Glyceraldehyde fructose phosphate formation from

The transaldolase functions primarily to make a useful glycolytic substrate from the sedoheptulose-7-phosphate produced by the first transketolase reaction. This reaction (Figure 23.35) is quite similar to the aldolase reaction of glycolysis, involving formation of a Schiff base intermediate between the sedohep-tulose-7-phosphate and an active-site lysine residue (Figure 23.36). Elimination of the erythrose-4-phosphate product leaves an enamine of dihydroxyacetone, which remains stable at the active site (without imine hydrolysis) until the other substrate comes into position. Attack of the enamine carbanion at the carbonyl carbon of glyceraldehyde-3-phosphate is followed by hydrolysis of the Schiff base (imine) to yield the product fructose-6-phosphate. 

There are two distinct groups of aldolases. Type I aldolases, found in higher plants and animals, require no metal cofactor and catalyze aldol addition via Schiff base formation between the lysine s-amino group of the enzyme and a carbonyl group of the substrate. Class II aldolases are found primarily in microorganisms and utilize a divalent zinc to activate the electrophiUc component of the reaction. The most studied aldolases are fructose-1,6-diphosphate (FDP) enzymes from rabbit muscle, rabbit muscle adolase (RAMA), and a Zn2+-containing aldolase from E. coll In vivo these enzymes catalyze the reversible reaction of D-glyceraldehyde-3-phosphate [591-57-1] (G-3-P) and dihydroxyacetonephosphate [57-04-5] (DHAP). 

The pentose phosphate pathway also catalyzes the interconversion of three-, four-, five-, six-, and seven-carbon sugars in a series of non-oxidative reactions. All these reactions occur in the cytosol, and in plants part of the pentose phosphate pathway also participates in the formation of hexoses from CO2 in photosynthesis. Thus, D-ribulose 5-phosphate can be directly converted into D-ribose 5-phosphate by phosphopentose isomerase, or to D-xylulose 5-phosphate by phosphopentose epimerase. D-Xylulose 5-phosphate can then be combined with D-ribose 5-phosphate to give rise to sedoheptulose 7-phosphate and glyceraldehyde-3-phosphate. This reaction is a transfer of a two-carbon unit catalyzed by transketolase. Both products of this reaction can be further converted into erythrose 4-phosphate and fructose 6-phosphate. The four-carbon sugar phosphate erythrose 4-phosphate can then enter into another transketolase-catalyzed reaction with the D-xylulose 5-phosphate to form glyceraldehyde 3-phosphate and fructose 6-phosphate, both of which can finally enter glycolysis. 

D-Fructose- 1,6-diphosphate (FDP) aldolase (E.C. 4.1.2.13) from rabbit muscle, catalyzes the equilibrium condensation of dihydroxyacetone phosphate (1 DHAP) with D-glyceraldehyde 3-phosphate (2 G-3-P) to form d-fructose 1,6-diphosphate (3 FDP Scheme l).42-44 The equilibrium constant for this reaction is K = 104 M-1 in favor of the formation of FDP. The stereoselectivity of the reaction is absolute the configuration of the vicinal diols at C-3 and C-4 is always threo (i.e. d-glycero). Although there is a significant discrimination (20 1) between the antipodes of the natural substrate (i.e. d- and l-G-3-P), this selectivity extends to only a few unnatural substrates.33... 

The third stage is a complex series of reactions involving C—C bond breakage and formation. The result of these reactions is the formation of two molecules of fructose-6-phosphate and one molecule of glyceraldehyde-3-phosphate from three molecules of pentose phosphate. 

Propose a mechanism for the formation of D-fructose-1,6-diphosphate from dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate, using HO as the catalyst. 

Fig. 3.8. Formation of glyceraldehyde-3-phosphate and dihydroxyacetone-1-phosphate from fructose-1,6-diphosphate during fermentation...

Aldolases have been studied as catalysts for monosaccharide synthesis for nearly 40 years. The best studied member of the group is a fructose-1,6-diphosphate (FDP) aldolase from rabbit muscle (RAMA, E.C. 4.1.2.13) (20), In vivo, this enzyme catalyzes the reversible condensation of D-glyceraldehyde-3-phosphate and dihydroxy acetone phosphate (DHAP) to generate FDP (Scheme 1). In the synthetic direction, the enzyme catalyzes the formation of two new stereogenic centers with absolute stereospecificity the stereochemistry of the new vicinal diol is always D-... 

We had already shown that sedoheptulose 7-phosphate was an intermediate in the conversion of pentose phosphate to hexosemonophosphate in crude liver extracts, since it accumulated early in the reaction and then disappeared, coincident with the formation of hexose phosphate. The enzyme that catalyzed the interconversion of sedoheptulose 7-phosphate and fructose 6-phosphate was discovered in my laboratory in 1953, and with Paul Marks and Howard Hiatt, we showed that it catalyzed a Cj-transfer from the substrate to a suitable aldehyde donor. This led to the discovery of another new phosphate ester, erythrose 4-phospWe, formed in the Cs-transfer from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate. Finally, erythrose 4-phosphate could be shown to be the acceptor for a second reaction catalyzed by transketolase, with xylulose 5-phosphate as the donor. All of the steps in the pentose phosphate pathway had now been elucidated (Fig. 2). Erythrose 4-phosphate was also shown to condense with glyceraldehyde 3-phosphate to... 

