Cell to cell contact is not required for bystander cell killing by Escherichia coli purine nucleoside phosphorylase

Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.     

Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway, which provides an alternative to the de novo pathway for the biosynthesis of purine nucleotides. PNP catalyzes the reversible phosphorolysis of 2'-deoxypurine ribonucleosides to the free bases and 2-deoxyribose 1-phosphate. Absence of PNP activity in humans is associated with specific T-cell immune suppression. Its key role in these two processes has made PNP an important drug design target. We have investigated the structural details of the PNP-catalyzed reaction by determining the structures of bovine PNP complexes with various substrates and substrate analogues. The preparation of phosphate-free crystals of PNP has allowed us to analyze several novel complexes, including the ternary complex of PNP, purine base, and ribose 1-phosphate and of the completely unbound PNP. These results provide an atomic view for the catalytic mechanism for PNP proposed by M. D. Erion et al. [(1997) Biochemistry 36, 11735-11748], in which an oxocarbenium intermediate is stabilized by phosphate and the negative charge on the purine base is stabilized by active site residues. The bovine PNP structure reveals several new details of substrate and inhibitor binding, including two phosphate-induced conformational changes involving residues 33-36 and 56-69 and a previously undetected role for His64 in phosphate binding. In addition, a well-ordered water molecule is found in the PNP active site when purine base or nucleoside is also present. In contrast to human PNP, only one phosphate binding site was observed. Although binary complexes were observed for nucleoside, purine base, or phosphate, ribose 1-phosphate binding occurs only in the presence of purine base.     

Mode of action of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl) cycloprop-1'-yl]methyl]guanine (A-5021) against herpes simplex virus type 1 and type 2 and varicella-zoster virus

The mode of action of (1'S,2'R)-9-([1', 2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl)guanine (A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) was studied. A-5021 was monophosphorylated at the 2' site by viral thymidine kinases (TKs). The 50% inhibitory values for thymidine phosphorylation of A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for penciclovir (PCV) and lower than those for acyclovir (ACV). Of these three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated more than ACV triphosphate but less than PCV triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5 cells had comparable levels of A-5021 and ACV triphosphates. The intracellular half-life of A-5021 triphosphate was considerably longer than that of ACV triphosphate and shorter than that of PCV triphosphate. A-5021 triphosphate competitively inhibited HSV DNA polymerases with respect to dGTP. Inhibition was strongest with ACV triphosphate, followed by A-5021 triphosphate and then (R,S)-PCV triphosphate. A DNA chain elongation experiment revealed that A-5021 triphosphate was incorporated into DNA instead of dGTP and terminated elongation, although limited chain extension was observed. Thus, the strong antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV and on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV.     

Purine nucleoside phosphorylase inhibitors: biochemical and pharmacological studies with 9-benzyl-9-deazaguanine and related compounds

Certain derivatives of 9-deazaguanine that contain arylmethyl, heteroarylmethyl or cycloalkylmethyl groups at the 9-position are potent inhibitors of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). To determine whether these agents can produce metabolically significant inhibition of PNP in cells and in animals, the authors performed pharmacological studies with a representative member of the series, 9-benzyl-9-deazaguanine (BzDAG). BzDAG was a potent inhibitor of PNP from calf spleen (Ki = 12 nM). It was also an effective inhibitor of PNP in cells and in animals as shown by the findings that it 1) inhibited the conversion of inosine to nucleotides in L1210 cells in culture at concentrations that had little effect on the utilization of hypoxanthine; 2) potentiated the toxicity of deoxyguanosine to CCRF-CEM cells in culture; 3) increased the pools of deoxy GTP in CCRF-CEM, Molt-3 and Molt-4 cells that had been treated with deoxyguanosine; 4) prevented the toxicity of 6-thioguanosine to HEp-2 cells in culture; 5) increased the plasma levels of endogenous inosine in rats; and 6) increased the plasma levels of 2',3'-dideoxyinosine in rats that had received BzDAG and dideoxyinosine in combination. Pharmacokinetic analysis of BzDAG in the rat showed it to be 48% orally bioavailable (at a dose of 5 mg/kg). About 95% of BzDAG was protein bound. After i.v. administration of BzDAG (5 mg/kg), more than 50% of the erythrocyte PNP was inhibited for 40 min. These results indicate that the 9-substituted-9-deazaguanines are potent orally active PNP inhibitors and are therefore of potential clinical interest as immunosuppressive and anti-inflammatory agents.     

