Carbon isotope effect study on orotidine 5'-monophosphate decarboxylase: support for an anionic intermediate

Orotidine 5'-monophosphate decarboxylase has been heavily examined in recent years due to its enzymatic proficiency, which provides a catalytic enhancement to a reaction rate approximately 1017 times greater than that of the nonenzymatic reaction. Several mechanisms proposed to explain this catalytic enhancement have included covalent addition, ylide or carbene formation, and most recently concerted protonation. All of these mechanisms have circumvented the formation of a high-energy vinyl anionic intermediate. To investigate the presence of an anionic intermediate, 13C isotope effect studies have been performed using the alternate substrate 5-fluoro-OMP (OMP = orotidine 5'-monophosphate). Isotope effects obtained for the wild-type enzyme with OMP and 5-fluoro-OMP are 1.0255 and 1.0106, respectively, corresponding to a decrease of approximately 1.5% for 5-fluoro-OMP. With the K59A enzyme, the intrinisic isotope effects show a similar decrease of approximately 1.9% from 1.0543 with OMP to 1.0356 with 5-fluoro-OMP. This decrease results from the inductive effect of the fluorine, which stabilizes the carbanion intermediate by electron withdrawal and produces a reaction with an earlier transition state. The isotope effect for the decarboxylation of the slow substrate 2'-deoxy-OMP produced a intrinsic isotope effect of nearly 1.0461.     

Dissecting the total transition state stabilization provided by amino acid side chains at orotidine 5'-monophosphate decarboxylase: a two-part substrate approach

Kinetic analysis of decarboxylation catalyzed by S154A, Q215A, and S154A/Q215A mutant yeast orotidine 5'-monophosphate decarboxylases with orotidine 5'-monophosphate (OMP) and with a truncated nucleoside substrate (EO) activated by phosphite dianion shows (1) the side chain of Ser-154 stabilizes the transition state through interactions with the pyrimidine rings of OMP or EO, (2) the side chain of Gln-215 interacts with the phosphodianion group of OMP or with phosphite dianion, and (3) the interloop hydrogen bond between the side chains of Ser-154 and Gln-215 orients the amide side chain of Gln-215 to interact with the phosphodianion group of OMP or with phosphite dianion.     

Orotidine-5'-monophosphate decarboxylase catalysis: kinetic isotope effects and the state of hybridization of a bound transition-state analogue

The enzymatic decarboxylation of orotidine 5'-monophosphate may proceed by an addition-elimination mechanism involving a covalently bound intermediate or by elimination of CO2 to generate a nitrogen ylide. In an attempt to distinguish between these two alternatives, 1-(phosphoribosyl)barbituric acid was synthesized with 13C at the 5-position. Interaction of this potential transition-state analogue inhibitor with yeast orotidine-5'-monophosphate decarboxylase resulted in a small (0.6 ppm) downfield displacement of the C-5 resonance, indicating no rehybridization of the kind that might have been expected to accompany 5,6-addition of an enzyme nucleophile. When the substrate orotidine 5'-monophosphate was synthesized with deuterium at C-5, no significant change in kcat (H/D = 0.99 +/- 0.06) or kcat/KM (H/D = 1.00 +/- 0.06) was found to result, suggesting that C-5 does not undergo significant changes in geometry before or during the step that determines the rate of the catalytic process. These results are consistent with a nitrogen ylide mechanism and offer no support for the intervention of covalently bound intermediates in the catalytic process.     

Isotope effect studies of the pyruvate-dependent histidine decarboxylase from Lactobacillus 30a

The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30a shows a carbon isotope effect of k12/k13 = 1.0334 +/- 0.0005 and a nitrogen isotope effect k14/k15 = 0.9799 +/- 0.0006 at pH 4.8, 37 degrees C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D2O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.     

Orotidine 5'-monophosphate decarboxylase: transition state stabilization from remote protein-phosphodianion interactions

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-(β-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.     

Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli

The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product gamma-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid. At pH 4.7, 37 degrees C, the isotope effect is k14/k15 = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid. Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation. This equilibrium isotope effect is k14/k15 = 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde. Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction [O'Leary, M.H., Yamada, H., & Yapp, C.J. (1981) Biochemistry 20, 1476] shows that decarboxylation and Schiff base formation are jointly rate limiting. The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state. The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.     

