Glutamine PRPP Amidotransferase Research Articles

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using 13C-labeled sugars and [15N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals.

Discoveries in Bacterial Nucleotide Metabolism

Discoveries in Bacterial Nucleotide Metabolism

Activation of the Hetero-octameric ATP Phosphoribosyl Transferase through Subunit Interface Rearrangement by a tRNA Synthetase Paralog

ATP phosphoribosyl transferase (ATP-PRT) joins ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP) in a highly regulated reaction that initiates histidine biosynthesis. The unusual hetero-octameric version of ATP-PRT includes four HisG(S) catalytic subunits based on the periplasmic binding protein fold and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. Here, we present the first structure of a PRPP-bound ATP-PRT at 2.9 A and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATP-PRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface and new contacts between the HisZ motif 2 loop and the HisG(S) dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. Through the oligomerization and functional re-assignment of protein domains associated with aminoacylation and phosphate binding, the HisZ-HisG octameric ATP-PRT acquired the ability to initiate the synthesis of a key metabolic intermediate in an allosterically regulated fashion.

Nuclear Localization and Mitogen-activated Protein Kinase Phosphorylationof the Multifunctional ProteinCAD

CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus.

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.

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.

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.

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.

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.

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.

Determination of the structure of a large protein by MAD phasing: glutamine PRPP amidotransferase

Determination of the structure of a large protein by MAD phasing: glutamine PRPP amidotransferase

De Novo Purine Nucleotide Biosynthesis

This chapter reviews current understanding of de novo purine nucleotide synthesis in Bacillus subtilis,with emphasis on recent developments in understanding gene organization and regulation and on the distinctive structural features of two enzymes of the pathway. Genes for de novo purine nucleotide synthesis are found in four positions on the B. subtilis chromosome. The possible role of gene overlaps is discussed in this chapter. Addition of purine compounds to the growth medium represses the levels of enzymes involved in de novo purine nucleotide synthesis. Adenosine or guanosine repress enzyme levels in the pathway to IMP in mutants unable to interconvert adenine and guanine nucleotides, whereas synthesis of purA-encoded adenylosuccinate synthetase and guaB-encoded IMP dehydrogenase is repressed specifically by adenosine and guanosine, respectively. Limitation of the growth rate resulting from starvation for guanine nucleotides is known to initiate sporulation. Information about the enzymes in the pathway for de novo purine nucleotide synthesis from B. subtilis is limited. Judging from deduced sequence similarities to E. coli genes, most enzymes in the pathway are likely similar to those from E. coli. There are, however, distinctive features for glutamine PRPP amidotransferase a n d 5'-phosphoribosyl- Af-formylglycinamidine (FGAM) synthetase.

Purine metabolism in high- and low-uric acid lines of chickens: hypoxanthine/guanine phosphoribosyltransferase activities.

The activity of hypoxanthine/guanine phosphoribosyltransferase (HGPRT) was examined in the livers and kidneys of two genetic lines of chickens selected for different plasma uric acid levels. Previous work demonstrated that the high-uric acid line (HUA) had significantly greater de novo uric acid synthesis rates in kidney tissue compared to the low-uric acid line (LUA). In addition, phosphoribosylpyrophosphate (PRPP) synthetase and xanthine dehydrogenase activities in livers and kidneys were significantly higher in the HUA compared to the LUA line. PRPP pool sizes were also significantly higher in both livers and kidneys of HUA birds. HGPRT activities in livers of HUA birds were significantly (P less than 0.05) greater than in LUA birds. The mean value of liver HGPRT was 7.36 +/- 0.25 pmole inosine-5'-monophosphate (IMP) and 6.05 +/- 0.27 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA lines. There were no significant differences (P greater than 0.05) in kidney HGPRT activities between the two groups. The mean value of kidney HGPRT was 52.87 +/- 1.62 pmole IMP and 50.72 +/- 1.62 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA line. Elevated liver HGPRT may serve to enhance the regeneration of PRPP in the HUA liver. Elevated liver PRPP synthetase and PRPP pool size suggest an increased flux through the de novo purine biosynthetic pathway in HUA birds. The resulting additional pyrophosphate from the glutamine PRPP amidotransferase reaction would stimulate recovery of PRPP and spare the system from a substantial loss of energy.

