Purine Biosynthesis Enzyme Research Articles

Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of pur B, purC, purL, purMN and guaBA expression in Escherichia coli

The purR gene encodes a repressor (PurR) controlling the synthesis of the enzymes of purine biosynthesis. The subunit of PurR was identified as a 38-kDa polypeptide by SDS/polyacrylamide gel electrophoresis. Analysis of a purR-lacZ transcriptional fusion indicated that purR expression is autoregulated. This was confirmed by gel retardation and DNaseI footprinting experiments, where two PurR-binding sites were identified in the transcribed part of purR. Introduction of a purR mutation in wild-type and pur-lac fusion strains was found to abolish purine repression of all genes of the purine biosynthetic pathway except for purA.

Genetic evidence for a repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli

Addition of purines to the growth medium of Escherichia coli represses synthesis of cytosine deaminase (codA) and enzymes of purine de novo synthesis. After Tn10 mutagenesis, mutants displaying derepressed levels of cytosine deaminase in the presence of hypoxanthine were isolated. One of these had simultaneously acquired resistance to the hypoxanthine analog 6-mercaptopurine. The mutation purR6::Tn10 was shown to affect de novo synthesis of the purine enzymes glutamine phosphoribosylpyrophosphate amidotransferase (purF) and phosphoribosyl glycinamide synthetase (purD). The mutation was mapped by P1 transduction at 36 min on the E. coli linkage map. A plasmid containing the purR region was obtained by complementation of the purR6::Tn10 mutation. By comparing the restriction maps of the cloned fragment and the E. coli chromosome, the purR gene was found to be located very close to the lpp gene (36.3 min).

Регуляция экзогенным аденином экспрессии генов ADE2 И ADE1, кодирующих структуру двух ферментов пуринового биосинтеза у дрожжей Saccharomyces cerevisiae

Регуляция экзогенным аденином экспрессии генов ADE2 И ADE1, кодирующих структуру двух ферментов пуринового биосинтеза у дрожжей Saccharomyces cerevisiae

Regulation of purine and pyrimidine metabolism by insulin and by resistance to tiazofurin

The purpose of this investigation was to clucidate the factors that regulate the pattern of gene expression in purine and pyrimidine metabolism in normal liver and hepatoma. For this purpose, the action of a hormone, insulin, and the development of resistance to a chemotherapeutic agent, tiazofurin, were studied. 1. 1.|This investigation brought detailed evidence showing that in the rat insulin exerted a profound effect on liver purine and pyrimidine metabolism by regulating the concentrations of nucleotides through controlling the activities of strategic enzymes involved in their biosynthesis. 2. 2.|When rats were made diabetic by alloxan treatment, in the average liver cell concentrations at ATP, GTP, UTP and CTP decreased to 66, 62, 54 and 63%, respectively, of those of normal liver. Administration of insulin for 2 days returned the hepatic nucleotide concentrations to normal range; further insulin treatment for an additional 5 days raised the concentrations of ATP, GTP, UTP and CTP to 197.352, 412 and 792% of values observed in the liver of diabetic rats. 3. 3.|In diabetic rats the hepatic activities of OMP decarboxylase, orotate phosphoribosyltransferase, uridine phosphorylase, uridine-cytidine kinase and uracil phosphoribosyltransferase decreased to 44, 48, 70, 36 and 41% of the activities of normalliver. Insulin treatment for 2 days returned activities to normal range. Continued insulin treatment for an additional 5 days increased the enzymic activities to 3.9- to 5.3-fold of those of the liver of the diabetic rats. The regulation by insulin treatment of the activities of enzymes of de novo and salvage synthesis of UMP should explain, in part at least, the decline and increase of the uridylate pool in diabetes and after insulin treatment. 4. 4.|In the diabetic rat hepatic CTP synthetase, the rate-limiting enzyme of CTP biosynthesis, decreased to 53% and insulin administration for 2 days restored activity to normal range. Insulin treatment for an additional 5 days increased the synthetase activity to 4-fold of the values of the diabetic liver. Thus, the behavior of liver CTP synthetase activity is tightly linked with that of the CTP pool. 5. 5.|In the diabetic rat liver, the activity of IMP dehydrogenase, the rate-limiting limiting enzyme of GTP biosynthesis, decreased to 24% of that of the normal liver. Insulin administration for 2 days returned the activity to normal range, yielding a 4.5-fold increase in the activity from the diabetic to the insulin-treated state. The decline of IMP dehydrogenase activity in diabetes and its restoration by insulin treatment indicates that the behavior of this enzyme is closely linked with that of the GTP pool. 6. 6.|Because the major action of insulin on liver has been thought to occur in carbohydrate metabolism, the impact of insulin administration in the rat was compared on carbohydrate-metabolizing and on purine and pyrimidine synthesizing liver enzymic activities. In the diabetic rat, the hepatic activities of glucokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase decreased to 20, 30 and 65% of those of the normal rat liver. The hepatic enzymes of purine and pyrimidine biosynthesis declined to 54 to 85% with the exception of IMP dehydrogenase, carbamoyl-phosphate synthetase II and aspartate carbamoyltransferase which decreased to 14 to 28% of the activity of normal rat liver. Insulin administration restored enzymic activities to normal range in 2 days. The insulin-induced increases in the carbohydrate enzymic activities were 2.5- to 6.5-fold. A similar extent of increases was observed for most of the nucleic acid synthetic enzymes. These results indicated that some of the insulin-induced rises inhepatic enzymic activities in purine and pyrimidine biosynthesis are comparable to those for the classically recognized inductions in glycolytic and pentose phosphate enzyme activities. 7. 7.|The reprogramming of gene expression in cancer cells was examined in a variant of hepatoma 3924A cells in tissue culture which was resistant to the C-nucleoside, tiazofurin. In the hepatoma cells a biochemical program emerged that was characterized by a decrease in the transport of tiazofurin, in the content of TAD, the active metabolite of tiazofurin, and in the de novo synthesis of IMP to 2-, 2- and 4.7-fold of the activities of sensitive cells. By contrast, the pools of guanylates, the activities of IMP dehydrogenase, the guanylate salvageand that of the ratio of salvage to de novo synthesis were elevated 3-, 2.6-, 2.8- and 4.8-fold of those of the sensitive cells. Thus, the resistance to tiazofurin in hepatoma cells is expressed by an integrated reprogramming of gene expression.

Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis.

Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine biosynthesis, has previously been shown to be rapidly inactivated and degraded in Bacillus subtilis cells at the end of growth. The loss of enzyme activity appears to involve the oxidation of an iron-sulfur cluster in the enzyme. The degradation of the inactive enzyme involves some elements of the stringent response because it is inhibited in relA and relC mutants. Intracellular pools of guanosine tetra- and pentaphosphate were measured by an improved extraction procedure in cells that had been manipulated in various ways to induce or inhibit amidotransferase degradation. The results are consistent with the hypothesis that one or both of these nucleotides stimulates the synthesis of a protein involved in degradation. An elevated level of these nucleotides was not required for the continued degradation of amidotransferase once it had begun.

Polyglutamyl folate coenzymes and inhibitors of chicken liver glycinamide ribotide transformylase

Chicken liver glycinamide ribotide transformylase (5,10-methenyltetrahydrofolate:5'phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an enzyme of purine biosynthesis de novo, has greater specificity for its poly-γ-glutamyl folate coenzymes, 5,10-methenyltetrahydropteroyl(glutamate) n, where n = 3, 4, 5, 6 or 7, when compared to the monoglutamyi folate coenzyme. The relative specificity constants ( V/ K m) for the coenzymes 5,10-methenyltetrahydropteroyl(glutamate) n are 1.0, 1.6, 2.6, 2.4, 2.4, 4.9 and 1.5 for n=1, 2, 3, 4, 5, 6 and 7, respectively. Pteroylpoly-γ-(glutamate) n, where n=3, 4, 5, 6 or 7, are much better inhibitors of this enzyme when compared to the pteroylmonoglutamate. The concentration of inhibitor required for 50% inhibition was found to be 490, 120, 58, 28, 16, 14 and 12 μM for n=1, 2, 3, 4, 5, 6 and 7, respectively. Inhibitors with four or more glutamic acid residues gave grossly non-linear Dixon plots, in contrast to the linear plots obtained using inhibitors with three or less glutamic acid residues. The above findings make it feasible for the activity of glycinamide ribotide transformylase to be regulated by alteration in the length of the poly-γ-glutamyl chain of its folate coenzymes and of folate inhibitors.

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.

