Aldolase reaction

FIGURE 16.8 (a) Phosphoglycolohydroxamate is an analog of the enediolate transition state of the yeast aldolase reaction, (b) Purine riboside, a potent inhibitor of the calf intestinal adenosine deaminase reaction, binds to adenosine deaminase as the 1,6-hydrate. The hydrated form of purine riboside is an analog of the proposed transition state for the reaction. 

FIGURE 19.13 (a) A mechanism for the fructose-l,6-bisphosphate aldolase reaction. The Schlff base formed between the substrate carbonyl and an active-site lysine acts as an electron sink, Increasing the acidity of the /3-hydroxyl group and facilitating cleavage as shown. (B) In class II aldolases, an active-site Zn stabilizes the enolate Intermediate, leading to polarization of the substrate carbonyl group. 

Subsequent action by fructose-l-phosphate aldolase cleaves fructose-l-P in a manner like the fructose bisphosphate aldolase reaction to produce dihy-droxyacetone phosphate and D-glyceraldehyde ... 

A good example of such a cleavage is the fructose bisphosphate aldolase reaction (see Chapter 19, Figure 19.14a). 

When carbon rearrangements are balanced to account for net hexose synthesis, five of the glyceraldehyde-3-phosphate molecules are converted to dihy-droxyacetone phosphate (DHAP). Three of these DHAPs then condense with three glyceraldehyde-3-P via the aldolase reaction to yield 3 hexoses in the form... 

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. 

Figure 23.10 Mechanisms of type I and type II aldolase reactions in glucose biosynthesis.

Application of an aldolase to the synthesis of the tricyclic microbial elicitor (-)-syringolide (Figure 10.34) is another excellent example that enzyme-catalyzed aldolizations can be used to generate sufficient quantities of enantiopure material in multistep syntheses of complex natural and unnatural products [159]. Remarkably, the aldolase reaction established absolute and relative configuration of the only chiral centers that needed to be externally induced in the adduct (95) from achiral precursor (94) during the subsequent cyclization events, all others seemed to follow by kinetic preference. 

A classical approach to driving the unfavorable equilibrium of an enzymatic process is to couple it to another, irreversible enzymatic process. Griengl and coworkers have applied this concept to asymmetric synthesis of 1,2-amino alcohols with a threonine aldolase [24] (Figure 6.7). While the equilibrium in threonine aldolase reactions typically does not favor the synthetic direction, and the bond formation leads to nearly equal amounts of two diastereomers, coupling the aldolase reaction with a selective tyrosine decarboxylase leads to irreversible formation of aryl amino alcohols in reasonable enantiomeric excess via a dynamic kinetic asymmetric transformation. A one-pot, two-enzyme asymmetric synthesis of amino alcohols, including noradrenaline and octopamine, from readily available starting materials was developed [25]. 

A syringolide 45, an elicitor of the bacterial plant pathogen Pseudomonas Siringae pv. tomato, has been synthesized in five steps via a fructose 1,6-diphosphate aldolase reaction (Scheme 95) <2000JOC4529>. 

The guilty party is the triose phosphate isomerase (TIM) reaction that interconverts DHAP and G3P. To be converted to pyruvate, the DHAP first has to be converted to G3P. TIM just moves the carbonyl group between the two carbons that don t have phosphate attached. TIM doesn t touch the phosphate. So, if the DHAP is labeled at the carbon that has the phosphate attached, the G3P that comes from DHAP will be labeled at the carbon with the phosphate attached. The carbon with the phosphate attached in the G3P that was produced directly by the aldolase reaction came from C-6 of glucose, but the carbon with the phosphate attached in the G3P that was produced from DHAP came from C-l of glucose. After TIM does it stuff, the carbon of G3P that has the phosphate will be... 

Figure 2. Mechanism of dihydroxyacetone/arsenate reaction with FDP aldolase. Both dihydroxyacetone and inorganic arsenate are not the inhibitor of the aldolase reactions. The rate constant for the arsenate ester formation is determined enzymatically (a plot of 1/v vs 1/E gives a non-zero intercept which is attributed to the rate at infinite enzyme concentration and that rate corresponds to the rate of nonenzymatic formation of the arsenate ester).

Figure 3. Selectivity of the FDP-aldolase reactions using DHAP vs. dihydroxyacetone/arsenate as a substrate. In the former case, the more stable sugar is obtained due to the reversible nature of the reaction. In the later case, both sugars were obtained in nearly equal amounts, because the reaction was found to be virtually irreversible and the formation of the arsenate ester was rate limiting.

Barbas et al offered the following mechanisms shown in Fig. 2 for the Class 1 aldolase reaction and Ab38C2 or Ab 33F12 antibody aldolase reactions, where R is 4-isobutyramidobenzyl or -butyl. Both mechanisms involve a chemically reactive lysyl e-amino group. 

Such intermediates are known to form between substrate and enzyme in the aldolase reaction and between pyri-doxal 5-phosphate and the amino group of enzyme or substrate in aminotransferase reactions. In the latter case, aldimine formation accounts for the high affinity of coenzyme binding to apotransaminases. 