Biochemical commitment to replication in cancer cells

The elucidation of the metabolic imbalance that underlies the biochemical commitment to continued replication of cancer cells is essential in the rational targeting of selective anti-cancer chemotherapy. The metabolic strategy of cancer cells entails an ordered pattern of imbalance in the activities of key enzymes and metabolic pathways and in the concentrations of intermediary metabolites, ribonucleotides and deoxyribonucleotides. A meaningful metabolic program was identified which is displayed in neoplastic cells as a result of a reprogramming of gene expression that is manifested in a transformation- and progression-linked fashion. The transition of the non-proliferating, resting cell culture into the proliferating phase provides a particularly suitable biological system of altered gene expression where it is possible to pinpoint the display of enzymic programs that illuminates the biochemical commitment of cancer cells to replication. In the proliferating hepatoma 3924A cells the activity of the pyrimidine synthetic enzymes, ribonucleotide reductase, thymidine kinase, CTP synthetase, uracil phosphoribosyltransferase, OMP decarboxylase, orotate phosphoribosyltransferase and uridine kinase were increased 23-, 13-, 4.9-, 3.1-, 3.5-, 3.6- and 2.1-fold over those of the resting phase cells. Concurrently, the activity of the rate-limiting catabolic enzyme, dihydrothymine dehydrogenase, was decreased to 42%. In consequence, the ratio of the activities of the synthetic to catabolic enzymes of thymidine metabolism increased 31-fold. In purine metabolism, the activity of the rate-limiting enzyme of de novo purine biosynthesis, glutamine PRPP amidotransferase, was increased 1.5-fold and that of the rate-limiting catabolic enzyme, xanthine oxidase, decreased to 75%. In consequence, the ratio of the synthetic to the catabolic enzyme was elevated 2-fold. The activities of the key enzymes of pentose phosphate synthesis and of carbohydrate catabolism rose 1.4- and 1.3-fold, respectively. The extensive rise in the activities of enzymes of pyrimidine and purine biosynthesis indicates that the behavior of these enzymes is more stringently linked with replication than that of other enzymes. The results are in line with earlier ones in this Laboratory on solid tumors indicating an integrated imbalance of the key enzymes of pyrimidine, purine and carbohydrate metabolism that should confer selective advantages to cancer cells. In studying the behavior of purine enzymes that may reveal a relationship with the commitment of cancer cells to replication, the behavior of inosine phosphorylase and guanine deaminase provided such examples. Inosine phosphorylase activity was decreased in all the hepatomas examined irrespective of growth rates of these neoplasms; thus, the decrease of this enzymic activity was transformation-linked. By contrast, the guanine deaminase activity fluctuated in the various lines of the hepatoma spectrum indicating that the behavior of the activity of this enzyme was coincidental to transformation or progression in liver tumors. The relationship of 4 key enzymes with the progression-linked increase in the deoxynucleoside triphosphate pools in hepatomas was examined. The activities of CTP synthetase, thymidine kinase, IMP dehydrogenase and ribonucleotide reductase markedly increased in parallel with tumor growth rates and with the enlargement of the deoxyribonucleotide pools. The applicability of alterations of enzymic programs to 14 different tumor types was shown. There is a shared enzymic program that applies to rodent, avian and human neoplasms. The pattern was observed in carcinomas, sarcomas, leukemias and in human colon tumor xenografts carried in the nude mouse. The biochemical programs displayed were independent from the carcinogenic agent that caused the neoplasms because there was a shared enzymic program in chemically-induced, transplantable rat and mouse tumors, in viral-induced, transplantable avian neoplasms and in primary liver, kidney, lung and colon tumors in human. These observations indicate that strategic aspects of the biochemical commitment of the cancer cells to replication have now been identified. These novel insights should assist in the targeting of selective chemotherapy against cancer cells.

