PRPP Amidotransferase Research Articles

Investigations on the Mechanism of Action of the Novel Antitumor Agents 2-Benzothiazolyl, 2-Benzoxazolyl, and 2-Benzimidazolyl Hydrazones Derived from 2-Acetylpyridine

2-Acetylpyridine hydrazone derivatives of benzothiazole, benzoxazole, and benzimidazole were found to exhibit potent cytotoxic activity against the growth of suspended leukemia and lymphomas. They were also active in a number of solid tumor screens, e.g. HeLa uterine carcinoma, SOS bone osteosarcoma, lung MB9812, lung A549, Mcf-7 breast growth. In L1210 lymphoid leukemia cells the compounds preferentially inhibited RNA synthesis followed by DNA synthesis at 100 μM after 60 min. The reduction of de novo purine synthesis by the compounds at the regulatory sites PRPP-amido transferase, IMP dehydrogenase and dihydrofolate reductase was responsible for the suppression of nucleic synthesis. Other minor sites where the agents have metabolic effects were thymidylate synthetase and thymidine kinase which would be additive with the overall inhibition of cell growth. The ct-DNA studies suggest that the compounds also interacted with the DNA molecule itself, probably affecting template activity.

Substituted 4-hydroxyproline di- and tri-peptides as cytotoxic agents

4-Hydroxyproline di- and tri-peptides and N-cbz-hydroxypropyl- glycinamides were observed to be potent cytotoxic agents against the growth of suspended single cells, L-1210, Tmolt3, and HeLa-S3. The agents were not as potent against the growth of cultured solid tumor cells. Selected derivatives were investigated for their mode of action in Tmolt3 leukemia cells. The compounds selectively inhibited DNA synthesis at 50 and 100 microM. The target site of action of the agents appeared to be the purine de novo pathway with marked inhibition of the activities of the two regulatory enzymes of the pathway, i.e. PRPP amido-transferase and IMP dehydrogenase. d[NTP] pools were reduced by the agents consistent with their overall reduction of DNA synthesis. Other marginally inhibited targets of the agents were r-RNA polymerase and TMP-kinase activities. The DNA molecule itself did not appear to be a target of these agents.

Glutamine PRPP amidotransferase: snapshots of an enzyme in action

Recent studies of glutamine PRPP amidotransferase have provided a new understanding of the function and mechanism of this rather complicated enzyme that may be a paradigm for other complex enzymes. New insights have been gained into the mechanisms of catalysis in the active sites of the two half-reactions, catalytic coupling, allosteric control by feedback inhibitors and the channeling of reaction and metabolic intermediates.

Cytotoxicity of Substituted Alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylates in L1210 Lymphoid Leukemia Cells

Two alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylates proved to be potent cytotoxic agents in the murine L1210 lymphoid leukemia screen. DNA synthesis was preferentially inhibited with the major target of the agents being de novo purine biosynthesis at the regulatory enzyme sites of PRPP-amido transferase and IMP dehydrogenase. Other enzymatic activities which were suppressed by the drugs were DNA polymerase α, RNA polymerases, ribonucleoside reductase and dihydrofolate reductase. The d[NTP] pools, nucleoside kinase and the pyrimidine pathway were not affected by the presence of drugs. The DNA molecule itself was not the target of the agents, i.e. no alkylation of nucleotide bases, intercalation between bases or cross-linking of DNA strands occurred. The agents did cause L1210 DNA fragmentation after 24 h incubation at 100 μM.

The synthesis and antitumor activity of metal complexes of amine–carboxyborane adducts

The amine carboxyborane metal complexes, tetrakis - μ - (trimethylamine - boranecarboxylato)acetonitrile dicopper(II) and his-μ-(morpholine-boranecarboxylato)zinc(II) dihydrate demonstrated cytotoxic activity against human Tmolt 3 , HeLa-S 3 and MB-9812 cell growth. Tetrakis-μ-(trimethylamine-boranecarboxylato)-acetonitrile dicopper(II) and bis - μ - (morpholine - boranecarboxylato)zinc (II) dihydrate inhibited L 1210 DNA, RNA and protein syntheses, with greatest inhibitory effects on DNA synthesis. The reduction in DNA synthesis correlates well with inhibition of de novo purine synthesis and the key enzymes involved in this pathway, i.e. IMP dehydrogenase and PRPP amido transferase. These compounds were also observed to induce DNA strand scission but did not appear to intercalate between base pairs of DNA, alkylate bases or cause cross-linking of the strands of DNA. Tetrakis-μ-(trimethylamine - boranecarboxylato)acetonitrile dicopper(II) also demonstrated the ability to inhibit L 1210 DNA topoisomerase II activity.

