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The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken–egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a “metabolism first” theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network., The evolutionary origins of metabolism are largely unknown. This study shows that the prebiotically available proteinogenic amino acid cysteine can promote the metabolism-like rate-limiting formation of ribose-5-phosphate, suggesting that early metabolic pathways could have emerged thought the stepwise enzymatization of non-enzymatic reaction sequences.

Accumulation of nucleotide building blocks prior to and during S phase facilitates DNA duplication. Herein, we find that the anaphase-promoting complex/cyclosome (APC/C) synchronizes ribose-5-phosphate levels and DNA synthesis during the cell cycle. In late G1 and S phases, transketolase-like 1 (TKTL1) is overexpressed and forms stable TKTL1-transketolase heterodimers that accumulate ribose-5-phosphate. This accumulation occurs by asymmetric production of ribose-5-phosphate from the non-oxidative pentose phosphate pathway and prevention of ribose-5-phosphate removal by depleting transketolase homodimers. In the G2 and M phases after DNA synthesis, expression of the APC/C adaptor CDH1 allows APC/CCDH1 to degrade D-box-containing TKTL1, abrogating ribose-5-phosphate accumulation by TKTL1. TKTL1-overexpressing cancer cells exhibit elevated ribose-5-phosphate levels. The low CDH1 or high TKTL1-induced accumulation of ribose-5-phosphate facilitates nucleotide and DNA synthesis as well as cell cycle progression in a ribose-5-phosphate-saturable manner. Here we reveal that the cell cycle control machinery regulates DNA synthesis by mediating ribose-5-phosphate sufficiency.

The pentose phosphate pathway (PPP) branches from glucose 6-phosphate (G6P), produces NADPH and ribose 5-phosphate (R5P), and shunts carbons back to the glycolytic or gluconeogenic pathway. The PPP has been demonstrated to be a major regulator for cellular reduction-oxidation (redox) homeostasis and biosynthesis. Enzymes in the PPP are reported to play important roles in many human diseases. In this review, we will discuss the role of the PPP in type 2 diabetes and cancer.

The 2-deoxy- d -ribose-5-phosphate aldolase (DERA) is a synthetically attractive enzyme because of its ability to perform C C-couplings stereoselectively, the enzyme uses acetaldehyde as nucleophile and thus produces true aldols rather than ketols, and may add two acetaldehyde molecules onto one electrophile. However, DERA produces crotonaldehyde as side reaction from acetaldehyde which is then an irreversible inhibitor forming a covalent Michael -adduct within the active site in particular with cysteine 47 (Dick et al., 2016). This inhibition can be resolved by mutating C47 to non-nucleophile amino acids. Still, the inhibition is not an on-off-feature and the present mutagenesis study illustrates that there must be a C47-independent inactivation mechanism. As a practical result: The virtually fully resistant mutant C47L was found, which shows no loss in stereoselectivity, − this renders this variant as promising catalyst.

[(13)C]Formaldehyde was selectively incorporated into the C-1 position of D-fructose 6-phosphate by condensation with D-ribulose 5-phosphate catalyzed by a partially purified enzyme system for formaldehyde fixation in Methylomonas aminofaciens 77a. Much of the [1-(13)C]D-fructose 6-phosphate produced in this reaction was converted to [1-(13)C]D-glucose 6-phosphate by the addition of glucose-6-phosphate isomerase. A fed-batch reaction with periodic additions of the substrates afforded 56.2 g/liter D-glucose 6-phosphate and 26.8g/liter D-fructose 6-phosphate. When [(13)C]methanol was used as the C1-donor, the yield of [1-(13)C]D-glucose 6-phosphate was high when alcohol oxidase was added. The optimum conditions for sugar phosphate production in the fed-batch reaction gave 45.6g/liter [1-(13)C]D-glucose 6-phosphate and 16.4g/liter [1-(13)C]D-fructose 6-phosphate in 165min. The molar yield of the total sugar phosphates to methanol added was 95%. The addition of H2O2 and catalase to the reaction system supplied molecular oxygen for methanol oxidation to formaldehyde by alcohol oxidase.

Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni(2+)-chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = β = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, β = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing.

Glycation of horse heart metmyoglobin with d-ribose 5-phosphate (R5P), d-2-deoxyribose 5-phosphate (dR5P), and d-ribose with inorganic phosphate at 37°C generates an altered protein (Myo-X) with increased SDS-PAGE mobility. The novel protein product has been observed only for reactions with the protein myoglobin and it is not evident with other common sugars reacted over a 1 week period. Myo-X is first observed at 1-2 days at 37°C along with a second form that is consistent in mass with that of myoglobin attached to several sugars. MALDI mass spectrometry and other techniques show no evidence of the cleavage of a peptide from the myoglobin chain. Apomyoglobin in reaction with R5P also exhibited this protein form suggesting its occurrence was not heme-related. While significant amounts of O(2)(-) and H(2)O(2) are generated during the R5P glycation reaction, they do not appear to play roles in the formation of the new form. The modification is likely due to an internal cross-link formed during a glycation reaction involving the N-terminus and an internal amine group; most likely the neighboring Lys133. The study shows the unique nature of these common pentose sugars in spontaneous glycation reactions with proteins.

The activity of ribose-5-phosphate isomerases (RpiB) from Clostridium difficile for D-ribose isomerization was optimal at pH 7.5 and 40 degrees C, while that from Thermotoga maritima for L-talose isomerization was optimal at pH 8.0 and 70 degrees C. C. difficile RpiB exhibited activity only with aldose substrates possessing hydroxyl groups oriented in the right-handed configuration (Fischer projections) at the C2 and C3 positions, such as D-ribose, D-allose, L-talose, L-lyxose, D-gulose, and L-mannose. In contrast, T. maritima RpiB displayed activity only with aldose substrates possessing hydroxyl groups configured the same direction at the C2, C3, and C4 positions, such as the D- and L-forms of ribose, talose, and allose.

Ribose 5-phosphate (R5P) undergoes the Maillard reaction with amines at significantly higher rates than most other sugars and sugar phosphates. The presence of an intramolecular phosphate group, which catalyzes the early stages of the Maillard reaction, provides the opportunity for the R5P molecule to undergo novel reaction paths creating unique Maillard products. The initial set of reactions leading to an Amadori product (phosphorylated) and to an alpha-dicarbonyl phosphate compound follows a typical Maillard reaction sequence, but an observed phosphate hydrolysis accompanying the reaction adds to the complexity of the products formed. The reaction rate for the loss of R5P is partially dependent on the pK(a) of the amine but also is correlated to the protonation of an early intermediate of the reaction sequence. In the presence of oxygen, a carboxymethyl group conjugated to the amine is a major product of the reaction of R5P with N-acetyllysine while little of this product is generated in the absence of oxygen. Despite lacking a critical hydroxyl group necessary for the Maillard reaction, 2-deoxyribose 5-phosphate (dR5P) still generates an Amadori-like product (with a carbonyl on the C-3 carbon) and undergoes phosphate cleavage. Two highly UV-absorbing products of dR5P were amine derivatives of 5-methylene-2-pyrrolone and 2-formylpyrrole. The reaction of dR5P with certain amines generates a set of products that exhibit an interesting absorbance at 340nm and a high fluorescence.

Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43: 7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii , we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6- 2 H 2 ]- and [U- 13 C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of d - arabino -3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the d - ara-bino -3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from d - arabino -3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.