Colon tumor: Enzymology of the neoplastic program

The biochemical strategy of colon tumor was investigated by comparing the enzymic programs of glycolysis, pentose phosphate production and purine and pyrimidine biosynthesis and degradation in liver, normal colon mucosa and transplantable colon adenocarcinoma in the mouse. In normal colon mucosa the carbohydrate and pentose phosphate enzymes were 2- to 9-fold higher in specific activity than those in liver. Among the enzymes of CTP synthesis, CTP synthetase was the rate-limiting one in both liver and colon. In colon tumor CTP synthetase, OMP decarboxylase, uracil phosphoribosyltransferase and thymidine kinase activities increased to 927, 863, 597 and 514% of activities of normal colon. In contrast, the activity of the catabolic enzymes, dihydrothymine dehydrogenase and uridine phosphorylase, decreased to 51 and 25%. The ratios of activities of uridine kinase/uridine phosphorylase and thymidine kinase/dihydrothymine dehydrogenase were elevated 6- and 10-fold. The activity of the key purine synthetic enzyme, glutamine PRPP amidotransferase, increased 7-fold and the opposing rate-limiting enzyme of purine catabolism, xanthine oxidase, decreased to 7%. The ratio of amidotransferase/xanthine oxidase was elevated to 8, 150%. Activities of glucose-6-phosphate dehydrogenase and transaldolase did not increase, but that of pyruvate kinase was elevated to 154%. Similar enzymic programs were observed in a transplantable adenocarcinoma of the colon in the rat. The alterations in gene expression in colon tumor manifested in an integrated pattern of enzymic imbalance indicate the display of a program, a segment of which is shared with rat and human liver and kidney tumors. These alterations in gene expression should confer selective advantages to colon tumor cells. The striking increases in the activities of CTP synthetase, OMP decarboxylase, glutamine PRPP amidotransferase and thymidine kinase mark out these enzymes as potentially sensitive targets for combination chemotherapy by specific inhibitors of these enzyme activities.

Enzymatic markers of neoplastic transformation and regulation of purine and pyrimidine metabolism

Enzymes that were identified as transformation-linked in the spectrum of hepatomas of different growth rates in the rat were used as markers of malignancy for liver tumors in rat and in man. The applicability of the enzymatic pattern was examined in kidney tumors of the rat, in lymphomas of the mouse and in hypernephroma-type kidney tumors in human. The impact of altered gene expression, as manifested in the enzymic imbalance, was further investigated by determining the concentration and behavior of purine and pyrimidine ribonucleotides in normal and neoplastic liver. The activity of transformation-linked enzymes was also studied in different organs of the rat and mouse to elucidate whether the activity might prove to be a predictor of the changes in activity observed in the different neoplasms. 1. 1.|In the hepatoma spectrum AMP deaminase and CTP synthetase activities were increased in all examined tumors and the rise correlated positively with the growth rate of the neoplasms. 2. 2.|A comparison of the in vivo concentrations (0-min sample) of ribonucleotides revealed that the level of adenine nucleotides was lower in rapidly-growing hepatoma 3924-A than in normal control liver. A further outstanding difference in ribonucleotide levels is the 5-fold increase in CTP content along with an overall elevation of the concentration of cytidine nucleotides. This biochemical imbalance in the hepatoma is attributed, in part at least, to the increased activity of UDP kinase and CTP synthetase in these tumors. 3. 3.|When ischemia was imposed, in normal liver there was a breakdown of the adenine, uridine, guanine and cytidine nucleotide triphosphates and a concurrent rise in the nucleoside monophosphate contents. It was particularly striking that the level of IMP increased 75-fold in 10-min ischemia. In contrast, no change was observed on ischemia in the hepatoma. When the hepatoma was injected with iodoacetamide and then subjected to ischemia the nucleoside triphosphates decreased as in the normal liver. 4. 4.|In measuring the activities of key carbohydrate, pentose phosphate, purine and pyrimidine enzymes we observed that frequently the high activity observed in the rat thymus was a predictor that the enzyme activity would be markedly increased in the rapidly growiing tumor. This relationship did not hoold without exception and did not apply to the mouse. 5. 5.|The integrated pattern of enzymatic alternations observed in rat, mouse and human neoplasms is specific to cancer cells, as no similar enzyme activity pattern was observed in the various normal organs and tissues examined in rat, mouse and human. 6. 6.|The present work demonstrates the applicability of the molecular correlation concept and much of the altered biochemical pattern discovered in the rat hepatoma spectrum to other tumors in rat, to lymphomas in mice and to primary kidney tumor in humans. Thus, increased activities of pyruvate kinase, glutamine PRPP amidotransferase, IMP dehydrogenase, AMP deaminase, adenylosuccinase and UDP kinase and decreased activity of xanthine oxidase were markers of malignancy in the three mouse tumors tested, plasma cell myeloma, Mecca lymphosarcoma and P1534 lymphocytic leukemia. In 15 cases of human hypernephromas, hexokinase and pyruvate kinase activities increased whereas those of glucose 6-phosphatase and fructose 1,6-diphosphatase decreased. Activities of glucose 6-phosphate dehydrogenase and glutamine PRPP amidotransferase increased, whereas that of xanthine oxidase decreased. IMP dehydrogenase, adenylosuccinase and AMP deaminase activities were elevated. Thus, these enzymes could be used in the biochemical diagnosis of human primary kidney tumors of the hypernephroma type. The biochemical imblance is similar to that observed in transplanted hepatomas and kidney tumors in the rat. 7. 7.|The applicability of the enzymatic imbalance observed in rodents to human tissue underlines the importance of such enzymatic markers in neoplastic transformation and these enzymes should be targets in the design of selective chemotherapy of neoplastic diseases.

