A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.     

Orotate phosphoribosyltransferase from Corynebacterium ammoniagenes lacking a conserved lysine

The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn²⁺ and Co²⁺. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The K_m values for the four substrates were determined to be 33 μM for orotate, 64 μM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 μM for orotidine-5-phosphate (OMP), and 36 μM for pyrophosphate. The K_m value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.     

Ternary complex formation and induced asymmetry in orotate phosphoribosyltransferase

Orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-alpha-d-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5'-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 A resolution); a ternary complex of enzyme, Mg2+-PRPP, and OA (1.74 A resolution); and the binary product complex of enzyme with OMP (1.89 A resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5 degrees in the OMP structure and an average of 24.6 degrees in the OA/Mg2+-PRPP ternary complex. As expected, the conserved loop, composed of residues 104-116, moves extensively and adopts a single stable conformation during the catalytic cycle in order to sequester the substrates from bulk solvent in the ternary complex. The OA and Mg2+-PRPP molecules bound in the ternary complex are oriented for proper attack of the N1 atom of OA onto the C1 atom of the ribose ring. This orientation of substrates, combined with the positioning of the flexible loop, provides a clear picture of a catalytically poised reaction complex for type I phosphoribosyltransferases. The structural asymmetry present in these structures, as well as that found in a recent structure of the S. typhimurium enzyme, combined with the closure of the flexible loop from one subunit into the active site of the opposing subunit in the ternary complex is consistent with the kinetic data [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342] that demonstrate induced nonequivalence and cooperativity of OPRTase.     

Orotate phosphoribosyltransferase and orotidylate decarboxylase from Crithidia luciliae: subcellular location of the enzymes and a study of substrate channeling

Orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) have been found to be particulate in the kinetoplastid protozoan, Crithidia luciliae. Sucrose density centrifugation indicated that these two enzymes are associated with the glycosome, a microbody which appears to be unique to the Kinetoplastida and which contains many of the glycolytic enzymes. The particulate location of OPRTase and ODCase was considered to be favorable for channeling of orotidine-5'-monophosphate (OMP), the product of the first enzyme and substrate for the second. The degree of channeling was determined by double radioactively labeled experiments designed to determine the relative efficiency of endogenous and exogenous OMP as substrates of ODCase. The efficiency of channeling was high, with an approximate 50-fold preference for endogenous OMP. By comparison, the degree of channeling for the yeast enzymes, which are soluble and unassociated, was less than 2-fold. The OPRTase-ODCase enzyme complex was solubilized using Triton X-100 in the presence of dimethyl sulfoxide, glycerol, and phosphoribosyldiphosphate. The percentage recovery of the overall enzyme activity was approximately 20%. The degree of channeling was reduced by approximately 10-fold for the solubilized complex. The Km for OMP changed from 7.5 (+/- 1.8) to 1.6 (+/- 0.3) microM in the ODCase reaction. There was no alteration in the Km for orotate in the OPRTase reaction.     

Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium

The chemical mechanism of the phosphoribosyltransferases (PRTases), although largely unknown, may proceed either via a concerted direct-transfer mechanism or with a two-step mechanism involving a carboxonium-like intermediate. To study this question, we have cloned the Salmonella typhimurium pyrE gene, coding for the enzyme orotate phosphoribosyltransferase (EC 2.2.4.10, OPRTase), and developed a bacterial strain that overproduces the enzyme, which we have purified to homogeneity. Initial velocity and product inhibition studies indicated that S. typhimurium OPRTase follows a random sequential kinetic mechanism. This result was further confirmed by equilibrium isotope exchange studies on two substrate-product pairs, PRPP-PPi and OMP-orotate. In addition, the rates of the individual equilibrium isotope exchanges allowed us to conclude that PPi release and PRPP release were the rate-determining steps in the forward and reverse reactions, respectively. Although partial reactions between the two substrate-product pairs, PRPP-PPi and OMP-orotate, were observed, further studies revealed that these exchanges were a result of contaminations. Our results are significant in that S. typhimurium OPRTase, like most PRTases but in contrast to its yeast homologue, follows sequential kinetics. The artifactual partial isotope exchanges found in this work may have implications for similar prior work on the yeast enzyme. In view of the careful isotope effect studies of Parsons and co-workers [Goitein, R.K., Chelsky, D., & Parsons, S.M. (1978) J. Biol. Chem. 253, 2963-2971] and the results obtained by us, we propose that PRTases may involve a direct-transfer mechanism but with low bond order to the leaving pyrophosphate moiety and attacking base.     

Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani

Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PP(i). Three-dimensional structures of the dimeric APRT enzyme from Leishmania donovani (LdAPRT) bear many similarities to other members of the type 1 phosphoribosyltransferase family but do not reveal the structural basis for catalysis (Phillips, C. L., Ullman, B., Brennan, R. G., and Hill, C. P. (1999) EMBO J. 18, 3533-3545). To address this issue, a steady state and transient kinetic analysis of the enzyme was performed in order to determine the catalytic mechanism. Initial velocity and product inhibition studies indicated that LdAPRT follows an ordered sequential mechanism in which PRPP is the first substrate to bind and AMP is the last product to leave. This mechanistic model was substantiated by equilibrium isotope exchange and fluorescence binding studies, which provided dissociation constants for the LdAPRT-PRPP and LdAPRT-AMP binary complexes. Pre-steady-state kinetic analysis of the forward reaction revealed a burst in product formation indicating that phosphoribosyl transfer proceeds rapidly relative to some rate-limiting product release event. Transient fluorescence competition experiments enabled measurement of rates of binary complex dissociation that implicated AMP release as rate-limiting for the forward reaction. Kinetics of product ternary complex formation were evaluated using the fluorophore formycin AMP and established rate constants for pyrophosphate binding to the LdAPRT-formycin AMP complex. Taken together, these data enabled the complete formulation of an ordered bi-bi kinetic mechanism for LdAPRT in which all of the rate constants were either measured or calculated.     

Loop Residues and Catalysis in OMP Synthase

Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.     

Significance of the enzyme complex that synthesizes UMP in ehrlich ascites cells

The de novo biosynthesis of pyrimidine nucleotides is completed by two sequential enzyme activities that convert orotate plus 5-phosphoribosyl-1-pyrophosphate to orotidine-5′-monophosphate (OMP) and PP_i and then decarboxylate OMP to produce 5′-uridylic acid. In mammalian cells the two enzyme activities, orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase, form a normally inseparable enzyme complex. It was previously reported that this complex is able to channel the intermediate product, OMP (Traut, T. W., and Jones, M. E., 1977, J. Biol. Chem.252, 8374–8381). The studies reported here indicate that one advantage of this channeling of OMP is to spare OMP from being degraded to orotidine by a potentially competitive nucleotidase activity. Yeast cells have two separate enzymes instead of an enzyme complex, and lack the ability to channel OMP. The OMP formed in yeast cells is not degraded because these cells lack significant nucleotidase activity. These results suggest that the capability for channeling OMP may have been important in evolving the enzyme complex found in mammalian cells.     

II. Transient phase of two-substrate enzyme systems

The time dependence in the transient phase of the two-substrate enzyme systems which evolve according to the random ternary-complex, ordered ternary-complex, ping-pong bi-bi and Theorell-Chance mechanisms has been studied. With this purpose, the equations derived in paper I have been applied. This has allowed to propose a method in order to obtain the rate constant values of these enzyme reactions. In addition, we show that, from experimental knowledge of the induction periods of the ligand species, it is possible to give a method which allows to discriminate between these mechanisms.     

Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 ? resolution. This structure resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99-109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, because of movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg(2+) in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substrate-assisted stabilization of the previously documented oxocarbenium transition state structure is proposed.     