FN3K (fructosamine 3-kinase) is a mammalian enzyme that catalyses the phosphorylation of fructosamines, which thereby becomes unstable and detaches from proteins. The homologous mammalian enzyme, FN3K-RP (FN3K-related protein), does not phosphorylate fructosamines but ribulosamines, which are probably formed through a spontaneous reaction of amines with ribose 5-phosphate, an intermediate of the pentose-phosphate pathway and the Calvin cycle. We show in the present study that spinach leaf extracts display a substantial ribulosamine kinase activity (approx. 700 times higher than the specific activity of FN3K in erythrocytes). The ribulosamine kinase was purified approx. 400 times and shown to phosphorylate ribulose-epsilon-lysine, protein-bound ribulosamines and also, with higher affinity, erythrulose-epsilon-lysine and protein-bound erythrulosamines. Evidence is presented for the fact that the third carbon of the sugar portion is phosphorylated by this enzyme and that this leads to the formation of unstable compounds decomposing with half-lives of approx. 30 min at 37 degrees C (ribulosamine 3-phosphates) and 5 min at 30 degrees C (erythrulosamine 3-phosphates). This decomposition results in the formation of a 2-oxo-3-deoxyaldose and inorganic phosphate, with regeneration of the free amino group. The Arabidopsis thaliana homologue of FN3K/FN3K-RP was overexpressed in Escherichia coli and shown to have properties similar to those of the enzyme purified from spinach leaves. These results indicate that the plant FN3K/FN3K-RP homologue, which appears to be targeted to the chloroplast in many species, is a ribulosamine/erythrulosamine 3-kinase. This enzyme may participate in a protein deglycation process removing Amadori products derived from ribose 5-phosphate and erythrose 4-phosphate, two Calvin cycle intermediates that are potent glycating agents.

To identify the genes involved in pyridoxine synthesis in yeast, auxotrophic mutants were prepared. After transformation with a yeast genomic library, a transformant (A22t1) was obtained from one of the auxotrophs, A22, which lost the pyridoxine auxotrophy. From an analysis of the plasmid harboured in A22t1, the RKI1 gene coding for ribose 5-phosphate ketol-isomerase and residing on chromosome no. 15 was identified as the responsible gene. This notion was confirmed by gene disruption and tetrad analysis on a diploid prepared from the wild-type and the auxotroph. The site of mutation on the RKI1 gene was identified as position 566 with a transition from guanine to adenine, resulting in amino acid substitution of Arg-189 with lysine. The enzymic activity of the Arg189→Lys (R189K) mutant of ribose 5-phosphate ketolisomerase was 0.6% when compared with the wild-type enzyme. Loss of the structural integrity of the protein seems to be responsible for the greatly diminished activity, which eventually leads to a shortage of either ribose 5-phosphate or ribulose 5-phosphate as the starting or intermediary material for pyridoxine synthesis.

NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U-13C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, ‘norpronyl-lysine’, as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) 13C–13C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of 13C5-ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products., NMR reveals numerous early and advanced glycation products, including a newly recognized ‘norpronyl-lysine,’ and cross links in solution, intact collagen and model systems. Solid state methods are directly applicable to in vitro and in vivo glycation pathway and product characterization.

The p53 tumour suppressor inhibits tumour development via various mechanisms such as apoptosis, inhibition of proliferation or the activation of senescence. Recently, several studies have indicated a novel role of p53 in the regulation of energy metabolism. Previously we have discovered TIGAR, a p53 target gene that acts as a fructose-2,6-bisphosphatase. TIGAR therefore can redirect glucose from the glycolytic pathway to the pentose phosphate pathway (PPP), which promotes NADPH production to generate reduced glutathione for protecting against ROS, and also ribose 5 phosphate production for nucleotide synthesis. In order to understand the function of TIGAR in vivo, we generated TIGAR deficient mice. We have determined a critical role of TIGAR in rapidly proliferating tissue, either for repair after damage or during tumor development.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1- related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg2+ or Mn2+ (being Ca2+ independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in hotosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells. Fil: Piattoni, Claudia Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Bustos, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

Phosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of thepurFgene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA independent of PurF are present inSalmonella enterica. Here, we describe mutant alleles of the essentialprsAgene, which encodes PRPP synthetase (E.C.2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation and allowed non-enzymic formation of PRA due to a build-up of ribose 5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network.

