Phosphoenolpyruvate labeled

By means of phosphoenolpyruvate labeled with both tritium and deuterium ( -[3- Hi, Hi]phosphoenolpyruvate), it is possible to determine the stereochemistry of both the addition and the elimination step involved in the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) (8) (Floss, 1986) (Fig. 7.3). The EPSP synthase reaction involves addition and elimination at the 2,3 double bond with opposite stereochemistry (i.e., either syn addition and anti elimination or anti addition followed by syn elimination) (Floss, 1986). 

A remarkable reaction of the phospho group is its transfer onto carbon in the biosynthesis of the C—P bond (Fig. 6). Incidentally, this leads to a difficulty with nomenclature, because the group -P03H2, which is known to chemists as phosphono, is called phospho only when on a heteroatom (64), so the transfer changes its name Despite much ealier guessing from labeling patterns that phosphoenolpyruvate was the source of the C—P bond, it was only in 1988 (65, 66) that the enzyme responsible was isolated. The difficulty proved to be that the equilibrium favors phosphoenolpyruvate by about 2000-fold (67), so that assays only detected the enzyme in the direction contrary to biosynthesis evidently the biosynthesis takes place because subsequent reac-... 

There will be no labeled carbons. The CO2 added to pyruvate (formed from the lactate) to form oxaloacetate is lost with the conversion of oxaloacetate into phosphoenolpyruvate. 

The specific and proximate precursor of the mCyN unit in ansamycin polyketides is 3-amino-5-hydroxybenzoic acid 59 (AHBA) [94]. The biosynthesis of AHBA has recently been described by Floss and co-workers from the initial branch point of the shikimic acid pathway prior to 3-deoxy-D-flra/jzno-heptulo-sonic acid 7-phosphate (DAHP) [95]. The pathway shown in Scheme 25 was delineated by feedings of the proposed AHBA precursors, in labelled forms, to cell-free extracts of both the rifamycin B producer A. mediterranei S699 and the ansatrienin A producer S. collinus Tul892. In these experiments each of the compounds 61-64 was converted into AHBA with generally increasing efficiency. Most importantly the shikimate pathway compound DAHP cannot replace phosphoenolpyruvate 61 and erythrose 4-phosphate 60, or aminoDAHP 62 as the precursor of AHBA 59. 

The equilibrium between phosphoenolpyruvate and phosphonopyruvate (19, Fig. 13-10) is catalyzed by a phosphomutase. The mechanism of the transformation of a phosphoryl into a phosphonoyl group has been studied with labeled and S-substituted analogs of the natural substrate ll53 158l... 

The use of alkaline phosphatase as an enzyme label allows enhancement of the sensitivity by using phosphoenolpyruvate as substrate and the utilization of a separate detection column in the ET unit for the determination of the product (pyruvate) by substrate recycling. This is accomplished by using the substrate recycling system described above [18] comprising the coimmobilized enzymes lactate dehydrogenase (reduces pyruvate to lactate under the consumption of... 

A series of early biochemical studies have identified the primary precursors of methanofuran as phosphoe-nolpyruvate, dihydroxyacetone phosphate, tyrosine, glutamate, 2-ketoglutarate, acetate, and CO2 as illustrated in Figure 2. Isotope incorporation studies with H- and C-labeled precursors have shown that the fiiran moiety of methanofuran is formed from phosphoenolpyruvate and dihydroxyacetone phosphate. Following dehydration, the next step in the biosynthesis of the fiiran would be a reduction of the carboxylic acid to an aldehyde. The final step in the biosynthesis of the fiiran moiety of methanofuran is expected to be a transamination reaction to form the 2-aminomethyI subunit. 

S)- 0, 0]-thiophosphoenolpyruvate, generated in situ from 2-[(5)- 0, 0]-thiophospho-D-glycerate in the presence of enolase, has been used as a substrate in the phosphoenolpyruvate carboxylase reaction carried out in [ 0]-labelled water. From the absolute configuration of the [ 0, 0/ 0]-thiophosphate produced, it was deduced that this reaction proceeds by a stepwise mechanism involving the intermediate formation of carboxyphosphate (Scheme 2). This deduction is, however, at variance with a mechanism proposed on... 

In the first case (A), phosphoenolpyruvate (124) derived from racemic [l,2- 3C2]glycerol would label C-7, C-1 , C-2 and erythrose 4-phosphate (125) would label C-4 , C-5 and C-6 , with C-3 remaining unlabelled. In the second case, 124 would label C-7, C-F and C-6 , and 125 would label C-2 , C-3 , C-4 , with C-5 remaining unlabelled. This would suggest that in the preceding case, C-1 of 125 was aminated, and in the latter case, C-3 of 125 was aminated. The coupling pattern in the enriched sample of P8/1-... 

OG (72) revealed satellites resulting from the incorporation of intact C2-units from racemic [l,2- 3C2]glycerol showing two labelled Cs-units, and an additional C2-unit in the side chain of 72. The fact that no satellites were observed for C-3 indicated that this C-atom was not labelled from a C2-unit of racemic [l,2- 3C2]glycerol. The missing satellites for C-3 were a clear confirmation of the first case (A) with C-7, C-1 and C-2 originating from phosphoenolpyruvate (124) and C-3 , C-4 , C-5 and C-6 originating from erythrose 4-phosphate (125). C-1 of erythrose 4-phosphate carries the N-atom or in the case of a cyclic intermediate, C-5 would be aminated. 

Radioactive acetyl CoA can be generated by direct synthesis from C-acetate or from (3 oxidation of radioactive fatty acids, such as uniformly labeled palmitate. Examination of the reactions of the citric acid cycle reveals that neither of the two carbons that enter citrate horn acetate is removed as carbon dioxide during the first pass through the cycle. Labeled carbon from C-methyl-labeled acetate appears in C-2 and C-3 of oxaloacetate, because succinate is symmetrical, with either methylene carbon in that molecule labeling C-2 or C-3 of oxaloacetate. The conversion of oxaloacetate to phosphoenolpyruvate yields PEP labeled at C-2 or C-3 as well. Formation of glyceraldehyde 3-phosphate and its isomer dihydroxyacetone phosphate gives molecules, both labeled at carbons 2 and... 

F i g. 5.11 Chemical reaction mechanism for an abbreviated model of the glycolytic pathway. The mechanism includes many of the known activations and inhibitions of enzymes by metabolites. Broken lines indicate activation (0) or inhibition (0) of enzymes by metabolites. The abbreviations for the enzymes are hexokinase (HK), phosphofrnctokinase (PFK), and pyruvate kinase (PK) the abbreviations for the five independent variables are fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (FDP), phosphoenolpyruvate (PEP), pyruvate (PYR), and ATP (in fig. 5.12, these are labeled 1-5, respectively). (From [1].)... 

A study of the kinetics of the labeling of alanine show that its rate of labeling reaches a maximum as soon as the intermediates of the carbon reduction cycle are saturated with C . Since no secondary products of carbon photosynthesis such as sucrose are approaching saturation at this time (3-5 minutes), it appears that alanine is formed directly from intermediates of the cycle. Presumably, alanine is formed from PGA by the transamination of pyruvic acid derived from phosphoenolpyruvic acid which in turn is derived from PGA [Eq. (28)]. 

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