Enolpyruvate phosphate

Most of the structural and biochemical work related to KDO is based on the estimation of the compound or its derivatives by the periodate-thiobarbituric acid (TBA) assay in its various modifications. Indeed, KDO (see Fig. 3) was discovered9 through the formation of a characteristic, purple, TBA chromophore (Xmax 549 nm) from its 8-phosphate (2), which is the product of the condensation of D-arabinose 5-phosphate with enolpyruvate phosphate, catalyzed by 3-deoxy-8-0-phosphonooctulosonate synthetase (EC 4.1.2.16) (see Scheme 1 and Section V,2). 

A comprehensive study of KDO 8-phosphate synthetase has been reported by Ray.137 The author purified the enzyme 450-fold from crude extracts of Escherichia coli B cells. The synthetase has a molecular mass of 90,000 6,000 daltons and is composed of three identical subunits having an apparent molecular mass of32,000 4,000 daltons. Two pH optima were observed, one being at pH 4.0-6.0 in succinate buffer, and the other, at pH 9.0 in glycine buffer. The isoelectric point of the enzyme is 5.1. The enzyme has an apparent KM for D-arabinose 5-phosphate of 20 pM and an apparent KM for enolpyruvate phosphate of 6 pM. 

Several observations regarding this aspect have been published, and are briefly mentioned here. 5,6-Dideoxy-6-C-phosphono-D-arabino-hexofuranose (135), an isosteric phosphonate analog of D-arabinose 5-phosphate, is apparently converted, in the presence of enolpyruvate phosphate, into 3,8,9-trideoxy-9-C-phosphono-D-mcmno-2-nonulosonic acid (136) under catalysis by KDO 8-phosphate synthetase from Escherichia coli K 235. Compound 136, an isosteric phosphonate analog of KDO 8-phosphate, is a product inhibitor of the synthetase, and, by the nature of the phosphonate group, is not subject to dephosphorylation as catalyzed by KDO 8-phosphate phosphatase156 (see Scheme 40). Compound 119 (see Scheme 33) is a weak inhibitor of KDO 8-phosphate synthetase.81 KDO inhibits KDO 8-phosphate phosphatase,139 and D-ribose 5-phosphate has an inhibitory... 

The occurrence of phosphoric ester groups at 0-9 is long established, as Neu5Ac9P was recognized as the condensation product of enolpyruvate phosphate (PEP) and ManNAc 6-phosphate in the biosynthetic pathway of sialic acids (see Section V,l). 

The synthesis of a 9-azido-9-deoxy derivative of Neu5Ac, namely, 5-acetamido-9-azido-3,5,9-trideoxy-D-gh/cero-D-ga/acto -2-nomdo-pyranosonic acid, from enolpyruvate phosphate and 2-acetamido-6-azido-2,6-dideoxy-D-mannose, using Neu5Ac synthase from A. meningitidis, has been reported.225... 

Two of the most frequent monosaccharide components of bacterial polymers belonging to this group have been the subjects of articles in this Series. They are 3-deoxy-D-manno-2-octulosonic acid,247 a normal constituent of the core region of bacterial lipopolysaccharides that is also present in some other polymers, and N-acetylneuraminic acid,248 found in several capsular polysaccharides. Enolpyruvate phosphate serves as the precursor of the C-l-C-3 fragment of the monosaccharides, with D-arabinose 5-phosphate or 2-acetamido-2-deoxy-D-mannose 6-phosphate being an acceptor for transfer of the three-carbon unit. Characteristic, activated forms of these monosaccharides are the CMP derivatives. 

In the biosynthesis of the capsular polysaccharide from Xanthomonas campestris, the modification was shown265 to occur at the level of a polyprenyl pentasaccharide diphosphate intermediate prior to polymerization of the repeating units, and enolpyruvate phosphate was a precursor of the pyruvic acid residues. A similar observation was made during a study of the biosynthesis of Rhizobium meliloti exopolysaccharide.266... 

Scheme 4.—Proposed Pathway for the Biosynthesis of Bacterial Xanthan Gum (PEP, enolpyruvate phosphate.)2 231...

Kdo-synthetase catalyzes the aldol addition of enolpyruvate phosphate with D-arabinose 5-phosphate (see Scheme 13), which gives 3-deoxy-r> manno-2-octulosonic add 8-phosphate (Kdo 8-phosphate). Kdo is an important component of oligosaccharides of Gram-negative bacteria. 

Kdo was prepared on the 38-mmol scale starting from D-arabinose, by the simultaneous operation of three enzymes in the same vessel. One is Kdo synthetase 56 hexokinase catalyzes the phosphorylation57 of D-arabinose by ATP (catalytic), and pyruvate kinase catalyzes58 the regeneration of ATP with enolpyruvate phosphate. Such systems are described in more detail in Section IV. In this preparation, enolpyruvate phosphate serves two very different purposes, acting as a source of high-energy phosphate, and as a three-carbon donor.59... 

Scheme 14.—The Aldol Addition of Enolpyruvate Phosphate with D-Erythrose 4-Phosphate, Catalyzed by DHAP-Synthetase.

Phosphorolysis of ribonucleic acid with polynucleotide phosphorylase gives a mixture of the diphosphates of the four common nucleosides, which are transformed into triphosphates with enolpyruvate phosphate and pyruvate kinase. This mixture may be used as such as a source of uridine triphosphate in the preparation of the nucleotide-sugar uridine 5 -(a-D-glucopy-ranosyl diphosphate) ( uridine-diphosphate-glucose, UDP-Glc), or as a... 