This rate law, for example, applies to the tritium exchange from labeled dihydroxyacetone phosphate to water in the aldolase reaction or the exchange from labeled malate to water in the fumarase reaction. 

MECHANISM FIGURE 14-5 The class I aldolase reaction. The reaction shown here is the reverse of an aldol condensation. Note that cleavage between C-3 and C-4 depends on the presence of the carbonyl group at C-2. and (2)The carbonyl reacts with an active-site Lys residue to form an imine, which stabilizes the carbanion generated by the bond cleavage—an imine delocalizes electrons even better than... 

Although the aldolase reaction has a strongly positive standard free-energy change in the direction of fructose 1,6-bisphosphate cleavage, at the lower concentrations of reactants present in cells, the actual free-energy change is small and the aldolase reaction is readily reversible. We shall see later that aldolase acts in the re-... 

C. Gautheron-Le Narvor, Y. Ichikawa, and C.-H. Wong, A complete change of stereoselectivity in sialic acid aldolase reactions A novel synthetic route to the KDO type of nine-carbon L sugars, J. Am. Chem. Soc. 113 1816 (1991). 

Fructose bisphosphate is cleaved by action of an aldolase (reaction 4) to give glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. These two triose phosphates are then equilibrated by triose phosphate isomerase (reaction 5 see also Chapter 13). As a result, both halves of the hexose can be metabolized further via glyceraldehyde 3-P to pyruvate. The oxidation of glyceraldehyde 3-P to the corresponding carboxylic acid, 3-phosphoglyceric acid (Fig. 17-7, reactions 6 and 7), is coupled to synthesis of a molecule of ATP from ADP and P . This means that two molecules of ATP are formed per hexose cleaved, and that two molecules of NAD+ are converted to NADH in the process. 

Cleavage of fructose-l,6-bisphosphate, an aldolase-catalyzed reaction. The aldolase reaction entails a reversal of the familiar aldol condensation. The first step involves abstraction of the hydrogen of the C-4 hydroxyl group, followed by elimination of an enolate anion. 

The transaldolase-catalyzed conversion of fructose-6-phosphate and erythrose-4-phosphate to glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate. This is a two-step conversion. The first step is similar to the aldolase reaction except that the dihydroxyacetone produced is held at the catalytic site while the aldose product diffuses away and is replaced by another aldose molecule. The second step involves an aldol condensation. 

The transketolase-catalyzed conversion of xylulose-5-phosphate and ribose-5-phosphate to glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate. Although the aldolase and ketolase reactions superficially resemble each other, they proceed by very different mechanisms. This is because in the aldolase reaction the carbon adjacent to a carbonyl... 

Although many aldolases have been characterized for research purposes, the four aldolase enzymes described in Scheme 19.32 have not been used commercially to any significant extent. This is likely a result of their availability and the need for dihydroxyacetone phosphate (DHAP) (54), the expensive donor substrate required in these aldolase reactions (Scheme 19.32). A number of chemical and enzymatic routes have been described for DHAP synthesis, which could alleviate these concerns.9,258... 

The use of reactive immunization to generate catalytic antibodies (or abzymes) that catalyze aldolase reactions has been described, offering additional utility for this synthetically useful transformation.260 Two such abzymes, 38C2 and 84G3, are available commercially and their respective, diverse activities have been described.261-262... 

Energetics of the Aldolase Reaction Aldolase catalyzes the glycolytic reaction... 

Answer Problem 1 outlines the steps in glycolysis involving fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, and dihydroxyacetone phosphate. Keep in mind that the aldolase reaction is readily reversible and the triose phosphate isomerase reaction catalyzes extremely rapid interconversion of its substrates. Thus, the label at C-l of glyceraldehyde 3-phosphate would equilibrate with C-l of dihydroxyacetone phosphate (AG ° = 7.5 kJ/mol). Because the aldolase reaction has AG ° = -23.8 kJ/mol in the direction of hexose formation, fructose 1,6-bisphosphate would be readily formed, and labeled in C-3 and C-4 (see Fig. 14-6). 

DHAP, a glycolysis intermediate, is reduced by NADH to glycerol-3-phosphate. Triose isomerase converts it to glyceraldehyde-3-phosphate, and it is a product of the fructose-1-phosphate aldolase reaction. 

Figure 7.6. The metabolic reactions involved in the conversion of glycerol to glucose, the required precursor in the formation of sophorose. Note Reaction 1 catalyzed by triose phosphate isomerase. Reaction 2 catalyzed by aldolase. Reaction 3 catalyzed by fructose 1,6-bisphosphatase. Reaction 4 catalyzed by phosphoglucose isomerase., Reaction 6 catalyzed by glucose 6-phosphatase.

Scheme 5.2. The four main groups of aldolase reactions classified by their donor substrate (1) Dihydroxyacetone phosphate (DHAP)- dependent aldolases, (2) phosphoenol pyruvate (PEP)-and pyruvate-dependent aldolases, (3) 2-deoxyribose-5-phosphate aldolase (DERA), a member of the acetaldehyde-dependent aldolases, and (4) glycine-dependent aldolases (GDA).

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