Intron-exon structure of the MET gene and cloning of an alternatively-spliced Met isoform reveals frequent exon-skipping of a single large internal exon

BACKGROUND AND AIMS: the purine nucleoside analogues cladribine (CdA), fludarabine (F-Ara-AMP) and pentostatin (dCf), are effective therapy for a range of T- and B-cell lymphoid malignancies. The effects upon nucleotide metabolism in human CCRF-CEM T-cell leukaemia and Raji B-cell lymphoma cell lines of these drugs have been compared to assess possible mechanisms of cytotoxicity. METHODS: Leukaemia cells were exposed to a purine nucleoside analogue and perchloric acid extracts were analysed by HPLC for 2'-deoxynucleoside-5'-triphosphates (dNTPs), nucleoside-5'-triphosphates (NTPs) and drug metabolites. RESULTS: After addition of a purine nucleoside analogue, CdA-TP and F-Ara-ATP accumulate in cells while the levels of dCf-TP formed were not detectable by ultra-violet absorbance. In response to accumulating concentrations of drug triphosphate, the cellular levels of dNTPs initially decrease (0-4 h), then accumulate above their initial levels (4-10 h) before slowly declining beyond 10 h. NTPs also accumulate during the period 4-10 h before declining at later times. CONCLUSION: The temporal effects on the levels of dNTPs and NTPs of the 3 purine nucleoside analogues are similar against CCRF-CEM and Raji cells. However, CdA induces major depletions of dTTP, dGTP and dATP in CCRF-CEM cells and F-Ara-A induces a major accumulation of dATP in Raji cells.     

The use of computed tomography in stereotaxic operations in dyskinesia patients

The pool of free purine derivatives and activities of the key enzymes of purine metabolism (adenosine deaminase, purine nucleoside phosphorylase, and 5'-nucleotidase) in lymphocytes, erythrocytes, and epidermis homogenates were measured in 20 normal subjects and 15 patients with psoriasis by high-performance liquid chromatography. The levels of AMP, GMP, and IMP purine monophosphates are decreased in the epidermis and red cells of psoriasis patients, whereas the final products of hypoxanthine, xanthine, and uric acid metabolism are accumulating, and the activities of ADA and PNP are increased double in the skin, all this indicating purine derivatives catabolism.     

The antiproliferative and cell cycle effects of 5,6,7, 8-tetrahydro-N5,N10-carbonylfolic acid, an inhibitor of methylenetetrahydrofolate dehydrogenase, are potentiated by hypoxanthine

5,6,7,8-Tetrahydro-N5,N10-carbonylfolic acid (LY354899) has been demonstrated to inhibit the dehydrogenase activity of C1-tetrahydrofolate synthase. This compound was only moderately antiproliferative toward CCRF-CEM lymphocytic leukemia cells in culture, but induced apoptosis after long incubation times. Slightly greater potency was observed in CEM cells adapted to grow in low folate media. Cell cycle alterations induced by LY354899 were unique relative to antifolates that inhibit either the purine or thymidine de novo biosynthetic pathways. Based on the observed changes in DNA content, we hypothesized that inhibition of the dehydrogenase resulted in two temporally distinct events: the first was a purineless-like effect and the second was a thymineless-like effect that resulted in apoptosis. To test this hypothesis, we combined LY354899 with the purine salvage metabolite, hypoxanthine. This combination resulted in an earlier and more dramatic apoptotic response, indicating that the thymineless effect had been potentiated. Biochemical analysis of ribo- and deoxyribonucleoside triphosphates confirmed that inhibition of the dehydrogenase activity initially resulted in decreased pools of deoxypurines and deoxypyrimidines, followed 16 hr later by an increase in deoxyadenosine triphosphate (dATP) and a further decrease in deoxythymidine triphosphate (dTTP). These studies demonstrate that the inhibition of the dehydrogenase activity of C1-tetrahydrofolate synthase may represent a viable target for the development of novel antifolates. The results are discussed in terms of deoxypurine and deoxypyrimidine biosynthesis.     

