Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.     

Catalytic mechanism of the cyclohydrolase activity of human aminoimidazole carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Using recently determined crystal structures of ATIC as a guide, four candidate residues, Lys66, Tyr104, Asp125, and Lys137, were identified for site-directed mutagenesis to study the cyclohydrolase activity of this bifunctional enzyme. Steady-state kinetic experiments on these mutants have shown that none of these residues are absolutely required for catalytic activity; however, they strongly influence the efficiency of the reaction. Since the FAICAR binding site is made up mostly of backbone interactions with highly conserved residues, we postulate that these conserved interactions orient FAICAR in the active site to favor the intramolecular ring closure reaction and that this reaction may be catalyzed by an orbital steering mechanism. Furthermore, it was shown that Lys137 is responsible for the increase in cyclohydrolase activity for dimeric ATIC, which was reported previously by our laboratory. From the experiments presented here, a catalytic mechanism for the cyclohydrolase activity is postulated.     

Crystal structures of human bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase/IMP cyclohydrolase in complex with potent sulfonyl-containing antifolates

Aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (ATIC) is a bifunctional enzyme with folate-dependent AICAR transformylase and IMP cyclohydrolase activities that catalyzes the last two steps of purine biosynthesis. The AICAR transformylase inhibitors BW1540 and BW2315 are sulfamido-bridged 5,8-dideazafolate analogs with remarkably potent K(i) values of 8 and 6 nm, respectively, compared with most other antifolates. Crystal structures of ATIC at 2.55 and 2.60 A with each inhibitor, in the presence of substrate AICAR, revealed that the sulfonyl groups dominate inhibitor binding and orientation through interaction with the proposed oxyanion hole. These agents then appear to mimic the anionic transition state and now implicate Asn(431') in the reaction mechanism along with previously identified key catalytic residues Lys(266) and His(267). Potent and selective inhibition of the AICAR transformylase active site, compared with other folate-dependent enzymes, should therefore be pursued by further design of sulfonyl-containing antifolates.     

The kinetic mechanism of the human bifunctional enzyme ATIC (5-amino-4-imidazolecarboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase). A surprising lack of substrate channeling

5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of substrate channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.     

Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5''-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.

With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5'-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements producing the cytotoxic effect of this drug by (1) producing tighter binding of methotrexate to folate-dependent enzymes, (2) producing inhibitors of folate-dependent enzymes from their tetrahydrofolate coenzymes, and (3) trapping toxic amounts of adenine nucleosides and nucleotides as a result of inhibition of adenosine deaminase and 5'-adenylate deaminase respectively.     

Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay.

The role of insulin in modulating phosphoinositide breakdown and accumulation of inositol phosphates was investigated in isolated rat pancreatic islets by using GPAIS (guinea-pig anti-insulin antiserum) that neutralizes effects of insulin in the medium. At either 3.0 mM- or 16.7 mM-glucose or 3.0 mM-glucose plus 10 microM-arecaidine propargyl ester (muscarinic receptor agonist), GPAIS (but not control serum) was able to increase InsP2 and InsP3, but not InsP, in myo-[3H] inositol-prelabelled islets. The effect of GPAIS on 3H incorporation into InsP3 was dose-dependent, with a half-maximal effect at a concentration able to bind 4004 +/- 163 microunits of insulin. A specific mass assay of the biologically relevant isomer Ins (1,4,5)P3 revealed a huge increase (greater than 3-folf). Formation of PtdIns, PtdInsP and PtdInsP2 was not affected by GPAIS. This is indirect evidence for an effect of insulin on inositide metabolism, and therefore endogenously released insulin may have led to an underestimation in earlier studies of effects of insulinotropic substances on inositol phosphate accumulation.     

Antifolates induce primary inhibition of the de novo purine pathway prior to 5-aminoimidazole-4-carboxamide ribotide transformylase in leukemia cells.

