalyzed decarboxylation of OMP.65 This value differs substantially from the experimental value of 1.013 measured by Singleton for the decarboxylation of orotic acid in sulfolane, implying that the uncatalyzed and catalyzed reactions are quite different. 

For ODCase, non-covalent mechanisms have often been proposed, as reflected in three of the mechanisms shown in Fig. 2. This is the crux of the attention showered on ODCase how can this enzyme achieve its rate acceleration without the use of cofactors, metals, or acid-base catalysis From Wolfenden s measurements of the uncatalyzed reaction of 1-methylorotic acid in water, he calculated the rate enhancement (kcat/kun) in the enzyme to be 1.4x10, corresponding to a reduction of AG of 24 kcal/mol [1]. He also reported the catalytic proficiency to be 2x10 meaning that the enzyme-transition state complex is an impressive 32 kcal/mol more stable than the fi-ee enzyme and transition state in water (i.e., the effective binding free energy of the transition state out of water is 32 kcal/mol) [1] The experimental free energy of activation is 15 kcal/mol for this decarboxylation in ODCase. 

Additional density functional calculations on orotate derivatives by Singleton et al. indicated that the major factor favoring decarboxylation via 04 protonation is likely an inherent preference for 04 protonation in uracil derivatives, rather than strong selective stabilization of the 04-protonated decarboxylated product and the transition state for its formation [28]. The authors also compared experimental (in solution without enzyme present) and theoretical kinetic isotope effects, finding that those computed for the 04 protonation pathway matched best the experimentally determined values. It was also noted, though, that differences between isotope effects measured for the carboxylate carbon in the uncatalyzed [28] and enzyme-catalyzed [29] decarboxylations may indicate that the mechanisms in these two environments differ considerably. More recent isotope effect calculations performed by Phillips and Lee [30], however, indicate that protonation of either 04 or 02 is consistent with the reported experimental isotope effects for the ODCase-catalyzed reaction [31]. 

In analyzing the origin of enzyme catalysis, Warshel and others have advocated the importance of comparing the enzymatic reaction with a reference reaction in water [32]. In addition, it is also necessary to study the reference reaction in the gas phase in order to understand the intrinsic reactivity and the effect of solvation. Thus, to understand enzyme catalysis fully, we must compare results for the same reaction in the gas phase (intrinsic reactivity), in aqueous solution (solvation effects), and in the enzyme (catalysis). This is not possible when there is no model reaction for the uncatalyzed process in the gas phase and in water, or if the uncatalyzed reaction is a bimolecular process as opposed to a unimolecular reaction in the enzyme active site. None of these problems apply to the ODCase reaction. Furthermore, OMP decarboxylation is a unimolecular process, both in water and the enzyme, providing an excellent opportunity to compare directly the computed free energies of activation [1] this is the approach that we have undertaken [16]. Warshel et al. used an ammonium ion-orotate ion pair fixed at distances of 2.8 or 3.5 A as the reference reaction in water to mimic an active site lysine residue [32]. 

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