Crystal structure of quinolinic acid phosphoribosyltransferase from Mmycobacterium tuberculosis: a potential TB drug target.

BACKGROUND: . Mycobacterium tuberculosis is the single most deadly human pathogen and is responsible for nearly three million deaths every year. Recent elucidation of the mode of action of isoniazid, a frontline antimycobacterial drug, suggests that NAD metabolism is extremely critical for this microorganism. M. tuberculosis depends solely on the de novo pathway to meet its NAD demand. Quinolinic acid phosphoribosyltransferase (QAPRTase), a key enzyme in the de novo biosynthesis of NAD, provides an attractive target for designing novel antitubercular drugs. RESULTS: . The X-ray crystal structure of the M. tuberculosis QAPRTase apoenzyme has been determined by multiple isomorphous replacement at 2.4 A resolution. Structures of the enzyme have also been solved in complex with the substrate quinolinic acid (QA), the inhibitory QA analog phthalic acid (PA), the product nicotinate mononucleotide (NAMN), and as a ternary complex with PA and a substrate analog, 5-phosphoribosyl-1-(beta-methylene)pyrophosphate (PRPCP). The structure of the nonproductive QAPRTase-PA-PRPCP Michaelis complex reveals a 5-phosphoribosyl-1-pyrophosphate-binding site that is different from the one observed in type I phosphoribosyltransferases (PRTases). The type II PRTase active site of QAPRTase undergoes conformational changes that appear to be important in determining substrate specificity and eliciting productive catalysis. CONCLUSIONS: . QAPRTase is the only known representative of the type II PRTase fold, an unusual alpha/beta barrel, and appears to represent convergent evolution for PRTase catalysis. The active site of type II PRTase bears little resemblance to the better known type I enzymes.

A new function for a common fold: the crystal structure of quinolinic acid phosphoribosyltransferase.

BACKGROUND: Quinolinic acid (QA) is a neurotoxin and has been shown to be present at high levels in the central nervous system of patients with certain diseases, such as AIDS and meningitis. The enzyme quinolinic acid phosphoribosyltransferase (QAPRTase) provides the only route for QA metabolism and is also an essential step in de novo NAD biosynthesis. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide (NAMN) from QA and 5-phosphoribosyl-1-pyrophosphate (PRPP). The structures of several phosphoribosyltransferases (PRTases) have been reported, and all have shown a similar fold of a five-strandard beta sheet surrounded by four alpha helices. A conserved sequence motif of 13 residues is common to these 'type I' PRTases but is not observed in the QAPRTase sequence, suggestive of a different fold for this enzyme. RESULTS: The crystal structure of QAPRTase from Salmonella typhimurium has been determined with bound QA to 2.8 A resolution, and with bound NAMN to 3.0 A resolution. Most significantly, the enzyme shows a completely novel fold for a PRTase enzyme comprising a two-domain structure: a mixed alpha/beta N-terminal domain and an alpha/beta barrel-like domain containing seven beta strands. The active site is located at the C-terminal ends of the beta strands of the alpha/beta barrel, and is bordered by the N-terminal domain of the second subunit of the dimer. The active site is largely composed of a number of conserved charged residues that appear to be important for substrate binding and catalysis. CONCLUSIONS: The seven-stranded alpha/beta-barrel domain of QAPRTase is very similar in structure to the eight-stranded alpha/beta-barrel enzymes. The structure shows a phosphate-binding site that appears to be conserved among many alpha/beta-barrel enzymes including indole-3-glycerol phosphate synthase and flavocytochrome b2. The new fold observed here demonstrates that the PRTase enzymes have evolved their similar chemistry from at least two completely different protein architectures.

Primary structure and crystallization of orotate phosphoribosyltransferase from Salmonella typhimurium.

Orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) catalyzes phosphoribosyl group transfer between alpha-D-5-phosphoribosyl-1-pyrophosphate and orotate to form orotidine-5'-monophosphate and pyrophosphate, the nucleotide-forming step in pyrimidine biosynthesis. It is one of ten PRTases that perform vital roles in de novo and salvage pathways for purine, pyrimidine and pyridine nucleotides. Although the PRTases are important drug targets, they are poorly understood mechanistically, and no three-dimensional structures exist. Here, we report the complete sequence of the Salmonella typhimurium pyrE gene and the deduced sequence of the OPRTase gene product. OPRTase forms tetragonal crystals from polyethylene glycol solutions; these crystals diffract to better than 2 A resolution, and are stable to radiation damage. The space group is P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 48.5 A, c = 210.5 A, and alpha = beta = gamma = 90 degrees. A crystalline form of the selenomethionine derivative of the protein is also reported.

ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS.

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by "aging" in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. "Aged" or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO(3), which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or "aged" submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO(3) (-), but inhibited by Cl(-), indicating that Cl(-) stimulation is a distinguishing property of the pea mitochondrial ATPase complex.

Oligomycin-sensitive ATPase of Submitochondrial Particles from Corn.

To test the hypothesis (Plant Physiology 59: 155-157) that monocotyledons contain a unique oligomycin-insensitive ATPase, we prepared submitochondrial particles and a soluble fraction from sonicated corn mitochondria (Zea mays L. cv. Earliking). Although the ATPase activity of the whole sonicate was relatively insensitive to oligomycin, the corn submitochondrial particles possessed an ATPase activity that was nearly completely inhibited by oligomycin, and was activated by trypsin. This ATPase is similar to that from other sources (plants, animals, and microorganisms). The soluble fraction also contained an active ATPase, which was inhibited by azide and stimulated by sodium chloride and trypsin. The soluble fraction differed from other F(1)-ATPases in that it was cold-stable.

Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid/base.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile of kcat for IMP pyrophosphorolysis revealed an essential acidic group with pKa of 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease in kcat for nucleotide formation with Hx as substrate, a 275-fold decrease in kcat with Gua, and a 500-fold decrease in kcat for IMP pyrophosphorolysis. D137N also showed lower KD values for nucleotides and PRPP. The pH profiles of kcat for D137N were severely altered. In contrast to D137N, the kcat for K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. The Km and KD values showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.

Mechanism of Salmonella typhimurium histidinol dehydrogenase: kinetic isotope effects and pH profiles.

L-Histidinol dehydrogenase catalyzes the biosynthetic oxidation of L-histidinol to L-histidine with sequential reduction of two molecules of NAD. Previous isotope exchange results had suggested that the oxidation of histidinol to the intermediate histidinaldehyde occurred 2-3-fold more rapidly than overall catalysis. In this work, we present kinetic isotope effects (KIE) studies at pH 9.0 and at pH 6.7 with stereospecifically mono- and dideuterated histidinols. The data at pH 9.0 support minimal participation of the first hydride transfer and substantial participation of the second hydride transfer in the overall rate limitation. Stopped-flow experiments with protiated histidinol revealed a small burst of NADH production with stoichiometry of 0.12 per subunit, and 0.25 per subunit with dideuterated histidinol, indicating that the overall first half-reaction was not significantly faster than the second reaction sequence. Results from kcat and kcat/KM titrations with histidinol, NAD, and the alternative substrate imidazolyl propanediol demonstrated an essential base with pKa values between 7.7 and 8.4. In KIE experiments performed at pH 6.7 or with a coenzyme analogue at pH 9. 0, the first hydride transfer became more rate limiting. Kinetic simulations based on rate constants estimated from this work fit well with a mechanism that includes a relatively fast, and thermodynamically unfavorable, hydride transfer from histidinol and a slower, irreversible second hydride transfer from a histidinaldehyde derivative. Thus, although the chemistry of the first hydride transfer is fast, both partial reactions participate in the overall rate limitation.

Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues.

The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD. An essential base, with pKa about 8, is involved in catalysis. Here we report site-directed mutagenesis studies to replace each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine. In all cases, the overexpressed enzymes were readily purified and behaved as dimers. Substitution of His-261 and His-326 by asparagine caused about 7000- and 500-fold decreases in kcat, respectively, with little change in KM values. Similar loss of activity was also reported for a H261N mutant Brassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis. Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.

A new paradigm for biochemical energy coupling. Salmonella typhimurium nicotinate phosphoribosyltransferase.