The fructose 6-phosphate aldolase (ESA) from E. coli is a novel class I aldolase that catalyzes the reversible formation of fructose 6-phosphate from dihydroxyacetone and o-glyceraldehyde 3-phosphate it is, therefore, functionally related to transaldolases [245]. Recent determination of the crystal structure of the enzyme sho ved that it also shares the mechanistic machinery [246]. The enzyme has been sho vn to accept several aldehydes as acceptor components for preparative synthesis. In addition to dihydroxyacetone it also utilizes hydroxyacetone as an alternative donor to generate 1-deoxysugars, for example 118, regioselectively (Eigure 5.52) [247]. 

An indirect enzymatic pathway for the formation of uctose-6-phosphate from fructose-l-phosphate has recently been described by Leuthardt et al. Fructose-l-phosphate is cleaved to D-glyceraldehyde and dihydroxyacetone phosphate. The latter isomerizes to form glyceraldehyde-3-phosphate. The triose phosphates condense to fructose-1,6-diphosphate which is dephosphorylated at the 1 position. In the presence of ATP, the glyceraldehyde may be converted by means of a triosekinase to glyceraIdehyde-3-phosphate. 

The aldehyde substrates may be used as racemic mixtures in many cases, as the aldolase catalyzed reactions can concomitantly accomplish kinetic resolution. For example, when DHAP was combined with d- and L-glyceraldehyde in the presence of FDP aldolase, the reaction proceeded 20 times faster with the D-enantiomer. Fuc 1-P aldolase and Rha 1-P aldolase show kinetic preferences (greater than 19/1) for the L-enantiomer of 2-hydroxy-aldehydes. Alternatively, these reactions may be allowed to equilibrate to the more thermodynamically favored products. This thermodynamic approach is particularly useful when the aldol products can cyclize to the pyranose form. Since the reaction is reversible under thermodynamic conditions, the product with the fewest 1,3-diaxial interactions will predominate. This was demonstrated in the formation of 5-deoxy-5-methyl-fructose-l-phosphate as a minor product (Scheme 5.5).20a 25 The major product, which is thermodynamically more stable, arises from the kinetically less reaction acceptor. 

Although TA from yeast is commercially available, it has rarely been used in organic synthesis applications, and no detailed study of substrate specificity has yet been performed. This is presumably due to high enzyme cost and also since the reaction equilibrium is near unity, resulting in the formation of a 50 50 mixture of products. In addition the stereochemistry accessible by TA catalysis matches that of FruA DHAP-dependent aldolase and the latter is a more convenient system to work with. In one application, TA was used in the synthesis D-fructose from starch.113 The aldol moiety was transferred from Fru 6-P to D-glyceraldehyde in the final step of this multi-enzyme synthesis of D-fructose (Scheme 5.60). This process was developed because the authors could not identify a phosphatase that was specific for fructose 6-phosphate and TA offered an elegant method to bypass the need for phosphatase treatment. 

L-Fmctose l-Phosphate from L-Clyceraldehyde by in-situ Formation of DHAP. GPO (70 U), catalase (1000 U), and RhuA (50 U) was added to a solution of L-glycerol 3-phosphate (74, 1.0 mmol) and L-glyceraldehyde (110 mg, 1.2 mmol) in 10 mL oxygen-saturated water at pH 6.8. The mixture was shaken at 20 °C under an oxygen atmosphere at 100 rpm. Conversion was monitored by enzymatic assay for equivalents of 41 produced, and by H and NMR spectroscopy. After complete conversion and filtration through charcoal the pH was adjusted to 7.5 by addition of 1.0 m cyclo-hexylamine in ethanol and the solution was concentrated to dryness by rotary evaporation at <20 °C in vacuo. The solid residue was dissolved in 0.5 mL water and the resulting solution was filtered. Dry ethanol (2.5 mL) was added, then dry acetone until faint turbidity remained. Crystallization at 4 °C furnished L-fructose 1-phosphate bis(cyclohexylammonium) salt as colorless needles yield 370 mg (85%). 

Many other photosynthetic carbohydrates were identified and, by means of labeling and chemical degradation studies of the labeled compounds, their labeling patterns were determined, and the various reactions involved in the formation of several different carbohydrates were obtained. For example, the formation of d-fructose-l,6-bisphosphate showed that the label from CO2 was initially located only at C-3 and C-4, indicating that it was formed by the condensation of 3-phos-pho-D-glyceraldehyde and dihydroxyacetone phosphate in a reverse aldolase-catalyzed reaction (reaction 7 in Fig. 10.3). 

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