Cytotoxicity of Poly(Phenolic)Sulfonates and Their Sodium Salts in L1210 Lymphoid Leukemia Cells

Poly(phenolic)-sulfonates demonstrated very good cytotoxicity against the growth of tumor cell lines (L1210, Tmolt-3, HeLa-S3) and are comparable in potency with typical clinically used anticancer drugs. Four of the most active compounds, i.e. GL-2021, GL-2029, GL-2041 and GL-2063, were selected for a mode of action study in L1210 lymphoid leukemia cells at concentration of 25μM to 100μM for 60 min. The agents did not alkylate bases of ct-DNA, cause intercalation between base pairs, produce cross linking of ct-DNA strands or generate free radicals although L1210 DNA fragmentation was observed after 24 hr incubation. L1210 DNA synthesis was preferentially inhibited which was achieved by (1) suppressing DNA polymerase α activity which reduced the synthesis of new strands of DNA, (2) reducing of de novo purine synthesis at the regulatory enzyme PRPP amido transferase which reduced d(GMP) levels, and (3) inhibiting of nucleoside kinase activities which further reduced DNA synthesis. DNA template activity was altered by the poly(phenolic)sulfonates since they reduced DNA polymerase α and m-RNA and t-RNA polymerase activities. The kinetic studies at 50 μM over 2 hr demonstrated that the agents’ effect on PRPP-amido transferase activity is probably a major target of the compounds.

Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli.

Crystal structures of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli have been determined to 2.0-A resolution in the absence of ligands, and to 2.5-A resolution with the feedback inhibitor AMP bound to the PRPP catalytic site. Glutamine PRPP amidotransferase (GPATase) employs separate catalytic domains to abstract nitrogen from the amide of glutamine and to transfer nitrogen to the acceptor substrate PRPP. The unliganded and AMP-bound structures, which are essentially identical, are interpreted as the inhibited form of the enzyme because the two active sites are disconnected and the PRPP active site is solvent exposed. The structures were compared with a previously reported 3.0-A structure of the homologous Bacillus subtilis enzyme (Smith JL et al., 1994, Science 264:1427-1433). The comparison indicates a pattern of conservation of peptide structures involved with catalysis and variability in enzyme regulatory functions. Control of glutaminase activity, communication between the active sites, and regulation by feedback inhibitors are addressed differently by E. coli and B. subtilis GPATases. The E. coli enzyme is a prototype for the metal-free GPATases, whereas the B. subtilis enzyme represents the metal-containing enzymes. The structure of the E. coli enzyme suggests that a common ancestor of the two enzyme subfamilies may have included an Fe-S cluster.

The Synthesis and Antitumor Activity of the Sodium Salt and Copper(II) Complex of N‐[(Trimethylamineboryl)‐Carbonyl]‐L‐PhenylalanineMethyl Ester

Sodium N-[(trimethylamineboryl)-carbonyl]-L-phenylalanine 2 and {N-[(trimethylamineboryl)-carbonyl]-L-phenylalanyl- carbxylato}-bis-{N-[(trimethylaminebryl)-carbonyl]-L-phenylalanine} dicopper (II) 3 were successfully synthesized. The agents blocked L1210 leukemic cell DNA and RNA syntheses by inhibiting multiple enzyme activities for nucleic acid synthesis, e.g. PRPP amido transferase, IMP dehydrogenase, DNA polymerase α, thymidine kinase, and TMP kinase. The copper (II) complex 3 demonstrated improved ability to inhibit L1210 partially purified DNA topoisomerase II compared to the parent compound while the sodium salt was inactive at 100 μM.

Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides.

