ODCase reaction, isotope effects

Table 11.4 13C kinetic isotope effects on the ODCase reaction (Smiley,... 

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. 

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]. 

The Jones and O Leary groups then measured the carbon isotope effects at the substrate carboxylate under a number of different conditions [24]. If ODCase were using a mechanism where the substrate is sticky, the carbon isotope effects would also be near unity. However, under some conditions, the observed isotope effect approached the expected value of the intrinsic isotope effect (the isotope effect on the individual C-C bond-breaking step). The ODCase reaction is thus necessarily one in which substrate binding is freely reversible, and only small contributions to the overall rate are made by pre-decarboxylation steps. 

Rishavy and Cleland [30] addressed the possibility of nitrogen isotope effects on the ODCase reaction, with attention to N1 and any possible mechanism involving a secondary nitrogen isotope effect, such as mechanism 1 in Fig. 1. A kinetic equation describing this mechanistic model would be as follows ... 

Fig. 3 a Proposed mechanism of ODCase-catalyzed decarboxylation of OMP by 02 protonation. Both the protonation and decarboxylation steps would be expected to be slightly sensitive to isotopic substitution at Nl. b Model reactions used to assess the feasibility of the 02 protonation mechanism, or any mechanism with a pre-decarboxylation step that is isotopically sensitive at Nl, and the measured Nl equilibrium and kinetic isotope effects, a Data from [31]. b Data from [30]. c Model reactions for which Nl equilibrium and kinetic isotope effects were determined using computational approaches, and the computed values [32]... 

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