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Biochemistry. 2000 Dec 5;39(48):14728-38.

Remarkable rate enhancement of orotidine 5'-monophosphate decarboxylase is due to transition-state stabilization rather than to ground-state destabilization.

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1
Department of Chemistry, University of Southern California, Los Angeles, California 90089-1062, USA.

Abstract

The remarkable rate enhancement of orotidine 5'-phosphate decarboxylase (ODCase) has been attributed to ground-state destabilization (GSD) by desolvation and more recently to GSD by electrostatic stress. Here we reiterate our previous arguments that the GSD mechanisms are not likely to play a major role in enzyme catalysis and analyze quantitatively the origin of the rate enhancement of ODCase. This analysis involves energy considerations and computer simulations. Our energy considerations show that (i) the previously proposed desolvation mechanism is based on an improper reference state; (ii) a nonpolar active site cannot account for the catalytic effect of the enzyme; (iii) the focus on the role of the negatively charged protein residues in the electrostatic stress GSD mechanism overlooks the fact that the positively charged Lys72 strongly stabilizes the substrate; (iv) although the previous calculation of the actual enzymatic reaction correctly reproduced the observed rate enhancement, it could not obtain this rate enhancement from the calculated binding energies (which are the relevant quantities for determining GSD effects); (v) the GSD mechanism is inconsistent with the observed binding energy of the phosphoribosyl part of the substrate; and (vi) the presumably unstable substrate (orotate) can be stabilized, at equilibrium, by accepting a proton from the solvent. Our computer simulation studies involve two set of calculations. First, we study the catalytic reaction by using an empirical valence bond potential surface calibrated by ab initio calculations of the reference solution reaction. This calculation reproduces the observed catalytic effect of the enzyme. Next, we use free-energy perturbation calculations and evaluated the electrostatic contributions to the binding energies of the ground state and transition state (TS). These calculations show that the rate enhancement in ODCase is due to the TS stabilization rather than to GSD. The differences between our own and the previous theoretical analyses stem from both the selection of the reacting system and the treatment of the long-range electrostatic contributions to the binding energy. The reacting system was previously assumed to encompass only the orotate. However, this selection does not allow proper description of the reaction catalyzed by the enzyme (i.e., [Orotate(-) + LysH(+)] if [uracil + Lys + CO(2)]). Therefore, the reacting system should include both orotate and the general acid in the form of the protonated Lys72 protein residue. This selection leads to a simple and consistent interpretation of the catalytic effect where the electrostatic stabilization of the transition state is due to the fact that the two negatively charged aspartic residues are already placed near the reactive lysine so that they do not have to reorganize significantly during the reaction. Interestingly, even calculations with only orotate(-) as the reacting system do not produce sufficient destabilization to account for a GSD mechanism. In summary, we conclude, in agreement with previous workers, that ODCase catalyzes its reaction by electrostatic effects. However, we show that these effects are associated with TS stabilization due to a reduction in the protein-protein reorganization energy and not with protein-substrate destabilization effects.

PMID:
11101287
[Indexed for MEDLINE]
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