De novo purine nucleotide synthesis is regulated, at least in part, by end-product inhibition of glutamine PRPP amidotransferase. An important feature of this inhibition is the fact that certain synergistic nucleotide pairs give more than additive inhibition. The physiological importance of synergism is in amplifying regulation by the adenine and guanine nucleotide end products of de novo synthesis. Using a new method to quantitate synergism, ADP plus GMP were confirmed [Meyer, E., and Switzer, R. L. (1978) J. Biol. Chem. 254, 5397-5402] to give strong synergistic inhibition of Bacillus subtilis glutamine PRPP amidotransferase. An X-ray structure of the ternary enzyme.ADP.GMP complex established that ADP binds to the allosteric A site and GMP to the catalytic C site. GMP increased the binding affinity of ADP for the A site by approximately 20-fold. Synergism results from a specific nucleotide-nucleotide interaction that is dependent upon a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. Furthermore, synergism is enhanced by the competition between nucleotide inhibitor and PRPP substrate for the C site. Purine base specificity results from a backbone carbonyl interaction of Lys305' with the 6-NH2 group of adenine in the A site and a Ser347 Ogamma interaction with the 2-NH2 group of guanine in the C site. Steric considerations favor binding of the nucleoside diphosphate to the A site. Site-directed replacements of key residues increased the nucleotide concentrations needed for 50% inhibition and in some cases perturbed synergism. Mutations in either of the nucleotide sites perturbed function at both sites, supporting the important role of synergism.

Antineoplastic and Cytotoxic Activities of Nickel(II) Complexes of Thiosemicarbazones

Nickel(II) complexes of thiosemicarbazons were observed to be potent cytotoxic agents in human and rodent tissue cultured tumor cells. Each compound demonstrated a slightly different profile in the various histological types of tumors. The nickel complex of Appip demonstrated the most potent in vivo activity in the Ehrlich ascites carcinoma. This agent selectively inhibited L1210 DNA and purine syntheses, and DNA polymerase α, PRPP-amido transferase, IMP-dehydrogenase, dihydrofolate reductase, TMP-kinase and thymidylate synthetase activities. L1210 DNA strand scission was evident and DNA viscosity was reduced after 24 hr incubation. The nickel complexes were not L1210 DNA topoisomerase II inhibitors.

Autocatalytic processing of the 20S proteasome.

The Ntn (N-terminal nucleophile) hydrolases are enzymes with an unusual four-layer alpha + beta fold. The amino-terminal residue (cysteine, serine or threonine) of the mature protein is the catalytic nucleophile, and its side chain is activated for nucleophilic attack by transfer of its proton to the free N terminus, although other active-site residues may also be involved. The four currently known Ntn hydrolases (glutamine PRPP amidotransferase, penicillin acylase, the 20S proteasome and aspartylglucosaminidase) are encoded as inactive precursors, and are activated by cleavage of the peptide bond preceding the catalytic residue. It has been suggested that autocatalytic processing is a common feature of Ntn hydrolases, and proceeds by an intramolecular mechanism determined by their common fold. Here we show that propeptide processing in the proteasome from Thermoplasma acidophilum is indeed autocatalytic, but is probably intermolecular. Processing is not required for assembly, is largely unaffected by propeptide length and sequence, and occurs before beta-subunit folding is completed. Although serine is an acceptable active-site nucleophile for proteolysis, and cysteine for processing, only threonine is fully functional in both. This explains why threonine is universally conserved in active proteasome subunits.

Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 å crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase

Background: Amidotransferases use the amide nitrogen of glutamine in a number of important biosynthetic reactions. They are composed of a glutaminase domain, which catalyzes the hydrolysis of glutamine to glutamate and ammonia, and a synthetase domain, catalyzing amination of the substrate. To gain insight into the mechanism of nitrogen transfer, we examined the structure of the glutaminase domain of glucosamine 6-phosphate synthase (GLMS). Results The crystal structures of the enzyme complexed with glutamate and with a competitive inhibitor, Glu-hydroxamate, have been determined to 1.8 å resolution. The protein fold has structural homology to other members of the superfamily of N-terminal nucleophile (Ntn) hydrolases, being a sandwich of antiparallel β sheets surrounded by two layers of α helices. Conclusion The structural homology between the glutaminase domain of GLMS and that of PRPP amidotransferase (the only other Ntn amidotransferase whose structure is known) indicates that they may have diverged from a common ancestor. Cys1 is the catalytic nucleophile in GLMS, and the nucleophilic character of its thiol group appears to be increased through general base activation by its own α-amino group. Cys1 can adopt two conformations, one active and one inactive; glutamine binding locks the residue in a predetermined conformation. We propose that when a nitrogen acceptor is present Cys1 is kept in the active conformation, explaining the phenomenon of substrate-induced activation of the enzyme, and that Arg26 is central in this coupling.

