PEP synthase

Glycolysis provides the main source of ATP in Trypanosoma brucei, E histolytica, and G lamblia, which possess pyruvate kinase as well as a pyruvate phosphate dikinase for converting phosphoenolpyruvate (PEP) to pyruvate and generating ATP. Pyruvate phosphate dikinase is not a homolog of pyruvate kinase but is closely related to PEP synthase from bacteria. The enzyme catalyzes conversion of PEP to pyruvate accompanied by the synthesis of ATP from AMP and pyrophosphate. Genes encoding the enzyme have been isolated from E histolytica and G lamblia and have demonstrated considerable structural divergences. No specific inhibitor of this enzyme has yet been identified. 

This versatility is a direct consequence of the specificity of the PTS for PEP sugar transport is thereby harnessed to the stoicheiometric control mechanism for glycolysis vs. gluconeogenesis (i.e. control of the activity of pyruvate kinase and PEP synthase by the ATP concentration in the cell) and, as a consequence it will respond to the metabolic state within the cell. 

The enzyme catalyzes the condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 3-phospho-5-enolpyruvoylshikimate (PEPS) [Fig. 2 (6)]. PEPS synthase has been detected in mung bean seedlings (Koshiba, 1978b) properties of the enzyme were not studied, however. The enzyme was detected in bacteria and characterized as a freely reversible reaction (Levin and Sprinson, 1964). 

In its overall effect the new cycle is a reversal of the oxidative tricarboxylic add cycle, but endergonic in nature and hence includes two reactions (a-ketoglutarate synthase and citrate lyase) that can by-pass the irreversible steps of the KLrebs cycle. Photosynthetic bacteria also contain pyruvate synthase that brings about the direct synthesis of pyruvate from acetyl-CoA, COg and reduced ferredoxin, as well as PEP synthase that catalyzes the direct synthesis of PEP from pyruvate and ATP. 

Three types of synthases catalyze the addition of phosphoenolpyruvate (PEP) to aldoses or the corresponding terminal phosphate esters. By concurrent release of inorganic phosphate from the preformed enolate nucleophile, the additions are essentially irreversible. None of the enzymes are yet commercially available and little data are available oil the individual specificities for the aldehydic substrates. A bacterial NeuAc synthase (EC 4.1.3.19) has been used for the microscale synthesis of A -acetylncuraminic acid from Af-acetyl-D-mannosamine31 and its 9-azido analog from 2-acetamido-6-azido-2,6-dideoxy-D-mannose32. 

Deoxy-i>maw f)-2-octulosonic acid 8-phosphate can be obtained in gram quantities by using KDO synthase (F.C 4.1.2.16) for the addition of PEP to D-arabinose 5-phosphate33. DA HP synthase (EC 4.1.2.15) produces 3-deoxy-L>-arer/>/>io-heptulosonic acid 7-phosphate from PEP and D-erythrose 4-phosphate35. 

Figure 5 Model of phosphorus (P) deficiency-induced physiological changes associated with the release of P-mobilizing root exudates in cluster roots of white lupin. Solid lines indicate stimulation and dotted lines inhibition of biochemical reaction sequences or mclaholic pathways in response to P deliciency. For a detailed description see Sec. 4.1. Abbreviations SS = sucrose synthase FK = fructokinase PGM = phosphoglueomutase PEP = phosphoenol pyruvate PE PC = PEP-carboxylase MDH = malate dehydrogenase ME = malic enzyme CS = citrate synthase PDC = pyruvate decarboxylase ALDH — alcohol dehydrogenase E-4-P = erythrosc-4-phosphate DAMP = dihydraxyaceConephos-phate APase = acid phosphatase.

Figure 4. Reactions catalyzed by the broad-specificity DAHP synthase-Co of higher plant cytosol. Condensation of PEP and erythrose 4-P (top) yields 3-deoxy-D-arabino-heptulosonate 7-P (DAHP), whereas condensation of PEP and D-glyceraldehyde 3-P (G-3-P) yields 2-keto-3-deoxy-D-threo-hexulosonate 6-P (DTHP).

