ODCase

PRTase - phosphoribosyl transferase ODCase - OMP decarboxylase OMP - orotidine 5 -phosphate... 

Fig. 44 Pathways for uridylate biosynthesis. Mutants lacking enzymes PRTase or ODCase can complete a route to UMP provided by an antibody orotate decarboxylase in conjunction with the naturally occurring uracil PRTase. Decarboxylation of orotic acid [135] is thought to proceed through the transition state [136], for which the hapten [137] was developed (Smiley and Benkovic, 1994).

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

Fig. 1 Computed free energy reaction profiles for the decarboxylation of OMP in water and in the wild-type enzyme ODCase. Reprinted with permission from Reference 66. Copyright 2000 National Academy of Sciences.

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... 

Because of its essential role in nucleic acid biosynthesis and its unique mechanistic characteristics, ODCase has long been the subject of much study.6-14 Nonetheless, the catalytic mechanism remains unknown. 

Fig. 1 Experimentally derived binding free energies for the substrate (S) and transition state (TS) out of aqueous solution to form the ODCase substrate (E S) and ODCase transition state (E TS) complexes (AGSbmd and AGTSbind) and free energies of activation in aqueous solution and ODCase (AG q and AGoDCase), all in kcal mol-1.

The myriad mechanistic hypotheses have led to a plethora of studies - both experimental and theoretical - aimed at elucidating the ODCase mechanism. This review focuses on those studies which have employed computations as their primary mechanistic tool. These can be divided into two main categories quantum mechanical studies of small model systems (Section 2), and molecular mechanical studies of the entire enzyme (Section 3). These calculations are often intimately tied to experimental work, and where relevant, experimental studies are described in greater detail. 

Results. Lee and Houk were the first to model part of the ODCase active site when they calculated decarboxylation energetics for orotate in the presence of methylammonium ion as a mimic of the key active site lysine. Based on their conclusion that 4-protonation is an energetically favorable pathway (see above), they calculated the energy of reaction of orotate (la) plus Cf NHj to form a carbene-methylamine complex plus C02 (equation 1). 

The best low-energy models correspond to arrangements that are very unlikely given the crystallographically determined structure of the ODCase active site (Fig. 2). The calculated barriers never drop below 30 kcal mol-1, which is much higher than the experimentally observed barrier for decarboxylation by ODCase (AG = 15 kcal mol-1, Fig. I),1,6 prompting the authors to conclude that direct protonation is not a viable mechanism. 

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. 

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. 

Results. The experimental 15N isotope effect at N1 for the decarboxylation of OMP in ODCase (Scheme 1) was measured by Cleland et al. to be 1.0068.66 Comparison of this normal isotope effect with IEs measured for the model compounds picolinic acid (17) and A-methyl picolinic acid (18) led Cleland and coworkers to conclude that the normal IE observed for OMP decarboxylation is indicative of the lack of a bond order change at Nl. This conclusion was based on the following reasoning. The IE for the decarboxylation of picolinic acid (17) is 0.9955 this inverse value is due to the change in bond order incurred when the proton shifts from the carboxylate group to the N in order to effect decarboxylation (equation 2) the N is ternary in the reactant, but becomes quaternary in the intermediate, which results in the inverse IE. The decarboxylation of A-methyl picolinic acid (18) involves no such bond order change (equation 3), and the observed normal IE of 1.0053 reflects this. 

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. 

The remainder of Section 3 will discuss the free energy calculations reported so far on ODCase-catalyzed decarboxylation of OMP (Scheme 1). 

Gao and coworkers used QM/MM calculations74 to map out the reaction coordinate and predict the activation free energies for OMP decarboxylation by ODCase and for the decarboxylation of the 1-methylorotate anion (lb) in water.22 Free energies of binding were then computed for structures involved in the decarboxylation using FEP methods.71... 

The free energy changes accompanying the transfer of structures along the reaction coordinate from water to the ODCase active site were then computed using FEP methods.71 These computations employed a cutoff distance of 14 A for explicit electrostatic interactions, beyond which a shell (radius 14-16 A) of dielectric constant 4 was used to approximate the electrostatic properties of the remainder of the protein the area outside of this shell was treated with a dielectric constant of 78 to represent the electrostatic properties of the surrounding water. 

The overall energetics obtained by Gao and coworkers are consistent with previous calculations and experimental values. First, the reasonableness of using AMI, despite its semiempirical nature, is supported by the fact that it predicts an endothermicity of 35.5 kcal mol-1 for decarboxylation in the gas phase, which is extremely close to the values predicted previously with more involved computational methods (see Section 2). Second, the predicted activation free energy for decarboxylation in aqueous solution is 37.2 kcal mol-1, while the corresponding experimental value is 38.5 kcal mol-1 (Fig. I).1 Third, the QM/MM calculations predict a free energy of activation for OMP decarboxylation in ODCase of 14.8 kcal mol-1, while the experimental value is 15.2 kcal mol-1 (Fig. I).1... 

This proposal of a ground state destabilization mechanism for ODCase (this type of mechanism was referred to earlier by Fersht as electrostatic stress 81 and by Jencks as the Circe effect ) sparked considerable controversy. In some circles it was seen as a prime example of the catalytic power of ground state destabilization,83 but several groups immediately questioned its validity on the basis of theoretical objections and apparent inconsistencies with biochemical experiments.23 26... 

Warshel and coworkers used EVB72 and FEP71 calculations to predict the free energy of activation for ODCase-catalyzed decarboxylation and to examine its origins. This study differs from that of Gao and coworkers in two fundamental ways (besides in the details of the computational methods used in each). First, Warshel and coworkers explored the effects of changing the protonation states of several important residues in ODCase. Second, in some of their simulations, the ammonium group of Lys73 (Fig. 2) was treated quantum mechanically. 

After performing ab initio and solvation calculations to examine the decarboxylation reaction in water, the free energy surface of the enzyme-catalyzed reaction was explored. An initial ODCase-OMP complex was constructed from the structure of the ODCase-6-azaUMP complex reported by Pai and coworkers,22... 

While this study does apply a variety of computational methods to the problem of ODCase catalysis, the results obtained from the free energy calculations are at their best qualitative. Overall, this report indicates that a C5-protonation mechanism is possible, although its ability to overwhelm alternative mechanisms cannot be asserted. 

The optimized structure of the ODCase complex with the uracil anion (3) was used to argue for the importance of dynamic effects in transition state stabilization. When the C6-CO2 distance constraint was removed, CO2 was released and drifted away from the Asp-Lys-Asp-Lys tetrad, and at the same time, C6 of the resulting uracil anion moved towards Lys73. The Asp-Lys-Asp-Lys tetrad appeared to be... 

So far, the several reported studies in which free energy computations have been applied to the mechanism of OMP decarboxylation have not produced an answer to the question of where the rate acceleration provided by ODCase comes from. 

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