Structure and Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Glutamine Site and Communication with the Phosphoribosylpyrophosphate Site

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli exhibits a basal PRPP-independent glutaminase activity having a kcat/Km that is 0.3% of fully active enzyme. Binding of PRPP activates the enzyme by a structural change that lowers the Km for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5-phosphoribosylamine. By analysis of the x-ray structure of the glutamine site containing bound 6-diazo-5-oxonorleucine, a glutamine affinity analog, and by site-directed mutagenesis we have identified residues important for glutamine binding, catalysis, and coupling with PRPP. Tyr74 is a key residue in the coupling between the sites for glutamine in the NH2-terminal domain and PRPP in the COOH-terminal domain. Arg73 and Asp127 have roles in glutamine binding. The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys1 to participate as a proton acceptor in formation of the thiolate needed for nucleophilic attack on the carboxamide of glutamine, nor as a general acid for amide nitrogen transfer. Based on the x-ray model of the glutamine site and analysis of a mutant enzyme we propose that the free NH2 terminus of Cys1 functions as the proton acceptor and donor. The results indicate that the side chain of Asn101 and the backbone nitrogen of Gly102 function to stabilize a tetrahedral oxyanion resulting from attack of Cys1 on the glutamine carboxamide. Cys1, Arg73, Asn101, Gly102, and Asp127 are conserved in the NH2-terminal domain of a subfamily of amidotransferases that includes asparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes. Tyr74, on the other hand, is conserved only in glutamine PRPP amidotransferase sequences consistent with a specific role in interdomain coupling. The catalytic framework of key glutamine site residues supports the assignment of glutamine PRPP amidotransferase to a recently described Ntn (NH2-terminal nucleophile) hydrolase family of enzymes.

Circadian expression of genes involved in the purine biosynthetic pathway of the cyanobacterium Synechococcus sp. strain PCC 7942

Extensive circadian (daily) control over gene expression in the cyanobacterium Synechococcus sp. strain PCC 7942 is programmed into at least two differentially phased groups. The transcriptional activity of the smaller group of genes is maximal at about dawn and minimal at about dusk. We identified one of the genes belonging to this latter group as purF, which encodes the key regulatory enzyme in the de novo purine synthetic pathway, glutamine PRPP amidotransferase (also known as amidophosphoribosyltransferase). Its expression pattern as a function of circadian time was confirmed by both luminescence from a purF::luxAB reporter strain and the abundance of purF mRNA. By fusing sequences upstream of the purF coding region to promoterless luxAB genes, we identified a limited upstream region, which potentially regulates purF circadian expression patterns in vivo. We also identified the purL gene immediately upstream of purF. The purL gene encodes FGAM synthetase, the fourth enzyme in the purine nucleotide biosynthesis pathway. Although these genes are expressed as part of a larger operon in other bacteria, reporter gene fusions revealed that purF and purL are transcribed independently in Synechococcus and that they are expressed at different phases of the circadian cycle. This differential expression pattern may be related to the oxygen sensitivity of amidophosphoribosyltransferase.

Cytoxicity of [(5,6-dichloro-9a-n-propyl-2,3,9,9a-tetrahydro-3-oxo-1H fluoren-7-yl)oxy]acetic acid, an agent known to reduce brain edema

A known agent, [(5,6-dichloro-9a-n-propyl-2,3,9,9a-tetrahydro-3-oxo-1H fluoren-7-yl)oxy]acetic acid, which blocks brain edema, was also shown to be a potent cytotoxic agent in leukemia cells. The major site of action of the agents appears to be in the de novo purine synthetic pathway in L1210 leukemic cells. Both PRPP amido transferase and IMP dehydrogenase activities were suppressed by the agent. The inhibition of both regulatory enzymes of the pathway along with the reduction of dihydrofolate reductase activity would account for the observed suppression of DNA and RNA syntheses and subsequent cancer cell death.

A protein catalytic framework with an N-terminal nucleophile is capable of self-activation.

The crystal structures of three amidohydrolases have been determined recently: glutamine PRPP amidotransferase (GAT), penicillin acylase, and the proteasome. These enzymes use the side chain of the amino-terminal residue, incorporated in a beta-sheet, as the nucleophile in the catalytic attack at the carbonyl carbon. The nucleophile is cysteine in GAT, serine in penicillin acylase, and threonine in the proteasome. Here we show that all three enzymes share an unusual fold in which the nucleophile and other catalytic groups occupy equivalent sites. This fold provides both the capacity for nucleophilic attack and the possibility of autocatalytic processing. We suggest the name Ntn (N-terminal nucleophile) hydrolases for this structural superfamily of enzymes which appear to be evolutionarily related but which have diverged beyond any recognizable sequence similarity.