Key enzymes of IMP metabolism: Transformation- and proliferation-linked alterations in gene expression

The purpose of this investigation was to test the concept that key enzymes of IMP synthesis, degradation and utilization may show characteristic patterns in proliferative conditions. In the synthetic pathway of IMP the first enzyme committed to de novo purine biosynthesis, glutamine PRPP amidotransferase, was measured. In the pathway of IMP catabolism the rate-limiting enzyme, xanthine oxidase, and uricase, the final enzyme, were examined. In the synthetic utilization of IMP, IMP dehydrogenase, the enzyme involved in channeling IMP into the production of XMP and adenylosuccinate synthetase, the enzyme channeling IMP into SAMP biosynthesis, were studied. Investigations were also carried out on GMP synthetase and adenylosuccinase. The enzymes of IMP metabolism were examined in normal and neoplastic liver proliferation, such as in differentiating and regenerating liver and in a spectrum of hepatomas of different growth rates. The following main observations were made. 1. 1.|In differentiating liver the specific activities of IMP dehydrogenase and amidotransferase were high after birth and decreased during development to the levels observed in the adult rat liver. In contrast, the adenylosuccinate synthetase and xanthine oxidase activities were low after birth and rose to the adult levels during development. 2. 2.|In a spectrum of hepatomas of different growth rates the specific activity of IMP dehydrogenase was 2- to 3-fold increased in the slow growing hepatomas and it increased in parallel with tumor growth rates, reaching 12- to 13-fold increases in the most rapidly growing neoplasms. The behavior of IMP dehydrogenase belongs to Class 1 as grouped by the molecular correlation concept. 3. 3.|The activities of adenylosuccinate synthetase and glutamine PRPP amidotransferase were increased in all hepatomas to approximately 2- to 4-fold, and those of the adenylosuccinase 1.2- to 2-fold of the levels observed in the corresponding normal rat livers. In contrast, the activities of the catabolic enzymes, xanthine oxidase and uricase, were decreased in all hepatomas to 2- to 10-fold of the activity of the relevant control rat livers. The ratios of amidotransferase/xanthine oxidase were markedly increased in all hepatomas and they reached 20- to 27-fold increases in those of rapid growth rate. The behavior of adenylosuccinate synthetase, adenylosuccinase, amidotransferase, xanthine oxidase and uricase belongs to Class 2 of the molecular correlation concept where enzymes are grouped that exhibit alterations that occur in all hepatomas irrespective of growth rate and degree of differentiation. 4. 4.|Since the alterations in the activities of adenylosuccinate synthetase, adenylosuccinase, amidotransferase, xanthine oxidase and uricase are expressed in all the hepatomas, even in the slowest growing, most liver-like tumors, this reprogramming of gene expression appears to be linked with the malignant transformation per se. However, these enzyme activities do not signal a linkage with the degrees in the expression of malignancy and growth rate. In contrast, the activity of IMP dehydrogenase is an indicator both of malignant transformation and growth rate. 5. 5.|In the regenerating liver IMP dehydrogenase specific activity rapidly increased to 500% in 6 to 18 hours; it slowly returned towards normal range in 96 hours after operation. Amidotransferase specific activity reached a peak of 165% of the sham-operated value at 48 hours after operation and then returned to normal range by 96 hours. The activities of SAMP synthetase, adenylosuccinase, xanthine oxidase and uricase did not change in regeneration. Thus, IMP dehydrogenase appears to be the earliest enzyme to rise in the regenerating liver, whereas the elevation of amidotransferase is a delayed and a minor one. The increase in activities of these two enzymes in the regenerating liver is in the range of the activity observed in the newborn rat liver. However, the activities are markedly lower than those observed in the very malignant hepatomas that exhibit the same growth rate as the regenerating liver. 6. 6.|The discriminating power of the biochemical pattern of enzymes of purine metabolism indicates that the imbalance in the pattern of alteration of the key enzymes of IMP synthesis, degradation and utilization is specific to malignancy. The pattern of these enzyme activities also provides quantitative and qualitative discriminants for the identification of regenerating and differentiating liver and for distinguishing the enzyme patterns from those observed in normal and rapidly growing neoplastic liver. 7. 7.|These experiments indicate the operation of an antagonistic behavior for the key synthetic enzymes, glutamine PRPP amidotransferase and IMP dehydrogenase, that are high in differentiation, regeneration and neoplasia as compared to the rate-limiting catabolic enzyme, xanthine oxidase, that is low in differentiation and in neoplasia and is unaltered in the regenerating liver. These observations are in line with earlier studies that demonstrated an antagonistic behavior for the opposing key enzymes of gluconeogenesis and glycolysis and of the synthetic and catabolic pathways of the de novo and salvage pathways of pyrimidine metabolism.