Polyglutamated dihydrofolate, accumulated as a result of potent inhibition of dihydrofolate reductase (DHFR), has been postulated to directly inhibit the purine pathway at 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (reaction 9) in leukemia cells exposed to methotrexate (MTX). We have observed that 25 microM MTX or piritrexim, a "non-classical" antifolate, induce several-fold accumulations of AICAR and N-succino-AICAR to a combined cellular concentration of 89 microM in mouse L1210 leukemia cells after 2 h. By contrast, complete inhibition of reaction 4 by 25 microM azaserine results in accumulation of N-formyl-glycinamide ribotide (FGAR) polyphosphates to a combined cellular concentration of greater than 10 mM. MTX prevented azaserine-induced accumulation of FGAR polyphosphates. Hence, these antifolates induce primary inhibition of the de novo purine pathway at, or prior to, glycinamide ribotide transformylase (reaction 3).     

5-aminoimidazole-4-carboxamide riboside administration and purine release from the hypoxic/hypotensive rat cerebral cortex

The effects of the purine precursor 5-aminoimidazole-4-carboxamide riboside (AICAriboside) on the release of purines from cerebral cortices of normoxic and hypoxic/hypotensive rats was studied with the cortical cup technique. AICAriboside was administered either intravascularly (50 mg/kg) or intraperitoneally (500 mg/kg) to ascertain whether this agent can be used to enhance adenosine levels in the cerebral cortical interstitial fluid. Following its intraperitoneal administration AICAriboside appeared rapidly in the cortical superfusates at concentrations of up to 9 μM and remained at this level for a 90 min period. After intravascular administration, AICAriboside levels peaked at 2 μM, and then declined rapidly. No increases in basal (normoxic) or hypoxia-elicited adenosine levels in the cortical superfusates were observed. Increases did occur in the basal and hypoxia-evoked levels of hypoxanthine, xanthine and, especially, of uric acid. AICAriboside administration appears to have caused an increase in adenosine metabolite, rather than in adenosine, levels in the cerebral interstitial fluid and it may therefore be of little benefit as a precursor for adenosine formation and release in the treatment of cerebral ischemic damage.     

Occurrence and biosynthesis of 5-aminoimidazole-4-carboxamide ribonucleotide and N-(beta-D-ribofuranosyl)formamide 5'-phosphate in Methanobacterium thermoautotrophicum delta(H).

5-Aminoimidazole-4-carboxamide ribonucleotide (ZMP) and N-(beta-D-ribofuranosyl)formamide 5'-phosphate (FAR-P) have been identified as products of the metabolism of ATP and 5-phospho-alpha-D-ribosyl diphosphate by Methanobacterium thermoautotrophicum delta(H), a member of the domain Archaea. Evidence indicates that the first three steps in the pathway to the formation of these compounds are the same as the first three steps of histidine biosynthesis and lead to the generation of pro-phosphoribosyl formimino-5-aminoimidazole-4-carboxamide ribonucleotide (5'-proFAR). The 5'-proFAR then undergoes hydrolysis to ZMP and FAR-P. The reaction was detected by an unexpected high concentration of ZMP in cell extracts of M. thermoautotrophicum delta(H).     

Determination of 5-aminoimidazole-4-carboxamide in human plasma by ion-pair extraction and LC-MS/MS

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was established for the determination of 5-aminoimidazole-4-carboxamide (AICA) in human plasma. The method included a solvent extraction of AICA as an ion pair with 1-pentanesulfonate ion and a separation on a Hypersil ODS2 column with the mobile phase of methanol-water (68:32, v/v). Determination was performed using an electrospray ionization source in positive ion mode (ESI(+)). Multiple reaction monitoring (MRM) was utilized for the detection monitoring m/z at 127-->110 for AICA, and 172-->128 for IS. The calibration curve was linear within a range from 20 to 2000 ng/mL and the limit of quantity for AICA in plasma was 20 ng/mL. RSD of intra-assay and inter-assay were no more than 5.90% and 5.65%.     