The pncB gene of Salmonella typhimurium was used to develop an overexpression system for nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11), which forms nicotinate mononucleotide (NAMN) and PPi from nicotinate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAPRTase hydrolyzes ATP in 1:1 molar stoichiometry to NAMN synthesis. Hydrolysis of ATP alters the ratio of products/substrates for the reaction nicotinate + PRPP <--> NAMN + PPi from its equilibrium value of 0.67 to a steady-state value of 1100. The energy for the maintenance of this ratio must come from ATP hydrolysis. However, in contrast to other ATP-utilizing enzymes, when all ATP is hydrolyzed the unfavorable product/substrate ratio collapses. ATP/ADP exchange results suggest that the overall reaction involves a phosphoenzyme (E-P) arising from E.ATP. Km values for nicotinate and PRPP each decreased by 200-fold when ATP was present to phosphorylate the enzyme. PPi stimulated the ATPase activity of the enzyme to Vmax values, suggesting that PPi formation during catalysis provides a trigger for cleavage of the putative E-P in the overall reaction and regenerates the low affinity form of the enzyme. A model is presented in which alternation of high and low affinity forms of NAPRTase provides a "steady-state" coupling between ATP hydrolysis and NAMN formation.

Conserved cysteine residues of histidinol dehydrogenase are not involved in catalysis. Novel chemistry required for enzymatic aldehyde oxidation.

The 4-electron oxidoreductase L-histidinol dehydrogenase (HDH, EC 1.1.1.23) oxidizes the amino alcohol histidinol to histidine via an aldehyde-level intermediate at a single active site. The enzyme contains two Zn2+ per dimer, and treatment with metal chelators causes a metal-reversible inactivation. NAD-linked aldehyde oxidations, for which glyceraldehyde-3-phosphate dehydrogenase has served as the major paradigm, are thought to proceed via cysteine-based thiohemiacetals. Sequenced forms of HDH contain two conserved cysteine residues, Cys-116 and Cys-153 in the Salmonella typhimurium enzyme, and in previous work we have shown that HDH is inactivated by active site modification of Cys-116 by the reagent 4-nitro-7-chlorobenzadioxazole. Thus, Cys-116 is an excellent candidate for the active site nucleophile in HDH. In the current studies we show that treatment of HDH with the Zn2+ chelator 1,10-phenanthroline exposes Cys-116 to specific modification by iodoacetate, resulting in irreversible loss of activity. Site-specific mutagenesis was used to explore the roles of the conserved cysteine residues. The mutant enzymes C116S, C153S, C116A, and C153A and the double mutant C116,153A were each overproduced and purified to homogeneity. All mutant enzymes showed normal kcat and Km values for catalysis. The double mutant protein was unstable, and the single mutants also lose significant activities over a 3-h period during which wild-type enzyme retains full activity. The C116S mutant, and to a lesser extent the C116A mutant, were sensitive to the presence of EDTA in the assay medium, but the other mutants or wild-type enzyme were not, suggesting that Cys-116 may be near, but probably not liganded to, the bound metal ion. The results clearly indicate that HDH does not use a cysteine-based thiohemiacetal as a catalytic intermediate, requiring a new paradigm for NAD-linked aldehyde oxidation. Some models for the reaction are presented and discussed.

Active site lysines in orotate phosphoribosyltransferase.

Orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) catalyzes the formation of the nucleotide orotidine-5'-monophosphate from orotate and 5-phosphoribosyl-1-pyrophosphate (PRPP). The bacterial enzyme, unlike its mammalian homolog, is monofunctional and is a dimer of M(r) 23,000 subunits. The availability of large amounts of highly purified crystalline Salmonella typhimurium OPRTase have enabled us to being structure/function studies on the enzyme. Like other phosphoribosyltransferases, OPRTase binds the highly charged MgPRPP complex, as well as anionic orotate, suggesting an active site containing basic residues. The S. typhimurium sequence (Scapin, G., Sacchettini, J. C., Dessen, A., Bhatia, M., and Grubmeyer, C. (1993) J. Mol. Biol. 230, 1304-1308) contains 12 lysine and 13 arginine residues, of which Lys-26, Arg-99, Lys-100, Lys-103, and Arg-156 are conserved as identities among the sequences of OPRTases from other organisms, with Lys-19 and Arg-161 replaced by the alternative basic residue in one or more sequences. The lysine modifier 2,4,6-trinitrobenzene sulfonate inactivated S. typhimurium OPRTase in a pseudo first-order process, and OMP and PRPP provided good protection against inactivation. Spectral quantitation of trinitrophenyl (TNP) group incorporation showed that inactivation was correlated with incorporation of one TNP group per subunit. Surprisingly, tryptic proteolysis followed by high performance liquid chromatography and amino acid sequence analysis revealed that four peptides, containing three distinct lysines, had been modified. Peptides modified at Lys-26, Lys-100 and Lys-103, as well as a doubly modified peptide containing TNP groups at Lys-100 and Lys-103, were identified. Inactivation kinetics showed that the 3 lysine residues were modified at equal rates. Protection studies demonstrated that Lys-26, and to a lesser extent Lys-100 and Lys-103, were protected against modification by OMP, whereas PRPP protected Lys-26, Lys-100 and Lys-103. Pyrophosphate protected Lys-100 and Lys-103. The results suggest active site locations for the sequence-conserved and TNP-modified lysine residues, with Lys-26 interacting with the ribose-5-phosphate moiety of OMP and PRPP, and Lys-100 and Lys-103 interacting with the pyrophosphate moiety of PRPP.

The presence of two hydrolytic sites on beef heart mitochondrial adenosine triphosphatase.

The ribose-modified nucleotides 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP were used to probe the catalytic sites on soluble beef heart mitochondrial adenosine triphosphatase (F1). Both compounds were potent competitive inhibitors of ATP hydrolysis catalyzed by F1, Ki = 5.5 and 10 nM, respectively, and by submitochondrial particles, Ki (TNP-ATP) = 21 nM. Both compounds also were potent competitive inhibitors of ATP synthesis during oxidative phosphorylation (Ki = 1300 nM). Both analogs inhibited the 32Pi-ATP exchange reaction and the ATP-dependent reduction of NAD+ by succinate, catalyzed by submitochondrial particles. TNP-ATP and TNP-ADP were bound by F1. The presence of two binding sites on the enzyme for TNP-adenine nucleotides was determined by titrations of difference absorbance spectra, of the increase in fluorescence of the analog which occurred upon interaction with protein, and by titrations with the centrifuge column method using 32P-labeled TNP-adenine nucleotides. The first binding site bound the analogs with an affinity too high to be measured. The Kd for analog binding by the second site was 20 to 80 nM. In the presence of Mg2+, the 2 sites were filled with the TNP-ATP at a rate too rapid to be resolved by the procedure used. TNP-[gamma-32P]ATP was hydrolyzed by F1, Km = 0.2 microM, Vmax = 1.1 mol of 32Pi formed/mol of F1/s. It was shown, using the isotope trap technique as well as the inhibitor efrapeptin, that the 2 binding sites for TNP-ATP on F1 are hydrolytic sites.

Cooperatively between catalytic sites in the mechanism of action of beef heart mitochondrial adenosine triphosphatase.

Occupancy of only one of two hydrolytic sites on beef heart mitochondrial ATPase (F1) by the radioactive ATP analog, 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-[gamma-32P]-triphosphate (TNP-[gamma-32P]ATP) is associated with a low rate of hydrolysis of the substrate even under conditions otherwise favoring catalysis. Addition of excess nonradioactive TNP-ATP, in concentrations sufficient to fill catalytic Site 2 on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), accelerates the rate of hydrolysis of the radioactive substrate 15- to 20-fold. Since the excess nonradioactive substrate serves as an effective isotope trap, the involvement of medium TNP-[gamma-32P]-ATP as an intermediate is ruled out. These observations constitute direct evidence for catalytic cooperativity between active sites on F1. It is proposed that the use of high binding affinity substrates or substrate analogs, combined with the isotope trap technique, offers a new approach to the detection and study of catalytic site cooperativity in enzymes. The hydrolyzable nucleotides GTP, ITP, and ATP are excellent promoters of the hydrolysis of previously bound TNP-[gamma-32P]ATP whereas addition of nonhydrolyzable nucleotides such as TNP-ADP, ADP, and adenylyl imidodiphosphate result in a lower rate and extent of hydrolysis. AMP is without effect. Studies of the hydrolysis of [gamma-32P]ATP and TNP-[gamma-32P]ITP, under appropriate conditions, also provide evidence consistent with promoted catalysis. Based upon these findings, a model is presented for the mechanism of action of F1 in which site-site cooperativity reflects promoter-dependent hydrolysis of bound substrate.