Metal ion catalyzed hydrolysis of ethyl p-nitrophenyl phosphate

15N isotope effects in the nitro group and 18O isotope effects in the phenolic oxygen have been measured for the hydrolysis of ethyl p-nitrophenyl phosphate catalyzed by several metal ions. Co(III)-cyclen at pH 7, 50 degrees C, gave an 15N isotope effect of 0.12% and an 18O one of 2.23%, showing that P-O cleavage is rate limiting and the bond is approximately 50% broken in the transition state. The active catalyst is a dimer and the substrate is presumably coordinated to the open site of one Co(III), and is attacked by hydroxide coordinated to the other Co(III). Co(III)-tacn under the same conditions shows a similar 15N isotope effect (0.13%), but a smaller 18O one (0.8%). Zn(II)-cyclen at pH 8.5, 80 degrees C, gave an 15N isotope effect of 0.05% and an 18O one of 0.95%, suggesting an earlier transition state. The catalyst in this case is monomeric, and thus the substrate is coordinated to one position and attacked by a cis-coordinated hydroxide. Eu(III) at pH 6.5, 50 degrees C, shows a very large 15N isotope effect of 0.34% and a 1.6% 18O isotope effect. The large 15N isotope effect argues for a late transition state or Eu(III) interaction with the nitro group, and was also seen in Eu(III)-catalyzed hydrolysis of p-nitrophenyl phosphate.     

Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[alpha-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 37 degrees C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli [Abell, L. M., & O'Leary, M. H. (1988) Biochemistry 27, 3325], the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.     

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).     

Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects

Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large α-secondary 1'-(3)H k(cat)/K(m) kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787-8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition-state effects from the k(cat)/K(m) KIEs. The BIEs for interactions of [1'-(3)H]orotidine 5'-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp(3) bond hybridization of C1' toward the sp(2) geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1'-(3)H]OMP k(cat)/K(m) KIEs are approximately 1.20, half of the intrinsic k(cat)/K(m) KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1'-(3)H]orotidine BIEs. Thus, OPRTs are unable to initiate ground-state destabilization of orotidine by altered C1' hybridization because of the missing 5'-phosphate. However the k(cat)/K(m) KIEs for [1'-(3)H]orotidine are also approximately 1.20. The C1' distortion for OMP happens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1' distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.     

Aspartate beta-decarboxylase from Alcaligenes faecalis: carbon-13 kinetic isotope effect and deuterium exchange experiments

We have measured the 13C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D2O at pD 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D2O at pD 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The 13C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. We have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the 13C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier.     

A role for flexible loops in enzyme catalysis

Triosephosphate isomerase (TIM), glycerol 3-phosphate dehydrogenase, and orotidine 5'-monophosphate decarboxylase each use the binding energy from the interaction of phosphite dianion with a flexible phosphate gripper loop to activate a second, phosphodianion-truncated, substrate towards enzyme-catalyzed proton transfer, hydride transfer, and decarboxylation, respectively. Studies on TIM suggest that the most important general effect of loop closure over the substrate phosphodianion, and the associated conformational changes, is to extrude water from the enzyme active site. This should cause a decrease in the effective active-site dielectric constant, and an increase in transition state stabilization from enhanced electrostatic interactions with polar amino acid side chains. The most important specific effect of these conformational changes is to increase the basicity of the carboxylate side chain of the active site glutamate base by its placement in a 'hydrophobic cage'.     

Purification and characterization of yeast orotidine 5'-monophosphate decarboxylase overexpressed from plasmid PGU2

Orotidine 5'-monophosphate decarboxylase (ODCase) has been overexpressed in yeast 15C cells transformed with a plasmid carrying the URA3 gene that encodes ODCase. Twenty g of cells having ODCase activity equal to 30 mg of pure enzyme per liter of cell culture were obtained after 9 h of galactose induction. To remove yeast proteases, a 60-90% ammonium sulfate fractionation step plus the addition of EDTA as an inhibitor of metallopeptidases was necessary. The purification protocol yielded ODCase that was protease-free and stable to storage at 4 degrees C for 16 months. The pure enzyme had a specific activity of 40 units/mg in 50 mM phosphate buffer, pH 6, and could be stored at -20 degrees C in 20% glycerol with retention of full activity for more than 2 years. The enzyme had a Km for orotidine 5'-monophosphate of 0.7 microM at pH 6 and 25 degrees C. The molecular weight of the plasmid-derived ODCase monomer determined by electrophoresis on denaturing polyacrylamide gels was 29,500. ODCase sedimented through sucrose density gradients as a monomer of about 30 kDa at low protein concentration and in the absence of ligands that bind at the catalytic site. An increase in the sedimentation rate could be induced by increasing the ODCase concentration or by adding ligands that are competitive inhibitors. ODCase sedimented in a single band typical of a protein of 46 kDa at the highest protein concentration studied or in the presence of 50 mM phosphate or 933 microM substrate (orotidine 5'-monophosphate) or product (UMP). A dimer sedimenting as a protein of about 64 kDa occurred in the presence of 50 microM 6-azauridine 5'-monophosphate or 2 microM 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid, competitive inhibitors of ODCase. These results resemble the ligand-induced subunit association of the ODCase domain of bifunctional UMP synthase and support the use of yeast ODCase as a model for ODCases from other species.     