Folylpoly-.gamma.-glutamates as cosubstrates of 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase

N10-Formyltetrahydropteroylpoly-gamma-glutamates (N10-formyl-H4PteGlun) having n = 1, 3, 4, 5, 6, and 7 glutamyl residues have been tested as cosubstrates of the purine biosynthesis enzyme 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase (AICAR transformylase) of chicken liver. The cosubstrates were synthesized by solid-phase synthesis, reduced catalytically, and formylated; a purified enzyme preparation was used and assayed spectrophotometrically following the deltaOD at 298 nm resulting from conversion of the formylated folate to the free tetrahydro form. Km values and Vm values determined at saturating concentrations of AICAR and at 25 and 150 mM KC1 were used to calculate the relative specificity constants Vm/Km for the N10-formyl-H4PteGlun. At physiologic [K+] (150 mM) they were 1.0, 52, 250, 93, 120, and 59 and at the lower (25 mM) [K+] the relative specificity constants were 1.0, 64, 78,34, 48, and 37 for n = 1, 3, 4, 5, 6, and 7, respectively. The poly-gamma-glutamates are clearly the preferred cosubstrates, particularly when tested at physiologic [K+]. The maximal relative specificity constant observed with N10-formyl-H4PteGlu4 supports the hypothesis that regulation of certain pathways of one-carbon metabolism may operate via alterations of the poly-gamma-glutamyl chain length. No inhibition by the unnatural (d) isomers of the N10-formyl-H4PteGlun was observed.

Control of feedback repression of the enzymes of purine biosynthesis in Aerobacter aerogenes

Studies have been made of the regulation of the synthesis of six purine biosynthetic enzymes: P-ribosyl- PP amidotransferase (I), P-ribosyl glycinamide synthetase (II), P-ribosyl formyl glycinamide amidotransferase (IV), adenylosuccinate lyase (VIII-IIA), adenylosuccinate synthetase (IA), and IMP dehydrogenase (IG). Wild type Aerobacter aerogenes and two purine requiring mutants derived from it, were grown with limiting or excess adenine or guanine, cell extracts prepared, and enzyme activities measured. Using as a reference the levels of enzymes found in ghe wild type bacteria grown in unsupplemented medium, the results indicate that all of the enzymes are repressed by excess purine, and derepressed by purine starvation. However, these controls are not coordinate throughout the pathway, since each segment of the pathway shows a unique response, apparently to accommodate the special functions of the enzymes within it. The levels of three early enzymes of the pathway are primarily responsive to adenine supplementation and therefore it is concluded that these are controlled by the intracellular levels of adenine nucleotides, or perhaps those of the inosine (produced in cultures by deamination). Two of these (I and IV) are coordinately controlled, while the levels of the third enzyme (III) vary in parallel with the others, but with changes of a smaller magnitude. One enzyme (VIII-IIA) was shown to be subject to a dual or multivalent control, apparently being regulated both by the supply of adenine and guanine nucleotides. This enzyme has a dual function, catalyzing one step in IMP synthesis, and another in the conversion of IMP to AMP. Strikingly a subsequent enzyme in IMP formation, IMP cyclohydrolase, has been shown by others to be primarily controlled by guanine. In addition to its above mentioned function, this latter enzyme can be envisioned as necessary for an interconversion reaction in the pathway from adenine to guanine nucleotides. The synthesis of the enzymes studied subsequent to IMP formation, in the branches leading to AMP (IA) and GMP (IG), appear primarily controlled by their respective endproducts alone. However, the possibility that the levels of the first enzyme of GMP formation (IG) might be increased by the presence of substrate, as well as repressed by endproduct, was discussed.

Properties of 5′-phosphoribosylpyrophosphate amidotransferase in virus induced murine leukemia

Virus-induced 5′-phosphoribosylpyrophosphate amidotransferase (ribosylamine 5′-phosphate:pyrophosphate phosphoribosyltransferase (glutamate amidating). (EC 2.4.2.14), the first enzyme in purine biosynthesis, was isolated from spleens of mice infected with Friend leukemia virus, purified and certain of its properties were studied. The enzyme, which catalyzes the synthesis of 5′-phosphoribosylamine has a requirement for two substrates: 5′-phosphoribosyl pyropyhosphate as the phosphoribosyl donor, and for a nitrogen containing compound as a source of the amino group of 5′-phosphoribosylamine. Ribose 5-phosphate could not replace 5′ phosphoribosyl pyrophosphate. Nitrogen containing compounds found to be substrates were NH 4Cl, (NH 4) 2SO 4 and glutamine. The enzyme was sensitive to inhibition by purine ribonucleotides. Inhibition was competitive with 5′-ribosyl pyrophosphate and glutamine, but non competitive with respect to NH 4Cl.

Repression of an enzyme of purine biosynthesis in L cells

Repression of an enzyme of purine biosynthesis in L cells

Repression of an enzyme of purine biosynthesis in L cells

Repression of an enzyme of purine biosynthesis in L cells

Repression of an enzyme of purine biosynthesis in L cells

Repression of an enzyme of purine biosynthesis in L cells