Malignant transformation-linked imbalance: Decreased xanthine oxidase activity in hepatomas

Xanthine oxidase was decreased 2- to 10-fold in all examined rat hepatomas irrespective of the malignancy; growth rate and degrees of histological differentiation of the neoplasms. The affinity to substrate (KM=6-8 muM) and the pH optimum (8.0) of the liver and hepatoma enzymes were the same. The reprogramming of gene expression, as manifested in the decreased activity of this key purine metabolizing enzyme, appears to be specific to neoplastic transformation. Since glutamine PRPP amidotransferase activity was increased but the opposing enzyme, xanthine oxidase, was decreased in all the hepatomas, the reprogramming of gene expression results in an imbalance that favors synthesis against catabolism. This enzymatic imbalance should confer selective advantages to the cancer cells.

IMP dehydrogenase, an enzyme linked with proliferation and malignancy

PREVIOUS studies conducted in this laboratory demonstrated that in a spectrum of liver tumours with different growth rates there is an imbalance in the activities of key enzymes and competing pathways of carbohydrate, pyrimidine, DNA and ornithine metabolism1–4. Recent investigations on purine metabolism showed that in the hepatomas there was an increased capacity in the de novo pathway of biosynthesis of inosine 5′-monophosphate (IMP), as reflected in the increased activity of glutamine PRPP amidotransferase (EC 2.4.2.14) and a decrease in IMP catabolism5,6. These observations directed our attention to the metabolic fate of IMP because this purine nucleotide is at a strategic position in purine metabolism (Fig. 1). Control at such branching points is exerted primarily by modification of the activity or of the rate of synthesis of the first enzymes of the divergent pathways. In this instance, in normal conditions, a balance is maintained by both positive and negative feedback effects. Thus ATP is required for GMP biosynthesis and GTP for AMP biosynthesis; conversely, GMP and AMP each inhibit their own production7. In normal or malignant proliferation, or in response to a hormone stimulus, changes in control frequently involve reprogramming of gene expression. The theoretical framework and predictive value of generalisations about such biochemical changes (termed the molecular correlation concept) have been elaborated1–3.