Role of the Carboxylate in Enzyme-Catalyzed Decarboxylation of Orotidine 5'-Monophosphate: Transition State Stabilization Dominates Over Ground State Destabilization.

Kinetic parameters kex (s-1) and kex/Kd (M-1 s-1) are reported for exchange for deuterium in D2O of the C-6 hydrogen of 5-fluororotidine 5'-monophosphate (FUMP) catalyzed by the Q215A, Y217F, and Q215A/Y217F variants of yeast orotidine 5'-monophosphate decarboxylase (ScOMPDC) at pD 8.1, and by the Q215A variant at pD 7.1-9.3. The pD rate profiles for wildtype ScOMPDC and the Q215A variant are identical, except for a 2.5 log unit downward displacement in the profile for the Q215A variant. The Q215A, Y217F and Q215A/Y217F substitutions cause 1.3-2.0 kcal/mol larger increases in the activation barrier for wildtype ScOMPDC-catalyzed deuterium exchange compared with decarboxylation, because of the stronger apparent side chain interaction with the transition state for the deuterium exchange reaction. The stabilization of the transition state for the OMPDC-catalyzed deuterium exchange reaction of FUMP is ca. 19 kcal/mol smaller than the transition state for decarboxylation of OMP, and ca. 8 kcal/mol smaller than for OMPDC-catalyzed deprotonation of FUMP to form the vinyl carbanion intermediate common to OMPDC-catalyzed reactions OMP/FOMP and UMP/FUMP. We propose that ScOMPDC shows similar stabilizing interactions with the common portions of decarboxylation and deprotonation transition states that lead to formation of this vinyl carbanion intermediate, and that there is a large ca. (19-8) = 11 kcal/mol stabilization of the former transition state from interactions with the nascent CO2 of product. The effects of Q215A and Y217F substitutions on kcat/Km for decarboxylation of OMP are expressed mainly as an increase in Km for the reactions catalyzed by the variant enzymes, while the effects on kex/Kd for deuterium exchange are expressed mainly as an increase in kex. This shows that the Q215 and Y217 side chains stabilize the Michaelis complex to OMP for the decarboxylation reaction, compared with the complex to FUMP for the deuterium exchange reaction. These results provide strong support for the conclusion that interactions which stabilize the transition state for ScOMPDC-catalyzed decarboxylation at a nonpolar enzyme active site dominate over interactions that destabilize the ground-state Michaelis complex.

Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5'-Monophosphate Decarboxylase.

The caged complex between orotidine 5'-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5'-monophosphate (FOMP) undergoes decarboxylation ∼300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5'-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5'-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s(-1) for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.

The mechanism for activation of orotidine 5'-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters k(cat) and K(m) for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-ß-D-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (k(cat))(obs) for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (K(m))(obs).

Catalysis by orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.

The syntheses of two novel truncated analogs of the natural substrate orotidine 5'-monophosphate (OMP) for orotidine 5'-monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(ß-d-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5'-deoxy-5-fluoroorotidine (5'-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M⁻¹ s⁻¹) and 1-(ß-d-erythrofuranosyl)orotic acid (EO, 0.026 M⁻¹ s⁻¹) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO3²â»), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in the rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E•FEO•HPO3²â» complex for the reaction of FEO. The 4'-CH3 and 4'-CH2OH groups of 5'-dFO and orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4'-CH3 group of 5'-dFO and the 4'-CH2OH group of orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4'-CH3 substituent at 5'-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.

Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5'-phosphate decarboxylase-catalyzed reactions.

The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5'-monophosphate (OMP) decarboxylase (OMPDC) from Saccharomyces cerevisiae catalyzed reactions of OMP and 5-fluoroorotidine 5'-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5'-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(ß-d-erythrofuranosyl)orotic acid and 1-(ß-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, whereas the 7.2 kcal/mol side chain interaction with the transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number kex, whereas the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant Km. These results are rationalized by a mechanism in which the binding of OMP, compared with that for FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation.

Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme.

The binding of a ligand to orotidine 5'-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (E(o)) to a closed, active conformation (E(c)). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E(c)·S(‡) complex) are enforced. Focusing on the OMPDC from Methanothermobacter thermautotrophicus, we find the "remote" 5'-phosphate group of the substrate activates the enzyme 2.4 × 10(8)-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5'-phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the "mobile" and "fixed" half-barrel domains of the (ß/α)(8)-barrel structure. Our data support a model in which the IBE provided by the 5'-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the E(c)·S and E(c)·S(‡) complexes relative to the E(o)·S complex. The conclusion that the IBE of the 5'-phosphate group provides stabilization to both the E(c)·S and E(c)·S(‡) complexes, not just the E(c)·S(‡) complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions.

OMP decarboxylase: phosphodianion binding energy is used to stabilize a vinyl carbanion intermediate.

Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the exchange for deuterium from solvent D(2)O of the C-6 proton of 1-(ß-d-erythrofuranosyl)-5-fluorouracil (FEU), a phosphodianion truncated product analog. The deuterium exchange reaction of FEU is accelerated 1.8 × 10(4)-fold by 1 M phosphite dianion (HPO(3)(2-)). This corresponds to a 5.8 kcal/mol stabilization of the vinyl carbanion-like transition state, which is similar to the 7.8 kcal/mol stabilization of the transition state for OMPDC-catalyzed decarboxylation of a truncated substrate analog by bound HPO(3)(2-). These results show that the intrinsic binding energy of phosphite dianion is used in the stabilization of the vinyl carbanion-like transition state common to the decarboxylation and deuterium exchange reactions.

Enzyme architecture: on the importance of being in a protein cage.

Substrate binding occludes water from the active sites of many enzymes. There is a correlation between the burden to enzymatic catalysis of deprotonation of carbon acids and the substrate immobilization at solvent-occluded active sites for ketosteroid isomerase (KSI--small burden, substrate pKa=13), triosephosphate isomerase (TIM, substrate pKa≈18) and diaminopimelate epimerase (DAP epimerase, large burden, substrate pKa≈29) catalyzed reaction. KSI binds substrates at a surface cleft, TIM binds substrate at an exposed 'cage' formed by closure of flexible loops; and, DAP epimerase binds substrate in a tight cage formed by an 'oyster-like' clamping motion of protein domains. Directed evolution of a solvent-occluded active site at a designed protein catalyst of the Kemp elimination reaction is discussed.