Backbone 1H, 13C, 15N NMR assignments of yeast OMP synthase in unliganded form and in complex with orotidine 5′-monophosphate

The type I phosphoribosyltransferase OMP synthase (EC 2.4.2.10) is involved in de novo synthesis of pyrimidine nucleotides forming the UMP precursor orotidine 5′-monophosphate (OMP). The homodimeric enzyme has a Rossman α/β core topped by a base-enclosing “hood” domain and a flexible domain-swapped catalytic loop. High-resolution X-ray structures of the homologous Salmonella typhimurium and yeast enzymes show that a general compacting of the core as well as movement of the hood and a major disorder-to-order transition of the loop occur upon binding of ligands MgPRPP and orotate. Here we present backbone NMR assignments for the unliganded yeast enzyme (49 kDa) and its complex with product OMP. We were able to assign 212–213 of the 225 non-proline backbone ¹⁵N and amide proton resonances. Significant difference in chemical shifts of the amide cross peaks occur in regions of the structure that undergo movement upon ligand occupancy in the S. typhimurium enzyme.     

Orotate phosphoribosyltransferase from yeast: studies of the structure of the pyrimidine substrate binding site

The pH dependencies of both the forward and reverse orotate phosphoribosyltransferase (ORPTase)-catalyzed reactions have been examined and determined to be dissimilar, with maximal activity for the forward reaction near to pH 8. The maximal activity of the reverse pyrophosphorolysis was observed between pH 6.5 and 7.5. Appropriate pK values were determined using computer fitting exercises. One such pK value (equal to 8.6) suggested the presence of lysine residues at the OPRTase active site. Incubations of OPRTase with the substrate analog, uracil 6-aldehyde, in the presence of sodium borohydride, suggested that this compound is a covalent modifier of OPRTase lysine residues, and substrate protection studies provided evidence that the affected lysine residues were located near to both the phosphoribosyl 1-pyrophosphate (PRibPP) and the orotate binding sites. Similar studies with pyridoxal 5-phosphate and labeled sodium borohydride as modifiers have revealed that two modified active site lysine residues per OPRTase subunit account for the loss of 90% of the enzymatic activity with this reagent. We suggest that essential lysine residues, along with divalent metal ions, are located at the OPRTase active site, and form ion-pair bonds with anionic PRibPP and orotate as these substrates bind to the enzyme. We also report that 5-azaorotate is an alternate substrate for OPRTase (Km = 75.5 +/- 0.1 microM) leading to formation of an unstable nucleotide product).     

Multiple inhibitor analysis of the brequinar and leflunomide binding sites on human dihydroorotate dehydrogenase

Brequinar and the active metabolite of leflunomide, A77 1726, have been clearly shown to inhibit human dihydroorotate dehydrogenase (DHODH), but conflicting mechanisms for their inhibition have been reported. DHODH catalyses the conversion of dihydroorotate (DHO) to orotate concurrent with the reduction of ubiquinone. This study presents data that indicates brequinar is a competitive inhibitor versus ubiquinone; A77 1726 is noncompetitive versus ubiquinone and both are uncompetitive versus DHO. 2-Phenyl 5-quinolinecarboxylic acid (PQC), the core moiety of brequinar also shows competitive inhibition versus ubiquinone. Multiple inhibition experiments indicate that PQC (and thus brequinar) and A77 1726 have overlapping binding sites. Both PQC and A77 1726 are also mutually exclusive with barbituric acid (a competitive inhibitor versus DHO). In addition, we failed to observe brequinar binding to E.orotate by isothermal titration calorimetry (ITC). These results indicate that the E.DHO.inhibitor and E.orotate.inhibitor ternary complexes do not form. The absence of these complexes is consistent with the two-site ping-pong mechanism reported for DHODH. This kinetic data suggests that recent crystal structures of human DHODH complexed with orotate and A77 1726 or brequinar may not represent the relevant physiological binding sites for these inhibitors [Liu, S., Neidhardt, E. A., Grossman, T. H., Ocain, T., and Clardy J. (2000) Structure 8, 25-33].     

Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosis orotate phosphoribosyltransferase

Tuberculosis (TB) is a chronic infectious disease caused mainly by Mycobacterium tuberculosis. The worldwide emergence of drug-resistant strains, the increasing number of infected patients among immune compromised populations, and the large number of latent infected individuals that are reservoir to the disease have underscored the urgent need of new strategies to treat TB. The nucleotide metabolism pathways provide promising molecular targets for the development of novel drugs against active TB and may, hopefully, also be effective against latent forms of the pathogen. The orotate phosphoribosyltransferase (OPRT) enzyme of the de novo pyrimidine synthesis pathway catalyzes the reversible phosphoribosyl transfer from 5'-phospho-α-D-ribose 1'-diphosphate (PRPP) to orotic acid (OA), forming pyrophosphate and orotidine 5'-monophosphate (OMP). Here we describe cloning and characterization of pyrE-encoded protein of M. tuberculosis H37Rv strain as a homodimeric functional OPRT enzyme. The M. tuberculosis OPRT true kinetic constants for forward reaction and product inhibition results suggest a Mono-Iso Ordered Bi-Bi kinetic mechanism, which has not been previously described for this enzyme family. Absence of detection of half reaction and isothermal titration calorimetry (ITC) data support the proposed mechanism. ITC data also provided thermodynamic signatures of non-covalent interactions between substrate/product and M. tuberculosis OPRT. These data provide a solid foundation on which to base target-based rational design of anti-TB agents and should inform us how to better design inhibitors of M. tuberculosis OPRT.     

Orotate phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase from yeast: kinetic analysis with chromium (III) pyrophosphate

The reactions catalyzed by orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) from yeast differ in the kinetic mechanisms by which they are activated by divalent metal ions. Moreover, whereas OPRTase is activated specifically by Mg(II) or Mn(II), the reactions catalyzed by HGPRTase can utilize a wider range of divalent metal ions, including Mg(II), Mn(II), Co(II), and Zn(II). In this report we describe the results of a kinetic analysis of the effects of the addition of Cr(III) pyrophosphate (Cr-PPi) to the OPRTase and HGPRTase assay solutions, which delineates further the differences between these enzyme activations by metal ions. (1) Cr-PPi is an effective competitive inhibitor of the OPRTase catalysis, when the steady-state forward velocity of orotidine monophosphate (OMP) formation is examined over a range of phosphoribosyl alpha-pyrophosphate (PRibPP) concentrations, whereas pyrophosphate (PPi) has been reaffirmed to be a noncompetitive product inhibitor under the same conditions. (2) Cr-PPi itself serves as a substrate for the OPRTase-catalyzed reverse pyrophosphorolysis of OMP and does not inhibit the utilization of PPi as substrate during this reaction. (3) In contrast, Cr-PPi, at concentrations as high as 6 mM, has no effect on the HGPRTase-catalyzed formation of inosine monophosphate, whereas the inhibition exhibited by PPi during this reaction is noncompetitive but defined by two sets of lines in the double reciprocal plot of the initial velocity versus 1/PRibPP. (4) Cr-PPi is not a substrate for the HGPRTase-catalyzed pyrophosphorolysis of IMP under the conditions of these assay procedures.     

Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102+/-11 microM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N(alpha)(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT.     