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic l-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed.

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.

A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP), the ribulose monophosphate (RuMP) pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium) produce ribose-5-phosphate via the nonoxidative PPP (NOPPP), whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1) lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP), the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P) among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii) probably does not synthesize aromatic amino acids at all.

In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors. This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in Escherichia coli. Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent Kd measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase. Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or vice versa. Structural studies on the two genetically distinct isomerases in E. coli have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis. Sequence searches of completed genomes show ribose 5-phosphate isomerase B to be the sole isomerase in many bacteria, although ribose 5-phosphate isomerase A is a nearly universal enzyme. All genomes contain at least one of the two enzymes. These results confirm that both enzymes must be independently capable of supporting ribose metabolism, a fact that had not previously been established.

1. Glucose 5-phosphate was synthesized from ribose 5-phosphate by an enzyme extract prepared from an acetone-dried powder of rat liver. Three rates of ribose 5-phosphate utilization were observed during incubation for 17 h. An analysis of intermediates and products formed throughout the incubation revealed that as much as 20% of the substrate carbon could not be accounted for. 2. With [1-14C]ribose 5-phosphate as substrate, the specific radioactivity of [14C]glucose 6-phosphate formed was determined at 1, 2, 5 and 30 min and 3, 8 and 17 h. It increased rapidly to 1.9-fold the initial specific radioactivity of [1-14C]ribose 5-phosphate at 3 h and then decreased to a value approximately equal to that of the substrate at 6 h, and finally at 17 h reached a value 0.8-fold that of the initial substrate [1-14C]ribose 5-phosphate. 3. The specific radioactivity of [14C]ribose 5-phosphate decreased to approx. 50% of its inital value during the first 3 h of the incubation and thereafter remained unchanged. 4. The distribution of 14C in the six carbon atoms of [14C]glucose 6-phosphate formed from [1-14C]ribose 5-phosphate at 1, 2, 5 and 30 min and 3, 8 and 17 h was determined. The early time intervals (1–30 min) were characterized by large amounts of 14C in C-2 and in C-6 and with C-1 and C-3 being unlabelled. In contrast, the later time intervals (3–17 h) were characterized by the appearance of 14C in C-1 and C-3 and decreasing amounts of 14C in C-2 and C-6. 5. It is concluded that neither the currently accepted reaction sequence for the non-oxidative pentose phosphate pathway nor the ‘defined’ pentose phosphate-cycle mechanism can be reconciled with the labelling patterns observed in glucose 6-phosphate formed during the inital 3 h of the incubation.

Three forms (I, II, and III) of R 5-P ketol icomerase (EC 5.3,1.6) were separated and purified from Candida utilis. The Kms for R 5-P by I, II, and III were 0.77 mM, 0.67 mM and 0.16 mM, respectively. The isoelectric points of the forms, I, II, and III were 3.79, 3.87 and 3.99, respectively.The activation energies of the forms, I, II, and III were 10,800, 12,000 and 6360 cal/mole, respectively. The forms, I, II, and III were all inhibited with chelating reagent, EDTA (1 mM), but not affected with thiol reagents such as p-CMB, ICH2COOH, NEM, and DTNB. These properties are quite different from the other known R 5-P ketol isomerases, all of those are not affected with EDTA, but inhibited with the thiol reagents as described above.

Candida utilis Ribose 5-phosphate ketol isomerase (EC 5.3.1.6) was purified. The enzyme is homogeneous by ultracentrifugal analysis and disc electrophoresis. The isoelectric point of the enzyme is 3.8, Km for R 5-P is 0.108 mM and Kis by 5′-AMP and 6-P-G are 0.16 mM and 0.61 mM, respectively. The thermodynamic parameters, ΔH, ΔG (35°C), ΔS (35°C) and E are 4336 cal/mole, — 5629 cal/mole, 32.15 cal/deg-mole and 6360 cal/mole, respectively. The molecular weight of the enzyme is 183,000 and the enzyme is assumed to consist of three subunits, one with a molecular weight of 75,000 and two identical subunits each with a molecular weight of 54,000.