Structural basis for the catalytic mechanism of a proficient enzyme: orotidine 5'-monophosphate decarboxylase

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5'-monophosphate, the last step in the de novo synthesis of uridine 5'-monophosphate. ODCase is a very proficient enzyme [Radzicka, A., and Wolfenden, R. (1995) Science 267, 90-93], enhancing the reaction rate by a factor of 10(17). This proficiency has been enigmatic, since it is achieved without metal ions or cofactors. Here we present a 2.5 A resolution structure of ODCase complexed with the inhibitor 1-(5'-phospho-beta-D-ribofuranosyl)barbituric acid. It shows a closely packed dimer composed of two alpha/beta-barrels with two shared active sites. The orientation of the orotate moiety of the substrate is unambiguously deduced from the structure, and previously proposed catalytic mechanisms involving protonation of O2 or O4 can be ruled out. The proximity of the OMP carboxylate group with Asp71 appears to be instrumental for the decarboxylation of OMP, either through charge repulsion or through the formation of a very short O.H.O hydrogen bond between the two carboxylate groups.     

The mechanism of orotidine 5'-monophosphate decarboxylase: catalysis by destabilization of the substrate

The mechanism of orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) was studied using the decarboxylation of orotic acid analogues as a model system. The rate of decarboxylation of 1,3-dimethylorotic acid and its analogues as well as the stability of their corresponding carbanion intermediates was determined. The results have shown that the stability of the carbanion intermediate is not a critical factor in the rate of decarboxylation. On the other hand, the reaction rate is largely dependent on the equilibrium constant for the formation of a zwitterion. Based on these results, we have proposed a new mechanism in which ODCase catalyzes the decarboxylation of OMP by binding the substrate in a zwitterionic form and providing a destabilizing environment for the carboxylate group of OMP.     

13C and 15N isotope effects for conversion of L-dihydroorotate to N-carbamyl-L-aspartate using dihydroorotase from hamster and Bacillus caldolyticus

In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.     

Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specific peptide substrates

To probe the mechanistic origins of convex Eyring plots that have been observed for alpha-chymotrypsin (alpha-CT)-catalyzed hydrolysis of specific p-nitroanilide substrates [Case, A., and Stein, R. L. (2003) Biochemistry 42, 3335-3348], we determined the temperature-dependence of (15)N-kinetic isotope effects for the alpha-CT-catalyzed hydrolysis of N-succinyl-Phe p-nitroanilide (Suc-Phe-pNA). To provide an interpretational context for these enzymatic isotope effects, we also determined 15N-KIE for alkaline hydrolysis of p-nitroacetanilide. In 0.002 and 2 N hydroxide (30 degrees C), 15N-KIE values are 1.035 and 0.995 (+/-0.001), respectively, and are consistent with the reported [HO-]-dependent change in rate-limiting step from leaving group departure from an anionic tetrahedral intermediate in dilute base, to hydroxide attack in concentrated base. For the alpha-CT-catalyzed hydrolysis of Suc-Phe-pNA, 15N-KIE is on kc/Km and thus reflects structural features of transition states for all reaction steps up to and including acylation of the active site serine. The isotope effect at 35 degrees C is 1.014 (+/-0.001) and suggests that in the transition state for this reaction, departure of leaving group from the tetrahedral intermediate is well advanced. Significantly, 15N-KIE does not vary over the temperature range 5-45 degrees C. This result eliminates one of the competing hypotheses for the convex Eyring plot observed for this reaction, that is, a temperature-dependent change in rate-limiting step within the chemical manifold of acylation, but supports a mechanism in which an isomerization of enzyme conformation is coupled to active site chemistry. We finally suggest that the near absolute temperature-independence of 15N-KIE may point to a unique transition state for this process.     