7-Deazapurine 2'-deoxyribofuranosides are noncleavable competitive inhibitors of Escherichia coli purine nucleoside phosphorylase (PNP)

A series of 7-deazapurine 2'-deoxyribofuranosides were synthesized according to already known procedures and their substrate and inhibitor properties with purified E. coli purine nucleoside phosphorylase were examined. In agreement with previous findings, substrate activity was not detected for any of the compounds tested. Most of the nucleosides showed weak inhibition in the preliminary screening, i.e. at a concentration of about 100 microM. However some combinations of 6-chloro, 6-amino or 6-methoxy substituents with bulky hydrophobic groups at position 7 of the base and/or chloro, amino, methoxy or methylthio group at position 2 markedly enhanced affinity of such modified nucleosides for the E. coli enzyme. The most potent inhibition was observed for two nucleosides: 6-chloro- and 2-amino-6-chloro-7-deazapurine 2'-deoxyribofuranosides that show inhibition constants Ki = 2.4 and 2.3 microM, respectively. Several other compounds were also found to be good inhibitors, with inhibition constants in the range 5-50 microM. In all instances the inhibition was competitive vs. the nucleoside substrate 7-methylguanosine. Inhibition constants for 7-deazapurine nucleosides are in general several-fold lower than those observed for their purine counterparts. Therefore 7-deaza modification together with substitutions at positions 2, 6 and 7 of the base is a very promising approach to obtain competitive noncleavable inhibitors of E. coli PNP that may bind to the enzyme with inhibition constants in the microM range.     

Relative efficiency of tumor cell killing in vitro by two enzyme-prodrug systems delivered by identical adenovirus vectors

Enzyme-prodrug therapy for the treatment of cancer is an experimental procedure that is under intensive investigation. However, the relative merits of the various systems for use under specific conditions are still being determined. We have compared the efficacy of cell killing by the herpesvirus thymidine kinase (HSVTK)/ganciclovir and the purine nucleoside phosphorylase (PNP)/9-(beta-M-2-deoxy-erythropentofuranosyl)6-methylpurine enzyme/prodrug systems. These were chosen because of their differential dependence on DNA replication for their mechanism of action. The HSVTK and PNP genes, expressed from the identical prostate-specific antigen promoter, were transduced into human prostate and breast cancers cells using the same human adenovirus vector. The kinetics of cell killing in the presence of the respective prodrugs was monitored using a nondestructive assay that measured total cell bioactivity. The PNP/9-(beta-D-2-deoxy-erythropentofuranosyl)6-methylpurine system was clearly superior in its ability to cause cell death in vitro. Cells were killed in about half the time and at a 5-10-fold lower input of virus relative to the HSVTK/ganciclovir system. The PNP system may offer advantages for the treatment of slow-growing tumors in which the daily proliferative rate is low or in situations in which gene delivery or expression is inefficient.     

Structure-based design of inhibitors of purine nucleoside phosphorylase. 3. 9-Arylmethyl derivatives of 9-deazaguanine substituted on the methylene group

X-ray crystallography and computer-assisted molecular modeling (CAMM) studies aided in the design of a potent series of mammalian purine nucleoside phosphorylase (PNP) inhibitors. Enhanced potency was achieved by designing substituted 9-(arylmethyl)-9-deazaguanine analogs that interact favorably with all three of the binding subsites of the PNP active site, namely the purine binding site, the hydrophobic pocket, and the phosphate binding site. The most potent PNP inhibitor prepared during our investigation, (S)-9-[1-(3-chlorophenyl)-2-carboxyethyl]-9-deazaguanine (18b), was shown to have an IC50 of 6 nM, whereas the corresponding (R)-isomer was 30-fold less potent.     