The intracellular ribose 5-phosphate concentration was found to be an important determinant of rates of de novo purine synthesis. When ribose 5-phosphate production was reduced in cultured human lymphoblasts by glucose starvation, the intracellular phosphoribosylpyrophosphate concentration and rates of de novo purine synthesis decreased. Inosinate-guanylate:pyrophosphate phosphoribosyltransferase (HPR transferase)-deficient cells were relatively more resistant to glucose starvation. To minimize the effect of purine nucleotide feedback inhibition on the de novo pathway, cells were treated with inhibitors of IMP dehydrogenase and adenylosuccinate synthetase. In normal lymphoblasts, purine synthesis was stimulated only at glucose concentrations greater than 100 microM while in HPR transferase-deficient lymphoblasts, stimulation occurred even in the absence of glucose. The differences between the normal and HPR transferase-deficient cells were lost when ribose reutilization from endogenous nucleotide breakdown was impaired in the HPR transferase-deficient cells by incubation with 2'-deoxyinosine. Endogenous ribose reutilization for purine synthesis is, therefore, important when either glucose availability is limited or synthesis is stimulated. In the absence of glucose, exogenous purine nucleotides restored the intracellular concentrations of ribose 5-phosphate, phosphoribosylpyrophosphate, and purine nucleotides to almost 100% and rates of purine synthesis to 50-75% of those at 10 mM glucose. When ribose 5-phosphate production was increased in peripheral blood lymphocytes by phytohemagglutinin activation, the intracellular phosphoribosylpyrophosphate concentration and rates of de novo purine synthesis increased.

The reaction of hydroxyl radicals with ribose-5-phosphate in oxygenated aqueous solution leads to the following phosphate free products (G-values): Erythrodialdose (0.4), formic acid (0.4) and ribodialdose (0.1). In addition, inorganic phosphate (0.6) and some minor phosphate free products (total G≈ 0.08) are formed. Within the limits of experimental error material balance between dephosphorylation products and inorganic phosphate is obtained. A mechanism is proposed which can account for the main products. The primary steps of the dephosphorylation are H atom abstraction from C-5 of ribose-5-phosphate by OH radicals and subsequent addition of O2. The peroxy radicals formed decompose to ~80% by C-C bond cleavage followed by elimination of inorganic phosphate and only to ~20% by direct dephosphorylation. The relevance of these results with respect to the radiolysis of DNA is discussed.

An enzyme activity responsible for the formation of 5'-phosphoribosylamine from ribose 5-phosphate and a nitrogen donor was prepared from livers of ducks, chickens, and pigeons. This enzyme activity is distinct from ribosylpryophosphate 5-phosphate amidotransferase (EC 2.4.2.14), which catalyzes the formation of 5'-phosphoribosylamine from ribosylpyrophosphate 5-phosphate and glutamine. The enzyme was partially purified and completely separated from ribosylpyrophosphate 5-phosphate amidotransferase. For maximal synthesis of 5'-phosphoribosylamine, the reaction required ribose 5-phosphate, ammonium chloride, ATP, and magnesium ions. Prolonged heating at 80° destroyed the activity of the enzyme. It is proposed that an alternate pathway for the initial step of purine synthesis exists, namely, the direct conversion of ribose 5-phosphate to 5'-phosphoribosylamine. This reaction is enzymatic and is sensitive to inhibition by ribonucleotides and by ribose 5-phosphate.

