ODCase active site

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. 

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. 

ODCase active site. Wolfenden performed atomic absorption spectroscopy and initially concluded that two atoms of zinc were present per active ODCase monomer [9]. This finding was later withdrawn when atomic and x-ray absorption spectroscopy did not detect zinc in active ODCase samples [10]. 

Fig. 4a ODCase active-site (pyrimidine-binding region) with BMP bound from E. colL a Ball-and-stick drawing of polar and hydrophobic residues based on the crystallographi-cally determined coordinates. Bonds in BMP are darker than those in the active-site residues. b line drawing of polar active-site residues (bold) and their interactions with bound BMP...

Fig.5 ODCase active site (M. thermoautotrophicum) with CMP bound...

Hur and Bruice carried out classical molecular dynamics on the complex of ODCase with OMP before decarboxylation, as well as the putative intermediate (the C6 anion) formed by decarboxylation [39]. Based on these calculations, it was proposed that loop movement in ODCase may play a key role in catalysis a stable p hairpin structure appeared to form during decarboxylation that was not present before decarboxylation. In addition, the structure of OMP in aqueous solution was also simulated, and the similarity of the conformations of OMP in water and in the ODCase active site suggested that OMP is not bound in a particularly strained fashion, further arguing against a Circe effect. 

Fig. 4 Schematic representation of the yeast ODCase active site, showing contacts formed between the enzyme and bound 6-hydroxyuridine 5 -phosphate (BMP). Hydrogen bonding distances were measured between electronegative atoms... 

Fig. 6 Schematic representation of the yeast ODCase active site, depicting the effects on transition state binding affinity of replacing individual active site residues with alanine. Free energy contributions (AAG) of individual interactions were calculated from the decrease in catalytic efficiency (/Ccat/ m) produced upon mutation to alanine. The sum of individual mutations totals more than 45 kcal/mol of binding free energy...

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 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. 

Figure 3 compares the proficiencies (kcat/K]v[/kun) of ODCase, several other enzyme decarboxylases [2], and some antibody decarboxylases [3]. The proficiencies of the decarboxylase enzymes, including a variety of amino acid decarboxylases, are nearly equal. Many decarboxylases employ iminium intermediates formed by reaction of an amino acid with a cofactor such as pyruvoyl or pyridoxal, or by reaction of a -keto ester with an active-site lysine residue. These intermediates have been found to be so reactive that the... 

As noted above, many decarboxylases are known to exploit Schiff base formation in the active-site as a source of catalysis. Shostack and Jones explored this possibility in the case of ODCase [8]. They found that when the enzymatic reaction is performed in 0 water, the product does not incorporate from bulk solvent. For this reason, a covalent iminium mechanism for ODCase was abandoned, in spite of its attractive similarities to other decarboxylase mechanisms. 

To date, more than twenty X-ray crystal structures of ODCase have been reported, all by the groups of Ealick, Pai, Larsen, and Short and Wolfenden (see Table 1) [14-21]. The first six of these were reviewed previously by Houk, Lee, and coworkers [22] and Begley, Appleby, and Ealick [23]. As noted by these reviewers, the active-site structure of ODCase is extremely similar in all of the reported structures, boasting an Asp-Lys-Asp-Lys tetrad in the vicinity of the 6-position of the pyrimidine... 

After the initial crystal structures of ODCase were reported, a modified 04 protonation mechanism was proposed [22]. In the original Lee-Houk proposal, the active-site residue Lys93 (yeast numbering, analogous to Lys72 in E, coli Fig. 4 and Table 2), which was experimentally shown to be catalyt-ically important, was assumed to be the species that protonated 04 [27, 32]. However, in the reported crystal structures this lysine does not reside near 04. It was therefore proposed that proton transfer from Lys93 to 04 might be mediated by an active site water molecule (see Fig. 7). The viability of this scenario has not yet been established, however. 

Kollman and coworkers apphed a variety of computational methods to this mechanistic problem—including quantum mechanics on small model systems, molecular dynamics simulations with the AMBER force field on the whole ODCase-substrate system, and MM-PBSA free energy calculations on ODCase with bound OMP [38]. Based on their results, they proposed a decarboxylation mechanism for ODCase that involves C5 protonation. Their calculations at the MP2/6-31+G //HF/6-31+G level showed that C5 has a greater intrinsic proton affinity than C6, 02, and even 04. This, coupled with the fact that Lys72 (M. thermoautotrophicum numbering see Table 2) is near C5 and C6 in the inhibitor-bound crystal structures, prompted the authors to embrace a C5 protonation mechanism. However, the authors themselves acknowledged the uncertainties of their calculations because of approximations employed in representing the enzyme active site. 

Iminium ion formation often precedes decarboxylation and is essential for catalysis in many enzymes. As noted in the introduction, Shostack and Jones proposed a Schiff base mechanism for ODCase that involves iminium formation with an active-site lysine [8]. However, experiments have shown that no exchange between substrate and bulk solvent occurred when the enzymatic reaction was run in H2 0 [10]. 

Further efforts involving structural and kinetic experiments and making use of various mutants of ODCase have led to an improved understanding of the active site, even though it is still not possible to describe the exact chemical steps of the catalytic mechanism with complete certainty. In this review, the presently known crystal structures of ODCase, native as well as mutant forms, in their ligand-free form and in complex with various inhibitors will be discussed. The description will focus on the M. thermoautotrophicum ODCase because most of the structural work involves this enzyme also, it is the one the authors are most familiar with (and prejudiced towards). 

Fig. 4 Stereo representation of the interactions between active site residues in native M. thermoautotrophicum ODCase and the inhibitor 6-azaUMP. Several tightly bound water molecules are also shown. Blue hehces and yellow p-sheets belong to one monomer, red helices belong to the second monomer...

Fig. 5 Superposition (in stereo) of 6-azaUMP, UMP and BMP bound to the active site of native M. thermoautotrophicum ODCase. Carbon atoms of the BMP and 6-azaUMP complexes are colored in grey and those of the UMP complex are in black. The green contour (accessible area calculated with 1.2 A probe radius using VOIDOO [29]) represents the cavity located next to the pyrimidine bases and proposed as a temporary binding site for the product CO2...

Fig. 6a,b Electron density representing the inhibitor of 6-azaUMP in the active sites of base-recognition mutants, a shows the dual conformations adopted by the pyrimidine ring in S127A ODCase. b In the Q185A mutant, a chain of water molecules replaces the glutamine side chain... 

These water molecules cannot efficiently fix the side chain of Lys72, which therefore becomes quite mobile. The presence of a partial negative charge on N6 seems to prevent incorporation of a large Cl ion into the active site of D70 N ODCase. Now, all molecules have their pyrimidine rings rotated by 180° placing the more electron-rich 02 atom closer to the lysine residues (Fig. 7c). 

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