The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology

BACKGROUND: Purine nucleoside phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2'-deoxy)ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine nucleosides as substrates. The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme. The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction. Structural comparison of the active sites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. RESULTS: The crystal structure of E. coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E. coli uridine phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different. In E. coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar. The phosphate-binding site probably consists of two main-chain nitrogen atoms and three arginine residues. In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. CONCLUSIONS: The structures of human and E. coli PNP define two possible classes of nucleoside phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. coli PNP but not the human enzyme.     

Excess histidine enzymes cause AICAR-independent filamentation in Escherichia coli

High-level expression of the hisHAFI genes in Escherichia coli, cloned under the control of an IPTG-inducible promoter, caused filamentation, as previously reported in Salmonella typhimurium. We speculated that this filamentation might be produced by an action of the HisH and HisF enzymes on their product AICAR (amino-imidazole carboxamide riboside 5'-phosphate), a histidine by-product and normal purine precursor, possibly by favouring the formation of ZTP, the triphosphate derivative of AICAR. However, filamentation occurred even in the absence of carbon flow through the histidine and purine pathways, as observed in a hisG purF strain lacking the first enzyme in each pathway. Filamentation thus does not require either the normal substrate or products of the overproduced histidine enzymes and must reflect another activity.     

Reversed-phase gradient high-performance liquid chromatographic procedure for simultaneous analysis of very polar to nonpolar retinoids, carotenoids and tocopherols in animal and plant samples

Recently, we have demonstrated that adenosine and 2'-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2'-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2'-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2'-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2'-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2'-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2'-deoxyadenosine kinase with iodotubercidin and 5'-amino-5'-deoxyadenosine had no protective effect against 2'-deoxyadenosine toxicity. [3H]-thymidine incorporation was inhibited over 90% by 2'-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2'-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2'-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity.     

The effect of a nucleotide-nucleoside solution on hepatic regeneration after partial hepatectomy in rats

After hepatectomy, purine and pyrimidine metabolism is a key process in the synthesis of DNA and RNA and maintaining cellular energy metabolism. The purpose of this study is to evaluate changes in blood purine and pyrimidine levels after partial hepatectomy and the effect of purine and pyrimidine nucleoside solution injection on hepatic regeneration under the hypothesis that the rat after partial hepatectomy requires substrates for salvage nucleotide synthesis and changes blood nucleoside and nucleobase levels. Blood levels of nucleotides, nucleosides, and nucleobase by high-performance liquid chromatography method and liver ATP level by enzymatic analysis, and the effect of preoperative injection of nucleoside solution (OG-VI) on hepatic regeneration ratio and hepatocytes DNA synthesis, were assessed in rats after 70% partial hepatectomy. Decreased liver adenosine triphosphate and increased plasma xanthine and hypoxanthine after partial hepatectomy indicated an increase in catabolism of purine nucleotides in regenerating liver. Plasma thymidine and cytidine levels increased, then returned to the prevalue, suggesting that the thymidine and cytidine pool was enlarged. OG-VI increased labeling indices of hepatocytes at postoperative d 1 (POD) and hepatic regeneration ratio at POD 14. Blood purine nucleobase and pyrimidine nucleoside levels change after partial hepatectomy and preoperative supply of nucleoside solution is effective for increasing hepatocytes DNA synthesis and hepatic regeneration after partial hepatectomy.     