Cytotoxicity of imides-N-alkyl semicarbazones, thiosemicarbazones, acetylhydrazones and related derivatives

The semicarbazones, thiosemicarbazones and acetyl-hydrazones of phthalimide, o-benzosulfimide, naphthalimide and diphenimide demonstrated potent cytotoxicity against murine and human leukemia cell growth and cultured cell growth from human solid tumors. The major site of inhibition in L1210 leukemia cells was DNA synthesis after 60 min incubated with the agents at 25, 50 and 100 microM. De novo synthesis of purines at the regulatory enzyme sites of PRPP amidotransferase and IMP dehydrogenase were the major targets of the agent. Thymidylate synthetase, dihydrofolate reductase and ribonucleoside reductase activities were inhibited by the agents in a manner which would contribute to the overall reduction of DNA synthesis and cell death. d(NTP) pools were significantly reduced and the evidence suggests that the agents interacted with DNA affording DNA strand scission which would interfere with both template utilization by the polymerases and also ultimately reduce nucleic acid synthesis.

Cytotoxicity of boron containing dipeptide analogs

This work is an extension of our previous work (Hall et al., 1993) on the synthesis and cytotoxic activity of boronated peptides. The aim of this work was to carry out structural modifications of the amine terminal in compounds1 and2, to increase water solubility, and its effect on the cytotoxicity to tumor cell lines. Surprisingly, only compounds4,7 and8 were more water soluble than the parent compounds. With the exception of compound4, the new derivatives were generally less effective than the parent compounds (1 and2). There was no apparent correlation between structure and activity. Cytotoxic effect was more pronounced in single cell suspended cells. The growth of solid tumor cell lines was not significantly reduced. The most active derivative, (methanamine)dihydro[[[1-(phenylmethyl)-2-methylamino-2-oxoethyl]amino]carboxy]boron (4), inhibited DNA, RNA, and protein synthesis in Tmolt3 cells. Enzymatic activities, e.g., DNA polymeraseα, m-RNA polymerase, PRPP amidotransferase, carbamyl phosphate synthetase, TMP-kinase, TDP-kinase, dihydrofolate reductase, and ribonucleoside reductase were reduced after 60 min incubation with4. d(TTP) and d(CTP) pool levels were also reduced by 60 min incubation with4.

The cytotoxicity of N-pyridinyl and N-quinolinyl substituted derivatives of phthalimide and succinimide

The N-pyridinyl and N-quinolinyl substituted derivatives of phthalimides and succinimides demonstrated cytotoxicity against the growth of a number of cultured cell lines. The substituted succinimides were more effective than the unsubstituted succinimide derivative in reducing cell growth. On the other hand, phthalimide demonstrated more potent cytotoxicity than its N-substituted derivatives. Three representative examples N-[2-pyridinyl-1-oxide) methyl] phthalimide 8, 1-[N-2-phthalimidoethyl]-3,4-dihydroiso-quinoline 12, and 1-[N-(2-(1,2,3,4-tetrahydro-2-quinolinyl)] ethylphthalimide 14 were shown to inhibit L1210 leukemia DNA synthesis whereas RNA synthesis was not inhibited at 25-100 uM. All three agents inhibited the activities of DNA polymerase alpha, PRPP-amido transferase, nucleoside kinases, and dihydrofolate reductase. The cellular pool levels of d[GTP], d[CTP], and d[TTP] were reduced after 60 minutes incubation at 100 uM. The DNA molecule itself was not a target of these agents.

The cytotoxicity of N-substituted diphenimides and 6,7-dihydro-5H-dibenz[c, e]azepines

N-substituted diphenimides and 6,7-dihydro-5H-dibenz[c,e]azepines demonstrated significant cytotoxic activity against the growth of murine and human cells. These derivatives were active against leukemias, carcinomas and sarcomas. Different derivatives with N-substitutions showed specific activity against the growth of several tumor types. These agents inhibited L1210 leukemia IMP dehydrogenase and PRPP amido transferase activities; this was reflected in the inhibition of purine and DNA synthesis. Other sites inhibited to a minor degree by these agents included DNA polymerase alpha, r- and tRNA polymerases, ribonucleoside reductase, dihydrofolate reductase, pyrimidine synthesis, and nucleoside kinase. d(NTP) pool levels were reduced after 24 h incubation with these derivatives. L1210 DNA strand scission was evident after drug treatment.