Crystal structure and function of 5-formaminoimidazole-4-carboxamide ribonucleotide synthetase from Methanocaldococcus jannaschii

Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.     

Effect of nitrous oxide-induced inactivation of vitamin B12 on glycinamide ribonucleotide transformylase and 5-amino-4-imidazole carboxamide transformylase

Exposure to nitrous oxide (N2O) in vivo is accompanied by oxidation of cob[I]-alamin to the inactive cob[III]alamin [1]. There is loss of methionine synthetase activity [2] and evidence of depressed supply of single carbon units at the formate level of oxidation [3,4,5]. We measured the effect of inactivation of B12 on the folate-dependent transformylases concerned in purine synthesis. After 24 h exposure to N2O there was a significant fall in glycinamide ribonucleotide transformylase (EC 2.1.2.2) and a significant increase in 5-amino-4-imidazole carboxamide transformylase (EC 2.1.2.3).     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27?kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553?nm), in contrast to strain CE3 which produced a single 32?kDa c-type protein and did not produce the 553?nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553?nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli.     

Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia

Nodules of tropical legumes generally export symbiotically fixed nitrogen in the form of ureides that are produced by oxidation of de novo synthesized purines. To investigate the regulation of de novo purine biosynthesis in these nodules, we have isolated cDNA clones encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase from a mothbean (Vigna aconitifolia) nodule cDNA library by complementation of Escherichia coli purE and purC mutants, respectively. Sequencing of these clones revealed that the two enzymes are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide. As is the case in yeast, the mothbean AIR carboxylase has a N-terminal domain homologous to the eubacterial purK gene product. This PurK-like domain appears to facilitate the binding of CO₂ and is dispensable in the presence of high CO₂ concentrations. Because the expression of the mothbean PurE cDNA clone in E. coli apparently generates a truncated polypeptide lacking at least 140 N-terminal amino acids, this N-terminal region of the enzyme may not be essential for its CO₂-binding activity.     

Crystal structure of avian aminoimidazole-4-carboxamide ribonucleotide transformylase in complex with a novel non-folate inhibitor identified by virtual ligand screening

Aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase), one of the two folate-dependent enzymes in the de novo purine biosynthesis pathway, is a promising target for anti-neoplastic chemotherapy. Although classic antifolates, such as methotrexate, have been developed as anticancer agents, their general toxicity and drug resistance are major issues associated with their clinical use and future development. Identification of inhibitors with novel scaffolds could be an attractive alternative. We present here the crystal structure of avian AICAR Tfase complexed with the first non-folate based inhibitor identified through virtual ligand screening of the National Cancer Institute Diversity Set. The inhibitor 326203-A (2-[5-hydroxy-3-methyl-1-(2-methyl-4-sulfophenyl)-1H-pyrazol-4-ylazo]-4-sulfo-benzoic acid) displayed competitive inhibition against the natural cofactor, 10-formyl-tetrahydrofolate, with a K(i) of 7.1 mum. The crystal structure of AICAR Tfase with 326203-A at 1.8 A resolution revealed a unique binding mode compared with antifolate inhibitors. The inhibitor also accessed an additional binding pocket that is not occupied by antifolates. The sulfonate group of 326203-A appears to form the dominant interaction of the inhibitor with the proposed oxyanion hole through interaction with a helix dipole and Lys(267). An aromatic interaction with Phe(316) also likely contributes to favorable binding. Based on these structural insights, several inhibitors with improved potency were subsequently identified in the National Cancer Institute Compound Library and the Available Chemical Directory by similarity search and molecular modeling methods. These results provide further support for our combined virtual ligand screening rational design approach for the discovery of novel, non-folate-based inhibitors of AICAR Tfase.     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli. Received: 21 November 1996 / Accepted: 22 January 1997