Erythroid differentiation and growth inhibition of K562 cells by 2',5'-dideoxyadenosine: synergism with interferon-alpha

We found that 2',5'-dideoxyadenosine (DDA), a P-site specific adenylate cyclase inhibitor, inhibited the growth of K562 cells and caused them to become benzidine positive. The continuous exposure of cells to DDA was needed to recruit cells for growth inhibition and differentiation. Fetal calf or human sera were also necessary for DDA to induce differentiation. DDA at a concentration of 1.5 mM with serum induced 98% of the cells to produce hemoglobin and inhibited their growth to 15% of that of the control. An increase of epsilon-globin mRNA and a decrease of c-myc and c-myb mRNA occurred only during differentiation in the presence of fetal calf serum (FCS). An incubation with DDA and interferon-alpha (IFN-alpha) or hemin synergistically induced more benzidine-positive cells than in the presence of DDA alone, although IFN-alpha did not trigger differentiation by itself. The erythroid differentiation and growth inhibition were, however, not related to a decreased intracellular cyclic AMP (cAMP) concentration induced by DDA. The simultaneous incubation with dibutyryl cyclic AMP (dbc-AMP) and DDA enhanced the effects of DDA. Adenine, a possible metabolite of DDA digestion by purine nucleoside phosphorylase (PNP), also induced erythroid differentiation in K562 cells. However, it did not act synergistically with IFN-alpha.     

Transition state analogues for enzymes of nucleic acid metabolism

Knowledge of enzymatic transition states permits the logical design of transition state analogues. Kinetic isotope effects have been used to solve transition state structures for several N-ribosyltransferases. Nucleoside hydrolases from protozoan parasites show ribooxacarbenium ion character at their transition states, but with different extents of activation at the leaving group and the oxacarbenium ion. Transition state analogues are designed to capture these interactions and provide isozyme-specific inhibitors. Ricin A-chain is an RNA N-ribohydrolase for a single site on 28S rRNA. Its transition state resembles a fully dissociated ribooxacarbenium ion. A transition state analogue for ricin A-chain mimics the fully dissociated purine and cationic ribosyl transition state. The transition state for human purine nucleoside phosphorylase (PNP) is more dissociated than for the bovine enzyme. Immucillin-H, a powerful transition state inhibitor for human PNP, has entered clinical trials as an anti T-cell agent.     

The alpha-synuclein Ala53Thr mutation is not a common cause of familial Parkinson''s disease: a study of 230 European cases. European Consortium on Genetic Susceptibility in Parkinson''s Disease

Deoxyguanosine kinase (dGK) is an enzyme responsible for the phosphorylation of purine deoxynucleosides in mitochondria of mammalian cells. Its role in activation of pharmacologically used nucleoside analogs is not well understood, because of the low levels of dGK found in tissue extracts and its inactivation during purification. The cDNA for dGK was recently cloned and expressed in Escherichia coli. Here we present an improved procedure for expression and purification of a highly active form of human recombinant dGK. The enzyme showed a broad substrate specificity toward natural purine and pyrimidine deoxynucleosides as well as toward important nucleoside analogs. The Km and Vmax values for deoxyguanosine, deoxyinosine, deoxyadenosine, and deoxycytidine were 4, 13, 460, 330 microM and 43, 330, 430 and 60 nmol/min/mg of protein, respectively. Antileukemic purine analogs such as arabinosyl guanine, 2-chloro-2'-deoxyadenosine, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, and 2-fluoro-arabinosyl-adenine were phosphorylated as efficiently by dGK as the natural nucleoside substrates. This is the first report in which 2-fluoro-arabinosyl-adenine and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine were shown to be good substrates for dGK. The antiviral analogs dideoxyinosine and arabinosyl adenine also showed significant activity with dGK, as did several pyrimidine analogs (e.g., the cytostatic drugs 5-fluoro-2'-deoxycytidine and difluorodeoxycytidine). The broad specificity of dGK described here may change our understanding of the mechanisms responsible for the efficacy and mitochondrial toxicity of several nucleoside analogs.     

Refined crystal structures of guanine nucleotide complexes of adenylosuccinate synthetase from Escherichia coli

Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.