Mechanisms of enzymatic and acid-catalyzed decarboxylations of prephenate

The prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Escherichia coli catalyzes the oxidative decarboxylation of both prephenate and deoxoprephenate, which lacks the keto group in the side chain (V 78% and V/K 18% those of prephenate). Hydride transfer is to the B side of NAD, and the acetylpyridine and pyridinecarboxaldehyde analogues of NAD have V/K values 40 and 9% and V values 107 and 13% those of NAD. Since the 13C isotope effect on the decarboxylation is 1.0103 with deuterated and 1.0033 with unlabeled deoxoprephenate (the deuterium isotope effect on V/K is 2.34), the mechanism is concerted, and if CO2 has no reverse commitment, the intrinsic 13C and deuterium isotope effects are 1.0155 (corresponding to a very early transition state for C-C bond cleavage) and 7.3, and the forward commitment is 3.7. With deoxodihydroprephenate (lacking one double bond in the ring), oxidation occurs without decarboxylation, and one enantiomer has a V/K value 23-fold higher than the other (deuterium isotope effects are 3.6 and 4.1 for fast and slow isomers; V for the fast isomer is 5% and V/K 0.7% those of prephenate). The fully saturated analogue of deoxoprephenate is a very slow substrate (V 0.07% and V/K approximately 10(-5%) those of prephenate). pH profiles show a group with pK = 8.3 that must be protonated for substrate binding and a catalytic group with pK = 6.5 that is a cationic acid (likely histidine). This group facilitates hydride transfer by beginning to accept the proton from the 4-hydroxyl group of prephenate prior to the beginning of C-C cleavage (or fully accepting it in the oxidation of the analogues with only one double bond or none in the ring). In contrast with the enzymatic reaction, the acid-catalyzed decarboxylation of prephenate and deoxoprephenate (t1/2 of 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate, since 18O is incorporated into substrate and its epi isomer during reaction in H218O. pH profiles show that the hydroxyl group must be protonated and the carboxyl (pK approximately 4.2) ionized for carbonium ion formation. The carbonium ion formed from prephenate decarboxylates 1.75 times faster than it reacts with water (giving 1.8 times as much prephenate as epi isomer). The observed 13C isotope effect of 1.0082 thus corresponds to an intrinsic isotope effect of 1.023, indicating an early transition state for the decarboxylation step. epi-Prephenate is at least 20 times more stable to acid than prephenate because it exists largely as an internal hemiketal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Substrate binding induces domain movements in orotidine 5'-monophosphate decarboxylase

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyses the decarboxylation of orotidine 5'-monophosphate to uridine 5'-monophosphate (UMP). We have earlier determined the structure of ODCase from Escherichia coli complexed with the inhibitor 1-(5'-phospho-beta-d-ribofuranosyl)barbituric acid (BMP); here we present the 2.5 A structure of the uncomplexed apo enzyme, determined from twinned crystals. A structural analysis and comparison of the two structures of the E. coli enzyme show that binding of the inhibitor is accompanied by significant domain movements of approximately 12 degrees around a hinge that crosses the active site. Hence, the ODCase dimer, which contains two active sites, may be divided in three domains: a central domain that is fixed, and two lids which independently move 12 degrees upon binding. Corresponding analyses, presented herein, of the two Saccharomyces cerevisiae ODCase structures (with and without BMP) and the Methanobacterium thermoautotrophicum ODCase structures (with and without 6-aza UMP) show very similar, but somewhat smaller domain movements. The domain movements seem to be initiated by the phosphoryl binding to the enzyme and can explain why the binding of the phosphoryl group is essential for the catalytic function.     

Structural basis for the decarboxylation of orotidine 5'-monophosphate (OMP) by Plasmodium falciparum OMP decarboxylase

Orotidine 5'-monophoshate decarboxylase (OMPDC) catalyses the decarboxylation of orotidine 5'-monophosphate (OMP) to uridine 5'-monophosphate (UMP). Here, we report the X-ray analysis of apo, substrate or product-complex forms of OMPDC from Plasmodium falciparum (PfOMPDC) at 2.7, 2.65 and 2.65 A, respectively. The structural analysis provides the substrate recognition mechanism with dynamic structural changes, as well as the rearrangement of the hydrogen bond array at the active site. The structural basis of substrate or product binding to PfOMPDC will help to uncover the decarboxylation mechanism and facilitate structure-based optimization of antimalarial drugs.