Synthetic studies for sialic acid and its modifications have extensively used the catabolic enzyme N-acetylneuraminic acid aldolase (NeuA E.C. 4.1.3.3), which catalyzes the reversible addition of pyruvate (70) to N-acetyl-D-mannosamine (ManNAc, 11) to form the parent sialic acid N-acetylneuraminic acid (NeuSNAc, 12 Scheme 2.2.5.23) [1, 2, 45]. In contrast, the N-acetylneuraminic acid synthase (NeuS E.C. 4.1.3.19) has practically been ignored, although it holds considerable synthetic potential in that the enzyme utilizes phosphoenolpyruvate (PEP, 71) as a preformed enol nucleophile from which release of inorganic phosphate during... 

The availability of both the cataboHc aldolase and the uniquely synthetic anabolic synthase made it possible to assemble a novel continuous assay for the determination of the metabolite N-acetylneuraminic acid [46]. A combination of both enzymes, in the presence of an excess of PEP, will start a cycle in which the determinant sialic acid will undergo a steady conversion of cleavage and re-syn-thesis as a futile cycle (Scheme 2.2.5.24). With each progression, however, 1 equiv of pyruvate is liberated simultaneously, which causes time-dependent signal amplification. Pyruvate is quantified spectrophotometrically by a corresponding NADH consumption when the system is coupled to the standard pyruvate dehy-... 

Salgado J, Zabalegui N, Gil C et al. Polymorphisms in the thymidylate synthase and dihydropyrimidine dehydrogenase genes predict response and toxicity to capecitabine-raltitrexed in colorectal cancer. Oncol Pep 2007 17 325-328. 

Fig. 3.8.6 Spectrophotometric assays of complexes V, I III, II—III, and citrate synthase activity. DTNB Dithio-nitrobenzoic acid, LDH lactic dehydrogenase, ME medium E, MF medium F, MG medium G, MH medium H, PEP phosphoenol pyruvate, PK Pyruvate kinase, TNB thio-nitrobenzoic acid...

T Glycogen phosphorylase I Glycogen synthase I PFK-1 T FBPase-2 i Pyruvate kinase T PEP carboxykinase T Triacylglycerol lipase Perilipin phosphorylation T Acetyl-CoA carboxylase... 

The six carbons of the benzene ring of the aromatic amino acids are derived from the four carbons of erythrose 4-phosphate and two of the three carbons of phosphoenolpyruvate (PEP). The initial step in the pathway (Fig. 25-1, step a) is the condensation of erythrose 4-P with PEP and is catalyzed by 3-deoxy-D-arafrmo-heptulosonate-7-phosphate (DAHP) synthase. Closely analogous to an aldol condensation, the mechanism provides a surprise.10 When PEP containing lsO in the oxygen bridge to the phospho group reacts, the lsO is retained in the eliminated phosphate biochemical intuition would suggest that it should stay in the... 

Three types of lyases have been identified that catalyze the addition of phosphoenolpyruvate (PEP) to aldoses or to terminally phosphorylated sugar derivatives. With simultaneous release of inorganic phosphate from the preformed enolpyruvate nucleophile during C-C bond formation the additions are essentially irreversible and, therefore, these lyases are often referred to as synthases. The mechanistic details of these reactions, however, have yet to be elucidated but it seems obvious that the chances of variation on the part of the nucleophile will be strictly limited. Although the thermodynamic advantage makes these enzymes highly attractive for synthetic applications, none of them is yet commercially available and only few data have been reported concerning the individual specificities towards aldehydic substrates. 

The second enzyme in the sequence studied was KD0-8-phosphate synthase. This enzyme was purified to homogeneity (24). This enzyme catalyzes the condensation of D-arabinose-5-phosphate and PEP to yield KD0-8-phosphate and inorganic phosphate. One can assay this irreversible reaction either by measuring the formation of KD0-8-phosphate or the release of P. (2Jj). The latter is the method of choice, since a number of analogues were found to interfer with the thiobarbituric acid assay. The enzyme has an apparent K for PEP of 6 x 10 M and an apparent K for D-arabinose-5-phospftiate of 2 x 10 M. m... 

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