Pool and synthesis of phosphoribosylpyrophosphate in rat embryo cells infected with X14 or H-1 parvovirus

In rat embryo cell cultures infected with X14 or H-1 parvovirus the PRPP pool and the PRPP synthetase activity have been assayed. A radiometric method, prepared by Authors, based on the conversion of [6-14C) orotate to [6-14C) UMP by the mixed enzyme orotate phosphoribosyltransferase and orotidylate decarboxylase and on the separation of UMP by ascending chromatography, has been utilized. The PRPP pool and te PRPP synthetase activity appeared nearly unmodified in the cells infected with X14 or H-1 parvovirus compared to the mock-infected cells. Therefore, the lowered pyrimidine nucleotide synthesis in infected cells, shown in previous studies, may depend, rather than on the diminished PRPP pool, on the lower PRPP utilization; in fact, some inhibition by metabolites, that may be removed by added PRPP, might occur in the infected cells.     

Does the bifunctional uridylate synthase channel orotidine 5'-phosphate? Kinetics of orotate phosphoribosyltransferase and orotidylate decarboxylase activities fit a noninteracting sites model

Uridylate synthase is a bifunctional protein that first forms orotidine 5'-phosphate (OMP) from orotate via its orotate phosphoribosyltransferase activity (EC 2.4.2.10) and then converts OMP to uridine 5'-phosphate (UMP) via the OMP decarboxylase activity (EC 4.1.1.23). A computer modeling analysis of the experiments that led to the proposal [Traut, T.W., & Jones, M.E. (1977) J. Biol. Chem. 252, 8374-8381] that uridylate synthase channels intermediate OMP suggests that the experimental results do not demonstrate preferential use of OMP generated in the bifunctional complex as against exogenous OMP. This analysis shows that the experimentally observed amounts of [6-14C]UMP from [6-14C]orotate in the presence of various amounts of exogenous [7-14C]OMP agree well with the amounts predicted by the computer simulations. Thus we conclude that uridylate synthase does not channel OMP. Additionally, the subsequent suggestion that channeling of OMP occurs to protect the intermediate from degradation by a nucleotidase [Traut, T.W. (1980) Arch. Biochem. Biophys. 200, 590-594] seems unlikely. The appropriate computer simulation demonstrates that low transient levels of OMP and protection of the intermediate are provided for strictly by the kinetic parameters of orotate phosphoribosyltransferase, OMP decarboxylase, and the nucleotidase. Additionally, calculations show that, in both sets of published experiments, the concentration of transient OMP greatly exceeded the concentration of OMP decarboxylase active sites. Thus, channeling of OMP by the bifunctional complex cannot be invoked to explain the evolution of uridylate synthase, and that event must be the result of some other selective pressure.     

Uridine-5'-phosphate synthase: evidence for substrate cycling involving this bifunctional protein

Uridine 5'-phosphate (UMP) synthase contains two sequential catalytic activities for the synthesis of orotidine 5'-phosphate (OMP) from orotate (EC 2.4.2.10, orotate phosphoribosyltransferase) and the decarboxylation of OMP to form UMP (EC 4.1.1.23, OMP decarboxylase). Previous kinetic studies had indicated that partial channeling of OMP might occur [T.W. Traut and M.E. Jones (1977) J. Biol. Chem. 252, 8374-8381]; in the presence of a nucleotidase, there was no measurable formation of orotidine from OMP under conditions where OMP was maintained at a steady-state concentration [T.W. Traut (1980) Arch. Biochem. Biophys. 200, 590-594]. Recently claims were made that (i) the steady-state activities of UMP synthase could be modeled by Michaelis-Menten kinetics, and (ii) the nucleotidase activity in Ehrlich ascites cells was insufficient to degrade any significant amount of OMP [R.W. McClard and K.M. Shokat (1987) Biochemistry 26, 3378-3384]. The present studies show that UMP synthase has cooperative kinetics toward OMP, and that a substrate cycle involving orotate phosphoribosyltransferase, cytoplasmic nucleotidase, and uridine phosphorylase maintains the cyclic interconversion: orotate----OMP----orotidine----orotate, etc. It is therefore the complex steady-state kinetics of UMP synthase in the presence of OMP, and the existence of a substrate cycle that account for the results which were interpreted as channeling in the earlier studies.