Enzymes immobilized on PAN gel, unless stated otherwise. The actual phosphorylating agent, ATP, is regenerated either by acetyl phosphate and acetoldnase, or enolpyruvate phosphate and pyruvate kinase. Phosphorylations by a mixture of the common ribonucleotide triphosphates. Not isolated.4 Soluble enzymes. Enzymes immobilized on agarose.1 Enzymes enclosed in a dialysis bag. 

The conversion, by bacterial extracts, of D-oZtro-heptulose 1,7-diphosphate to shikimate, essentially without side reactions, greatly facilitated subsequent study of the intermediate steps in the synthesis. It was shown that the addition of iodoacetate or fluoride completely blocks this conversion. In the presence of iodoacetate, synthesis is restored by the addition of either D-glyceronic acid 3-phosphate or enolpyruvate phosphate. In the presence of fluoride, only enolpyruvate phosphate is able to restore shikimate synthesis. Neither D-fructose 1,6-diphosphate nor pyruvate reverses these inhibitions. These results suggested that the reactions of glycolysis, from triose phosphate to enolpyruvate phosphate (see Fig. 2), are involved in the conversion of D-oZfro-heptulose diphosphate to shikimate. The effect... 

Enolpyruvate phosphate and n-erythrose 4-phosphate are independent intermediates in metabolic pathways of n-glucose that are not directly concerned with aromatic biosynthesis. Their simultaneous requirement for shikimate formation therefore indicates the first specific, or branch-point,... 

Fig. 4.—The Condensation of Enolpyruvate Phosphate and n-Erythrose 4-Phosphate by 3-Deoxy-D-oro6mo-heptulosonic Acid 7-Phosphate Synthetase (DAHP synthetase).

The view that such a reaction is the initial one in the conversion of enolpyruvate phosphate plus D-erythrose 4-phosphate to shikimate, was supported by fractionation of the crude, bacterial extracts and by the chemical synthesis of 3-deoxy-D-ara6mo-heptulosonic acid 7-phosphate. The steps in... 

The synthetic 3-deoxy-D-arabmo-heptulosonic acid 7-phosphate was converted quantitatively to 5-dehydroquinic acid by bacterial extracts, as will be discussed below. It was also used as an aid in identifying the structure of the product formed from enolpyruvate phosphate plus D-erythrose... 

In extracts of a wild type of E. coli (Strain B), enolpyruvate phosphate and D-erythrose 4-phosphate are converted to a 3-deoxyheptulosonic acid (most probably of the arabino configuration) and two equivalents of orthophosphate. Since 3-deoxy-D-arai fno-heptulosonic acid 7-phosphate was readily dephosphorylated by these enzyme preparations, the conclusion was... 

The enzymic condensation of enolpyruvate phosphate and n-erythrose 4-phosphate is formulated in Fig. 4 as an attack by a nucleophilic group of the enzyme (here symbolized as OH ) on the enolphosphate, concerted with attack by carbon atom 3 of the enolpyruvate on the electrophilic carbon atom of the aldehyde, and protonation of the carbonyl oxygen atom by an acidic group. This results in the release of orthophosphate, or of a transient, phosphorylated enzyme, and the open-chain form of 3-deoxy-n-orobfno-heptulosonic acid 7-phosphate. In this mechanism, enolpyruvate phosphate could be replaced by an enolpyruvate-enzyme compound. The reaction is analogous to the irreversible reaction for the fixation of carbon dioxide (Reaction 7), found in plants, in which intermediates could also Enolpyruvate phosphate -f HCOi - oxaloacetate + HPO< (7)... 

It has previously been mentioned that brief treatment of the bacterial extracts with activated carbon abolished their ability to convert D-altro-heptulose 1,7-diphosphate to 5-dehydroquinate, whereas the conversion of enolpyruvate phosphate and n-erythrose 4-phosphate to this intermediate was unaffected. However, by using larger proportions of activated carbon, for longer periods of time, extracts were produced which could still carry out the condensation between enolpyruvate phosphate and D-erythrose 4-phosphate, but could not convert 3-deoxy-D-aro5fno-heptulosonic acid... 

Further study of this synthesis in partly purified extracts did not reveal any requirements for cofactor or metal. Shikimate 5-phosphate (alone) was extensively hydrolyzed in these extracts to shikimate and orthophosphate, but, when equimolar amounts of enolpyruvate phosphate were also added, only insignificant quantities of shikimate were produced, and, for every mole of Zl formed, two equivalents of orthophosphate were released. The phosphatase action on shikimate 5-phosphate was strongly inhibited when the amount of enolpyruvate phosphate present was equivalent to only 10 per cent of the shikimate 5-phosphate, but Zl added initially to shikimate 5-phosphate did not inhibit the phosphatase activity. 

Since shikimate 5-phosphate is a required precursor, and a 3-0-substi-tuted shikimate is the end product, it is suggested that the first intermediate in the reaction between shikimate 5-phosphate and enolpyruvate phosphate is the phosphorylated Zl (XIX) the latter is then hydrolyzed to Zl. Some evidence that this is, indeed, the case was obtained by the addi-... 

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