Two physically and genetically distinct forms of ribosephosphate isomerase have been identified in Escherichia coli K12. The constitutive ribosephosphate isomerase A has a Km for ribose 5-phosphate (4.4 +/- 0.5 mM) six times greater than that of the inducible ribosephosphate isomerase B (0.83 +/- 0.13 mM). Treatment of the enzymes with 1.25 mM iodoacetate resulted in 100% loss of activity for ribosephosphate isomerase B, whereas ribosephosphate isomerase A was unaffected. Various cellular metabolites were tested and found to be without significant effect on either enzyme. The two enzymes could be separated by filtration on Sephadex G75 superfine and their apparent molecular weights were 45000 for ribosephosphate isomerase A and 32000-34000 for ribosephosphate isomerase B. Under certain conditions the two enzymes showed different patterns of heat inactivation but the results with ribosephosphate isomerase A varied in an unusual way with the protein concentration. Ribosephosphate isomerase B was formed inducibly in a mutant lacking ribosephosphate isomerase A but there was no evidence for the production of ribosephosphate isomerase B in wild-type cells. The formation of ribosephosphate isomerase B was not a consequence of the ribosephosphate isomerase B mutation, since strains could be constructed which formed both enzymes constitutively in the anticipated amounts. The ribosephosphate isomerase formed by a secondary mutant obtained from a ribosephosphate-isomerase-A-negative strain was identified as ribosephosphate isomerase B on the basis of its Km, elution profile from Sephadex G75, inhibition of iodoacetate, and heat inactivation. The ribosephosphate isomerases of another Escherichia coli K12 strain, X289, were investigated, since their properties were reported to be different from many of these described here for ribosephosphate isomerases A and B. In our hands strain X289 contained two ribosephosphate isomerases apparently identical to ribosephosphate isomerases A and B. The evidence to date suggests that ribosephosphate isomerase A catalyses the formation of ribose 5-phosphate from ribulose 5-phosphate and also participates in the reverse reaction during ribose and adenosine catabolism. The normal physiological role of the inducible ribosephosphate isomerase B is still uncertain.

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3′-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce d-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce d-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.

Three enzyme reactions have been reported to catalyze the synthesis of phosphoribosylamine in eukaryotic cells. These activities are glutamine phosphoribosylpyrophosphate (P-Rib-P-P) amidotransferase [amidophosphoribosyl-transferase; 5-phosphoribosylamine: pyrophosphate phosphoribosyltransferase (glutamate-amidating) EC 2.4.2.14], ammonia P-Rib-P-P aminotransferase, and ammonia ribose-5-phosphate aminotransferase. A purine auxotroph derived from a cell line of Chinese hamster fibroblasts was shown to be deficient in catalytic activities of glutamine P-Rib-P-P amidotransferase and ammonia P-Rib-P-P aminotransferase. Extracts from this cell line had normal ammonia ribose-5-phosphate aminotransferase activity. The defect in purine biosynthesis in the mutant cell line was localized to the synthesis of phosphoribosylamine. These results indicate that glutamine P-Rib-P-P amidotransferase or ammonia P-Rib-P-P aminotransferase or both are important for phosphoribosylamine synthesis, but that ammonia ribose-5-phosphate aminotransferase activity probably does not play a significant role in this eukaryotic cell line. The simultaneous disappearance of both P-Rib-P-P-dependent activities suggests these two enzyme activities are closely related structurally or genetically.

A study was made to develop a new method for the production of ribose-5-phosphate (R-5-P) from uridine-5′-monophosphate (UMP) by the action of nucleotide- N -ribosidase of Pseudomonas oleovorans , and a suitable medium for the formation of nucleotide- N -ribosidase was established. For the enzymatic conversion of UMP to R-5-P, a cell suspension was employed as the enzyme source. Although degradation of R-5-P, the desired product, occurred during the course of the enzyme reaction, it was prevented by the addition of an appropriate amount of zinc ion and resulted in a stoichiometric conversion of UMP to R-5-P and uracil. Accumulated R-5-P was readily isolated by ion-exchange chromatography of the bacteria-free reaction mixture. Yield of isolated R-5-P was about 60% of the theoretical recovery.