Enzyme architecture: the activating oxydianion binding domain for orotidine 5'-monophophate decarboxylase.
ABSTRACT: Orotidine 5'-monophosphate decarboxylase catalyzes the decarboxylation of truncated substrate (1-?-D-erythrofuranosyl)orotic acid to form (1-?-D-erythrofuranosyl)uracil. This enzyme-catalyzed reaction is activated by tetrahedral oxydianions, which bind weakly to unliganded OMPDC and tightly to the enzyme-transition state complex, with the following intrinsic oxydianion binding energies (kcal/mol): SO3(2-), -8.3; HPO3(2-), -7.7; S2O3(2-), -4.6; SO4(2-), -4.5; HOPO3(2-), -3.0; HOAsO3(2-), no activation detected. We propose that the oxydianion and orotate binding domains of OMPDC perform complementary functions in catalysis of decarboxylation reactions: (1) The orotate binding domain carries out decarboxylation of the orotate ring. (2) The activating oxydianion binding domain has the cryptic function of utilizing binding interactions with tetrahedral inorganic oxydianions to drive an enzyme conformational change that results in the stabilization of transition states at the distant orotate domain.
Project description:The kinetic parameters for activation of yeast triosephosphate isomerase (ScTIM), yeast orotidine monophosphate decarboxylase (ScOMPDC), and human liver glycerol 3-phosphate dehydrogenase (hlGPDH) for catalysis of reactions of their respective phosphodianion truncated substrates are reported for the following oxydianions: HPO3(2-), FPO3(2-), S2O3(2-), SO4(2-) and HOPO3(2-). Oxydianions bind weakly to these unliganded enzymes and tightly to the transition state complex (E·S(‡)), with intrinsic oxydianion Gibbs binding free energies that range from -8.4 kcal/mol for activation of hlGPDH-catalyzed reduction of glycolaldehyde by FPO3(2-) to -3.0 kcal/mol for activation of ScOMPDC-catalyzed decarboxylation of 1-?-d-erythrofuranosyl)orotic acid by HOPO3(2-). Small differences in the specificity of the different oxydianion binding domains are observed. We propose that the large -8.4 kcal/mol and small -3.8 kcal/mol intrinsic oxydianion binding energy for activation of hlGPDH by FPO3(2-) and S2O3(2-), respectively, compared with activation of ScTIM and ScOMPDC reflect stabilizing and destabilizing interactions between the oxydianion -F and -S with the cationic side chain of R269 for hlGPDH. These results are consistent with a cryptic function for the similarly structured oxydianion binding domains of ScTIM, ScOMPDC and hlGPDH. Each enzyme utilizes the interactions with tetrahedral inorganic oxydianions to drive a conformational change that locks the substrate in a caged Michaelis complex that provides optimal stabilization of the different enzymatic transition states. The observation of dianion activation by stabilization of active caged Michaelis complexes may be generalized to the many other enzymes that utilize substrate binding energy to drive changes in enzyme conformation, which induce tight substrate fits.
Project description:Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with kcat/Km = 1.4 × 10-7 M-1 s-1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.
Project description: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-(?-d-erythrofuranosyl)orotic acid and 1-(?-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.
Project description:Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the exchange for deuterium from solvent D(2)O of the C-6 proton of 1-(?-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.
Project description:The mystery associated with catalysis by what were once regarded as protein black boxes, diminished with the X-ray crystallographic determination of the three-dimensional structures of enzyme-substrate complexes. The report that several high-resolution X-ray crystal structures of orotidine 5'-monophosphate decarboxylase (OMPDC) failed to provide a consensus mechanism for enzyme-catalyzed decarboxylation of OMP to form uridine 5'-monophosphate, therefore, provoked a flurry of controversy. This controversy was fueled by the enormous 1023-fold rate acceleration for this enzyme, which had " jolted many biochemists' assumptions about the catalytic potential of enzymes." Our studies on the mechanism of action of OMPDC provide strong evidence that catalysis by this enzyme is not fundamentally different from less proficient catalysts, while highlighting important architectural elements that enable a peak level of performance. Many enzymes undergo substrate-induced protein conformational changes that trap their substrates in solvent occluded protein cages, but the conformational change induced by ligand binding to OMPDC is incredibly complex, as required to enable the development of 22 kcal/mol of stabilizing binding interactions with the phosphodianion and ribosyl substrate fragments of OMP. The binding energy from these fragments is utilized to activate OMPDC for catalysis of decarboxylation at the orotate fragment of OMP, through the creation of a tight, catalytically active, protein cage from the floppy, open, unliganded form of OMPDC. Such utilization of binding energy for ligand-driven conformational changes provides a general mechanism to obtain specificity in transition state binding. The rate enhancement that results from the binding of carbon acid substrates to enzymes is partly due to a reduction in the carbon acid p Ka that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the p Ka = 29 for abstraction of the C-6 substrate hydrogen, due to stabilization of an enzyme-bound vinyl carbanion, which is also an intermediate of OMPDC-catalyzed decarboxylation. The protein-ligand interactions operate to stabilize the vinyl carbanion at the enzyme active site compared to aqueous solution, rather than to stabilize the transition state for the concerted electrophilic displacement of CO2 by H+ that avoids formation of this reaction intermediate. There is evidence that OMPDC induces strain into the bound substrate. The interaction between the amide side chain of Gln-215 from the phosphodianion gripper loop and the hydroxymethylene side chain of Ser-154 from the pyrimidine umbrella of ScOMPDC position the amide side chain to interact with the phosphodianion of OMP. There are no direct stabilizing interactions between dianion gripper protein side chains Gln-215, Tyr-217, and Arg-235 and the pyrimidine ring at the decarboxylation transition state. Rather these side chains function solely to hold OMPDC in the catalytically active closed conformation. The hydrophobic side chains that line the active site of OMPDC in the region of the departing CO2 product may function to stabilize the decarboxylation transition state by providing hydrophobic solvation of this product.
Project description: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-?-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).
Project description:The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.
Project description:A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of [6-(1)H]-uridine 5'-monophosphate (50%) and [6-(2)H]-uridine 5'-monophosphate (50%) from the decarboxylation of orotidine 5'-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae, Methanothermobacter thermautotrophicus, and Escherichia coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO(2) in a concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D, which is implied by the value of PIE = 1.0, is enforced by restricted C-N bond rotation of the -CH(2)-NL(3)(+) group of the side chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5'-monophosphate (5-FOMP) in 50/50 (v/v) HOH/DOD. Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A, and S154A/Q215A.
Project description:Mutants of orotidine 5'-monophosphate decarboxylase containing all possible single (Q215A, Y217F, and R235A), double, and triple substitutions of the side chains that interact with the phosphodianion group of the substrate orotidine 5'-monophosphate have been prepared. Essentially the entire effect of these mutations on the decarboxylation of the truncated neutral substrate 1-(?-d-erythrofuranosyl)orotic acid that lacks a phosphodianion group is expressed as a decrease in the third-order rate constant for activation by phosphite dianion. The results are consistent with a model in which phosphodianion binding interactions are utilized to stabilize a rare closed enzyme form that exhibits a high catalytic activity for decarboxylation.
Project description:African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.