A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase.

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.

Purification and in vitro complementation of mutant histidinol dehydrogenases.

The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the hisO1242 (constitutive overproducer) attenuator mutation and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl2, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Neither mutant protein showed negative complementation with wild-type enzyme. The Vmax for hybrid histidinol dehydrogenase was 11% of that for native enzyme, with only minor changes in Km values for substrate or coenzyme. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound 54Mn2+ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound 54Mn2+ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 2-mercaptoethanol was prevented by low levels of MnCl2. It thus appears that mutant histidinol dehydrogenase molecules bind metal ion poorly. The complementation procedure may allow for formation of a functional Mn2+-binding site, perhaps at the subunit interface.

Partial characterization of a soluble ATPase from pea cotyledon mitochondria.

A partially purified soluble ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pea cotyledon mitochondria was characterized. Inhibition patterns with azide, NaF, and cold, and a stimulation by 2,4-dinitrophenol were typical of F1-ATPases from mammalian mitochondria. The enzyme hydrolysed GTP, ITP, and ATP, but not CTP, UTP, ADP, or IDP. ATPase and ITPase activities were strongly inhibited by ADP and to a lesser extent by IDP. Distinctive properties of the pea mitochondrial enzyme were activation by high concentrations of CaCl2 and stimulation by NaCl.

The role of divalent magnesium in activating the reaction catalyzed by orotate phosphoribosyltransferase.

Orotate phosphoribosyltranferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate from the nitrogenous base orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). While it is known that Mg2+ is necessary for catalysis, the mechanism of activation of the phosphoribosyl transfer by Mg2+ remains unclear. The divalent cation may activate the phosphoribosyl transfer by binding to either or both substrates PRPP and orotate or/and the enzyme. In this work we chose to explore the role of divalent magnesium in activating the phosphoribosyl transfer in bacterial OPRTase. Studies on the effect of Mg2+ on the OPRTase-catalyzed reaction indicated that the divalent metal was necessary for catalysis. A maximal rate of 70 units/mg was achieved at 2 mM MgCl2. Mn2+ could replace Mg2+ as the divalent metal. Orotate methyl ester (OAME) and uracil, neither of which form chelates with divalent metal, were found to be substrates for OPRTase. The KM for OAME and uracil were 190 microM and 2.63 mM and kcat/KM were 0.91 x 10(5) and 6 M-1 s-1, respectively. These values compare with a KM of 27 microM for orotate, 44 microM for PRPP, and a kcat/KM of 1.3 x 10(6) M-1 s-1 for orotate. Spectroscopic studies failed to reveal the existence of Mg(2+)-orotate complexes. Thus we have concluded that an orotate-metal complex is not necessary for OPRTase catalysis. Metal-enzyme binding studies indicate that only weak metal-enzyme complexes may form in bacterial OPRTase. Thus the role of divalent metal in bacterial OPRTase must be to bind PRPP.

Transition state structure of Salmonella typhimurium orotate phosphoribosyltransferase.

Orotate phosphoribosyltransferase (OPRTase) catalyzes the magnesium-dependent conversion of alpha-D-phosphoribosylpyrophosphate (PRPP) and orotate to orotidine 5'-monophosphate (OMP) and pyrophosphate. We have determined kinetic isotope effects on the reaction of OMP with pyrophosphate and with the pyrophosphate analog phosphonoacetic acid. In the latter case, full expression of the kinetic isotope effects allowed us to calculate the structure of the transition state for the pyrophosphorylytic reaction. The transition state resembles a classical oxocarbonium ion. Using the recently reported three-dimensional structures of the OPRTase-OMP (Scapin et al., 1994) and the OPRTase-PRPP complexes (Scapin et al., 1995a), we have modeled the calculated transition state structure into the active site of OPRTase. We propose a detailed chemical mechanism which is consistent with these results.