Aldolase general reaction

The stereochemistry of the aldol reaction is highly predictable since it is generally controlled by the enzyme and does not depend on the structure or stereochemistry of the substrates. Aldolases generally show a very strict specificity for the donor substrate (the ketone), but tolerate a broad range of acceptor substrates (the aldehyde). Thus, they can be functionally classified on the base of the donor substrate accepted by the enzyme. 

Commercial A -acetylneuraminic acid aldolase from Clostridium perfringens (NeuAcA EC 4.1.3.3) catalyzes the addition of pyruvate to A-acetyl-D-mannosamine. A number of sialic acid related carbohydrates are obtained with the natural substrate22"24 or via replacement by aldose derivatives containing modifications at positions C-2, -4, or -6 (Table 4)22,23,25 26. Generally, a high level of asymmetric induction is retained, with the exception of D-arabinose (epimeric at C-3) where stereorandom product formation occurs 25 2t The unfavorable equilibrium constant requires that the reaction must be driven forward by using an excess of one of the components in order to achieve satisfactory conversion (preferably 7-10 equivalents of pyruvate, for economic reasons). 

As we look at some of the reactions of intermediary metabolism, we shall rationahze them in terms of the chemistry that is taking place. In general, we shall not consider here the involvement of the enzyme itself, the binding of substrates to the enzyme, or the role played by the enzyme s amino acid side-chains. In Chapter 13 we looked at specific examples where we know just how an enzyme is able to catalyse a reaction. Examples such as aldolase and those phosphate isomerase, enzymes of the glycolytic pathway, and citrate synthase from the Krebs cycle were considered in some detail. It may... 

An advantage of these enzymes is that they are stereocomplementary, in that they can synthesize the four possible diastereoisomers of vicinal diols from achiral aldehyde acceptors and DHAP (Scheme 4.2). Although this statement is generally used and accepted, it is not completely true since tagatose-l,6-bisphosphate aldolase (TBPA) from Escherichia coli-the only TBPA that has been investigated in terms of its use in synthesis-does not seems to control the stereochemistry of the aldol reaction when aldehydes different from the natural substrate were used as acceptors [7]. However, this situation could be modified soon since it has been demonstrated that the stereochemical course of TBPA-catalyzed C—C bond formation may be modified by enzyme-directed evolution [8]. 

When DHAP-dependent aldolases are used as catalyst of the aldol reaction, a phosphorylated azido or amino polyhydroxyketone is obtained. The phosphate may be cleaved enzymatically or reductively cleaved under the hydrogenation conditions of the next step in which the azide is reduced to the amine. Intramolecular imine formation occurs spontaneously when the azide is reduced. The intramolecular reductive amination is the second key step of the aldolase-mediated synthesis of iminocyclitols. In general, delivery of hydrogen onto five- and six-membered ring imines occurs from the face opposite to the C4 hydroxyl group. 

Enzymatic synthesis relying on the use of aldolases offers several advantages. As opposed to chemical aldolization, aldolases usually catalyze a stereoselective aldol reaction under mild conditions there is no need for protection of functional groups and no cofactors are required. Moreover, whereas high specificity is reported for the donor substrate, broad flexibility toward the acceptor is generally observed. Finally, aldolases herein discussed do not use phosphorylated substrates, contrary to phosphoenolpyruvate-dependent aldolases involved in vivo in the biosynthetic pathway, such as KDO synthetase or DAHP synthetase [18,19]. 

The enzymes which catalyze this reaction, the aldolases, are members of the general group called lyases (see Table I). They have been isolated from many living cells, and vary in specificity. The reader will find, in Methods of... 

In theory, the programmable stereoselectivities of catalytic antibodies makes them well suited for asymmetric synthesis. Several such transformations have been carried out on a preparative scale. Kinetic resolution of the epothilone precursor 19 with the aldolase antibody 38C2 is instructive (Scheme 4.9) [57]. The reaction proceeds in good yield (37 %) and high enantiomeric excess (90 %). However, so much catalyst is needed (0.5 g of IgG antibody was used for the resolution of 0.75 g 19) that large-scale production is likely to be impractical in many cases. As most antibody catalysts are much less efficient than the aldolases, catalyst costs will generally be appreciable. 

Enzymes turned out to be very helpful in the de novo synthesis of certain monosaccharides. Generally, two chiral carbonyl compounds are combined in an aldol-type reaction. In carbohydrate metabolism, aldolases catalyze the condensation of dihydroxyacetone phosphate (DHAP) and aldehydes to higher sugar components. To date, about thirty aldolases have been classified, but only... 

In reactions catalyzed by DHAP-aldolases, hydroxylated aldehydes are generally superior to unsubstituted aldehydes presumably because of their higher reactivity (electrophilicity), higher affinity to the enzyme active site (lower values), and the fact that the products are stabilized by the formation of cyclic isomers [42].Accordingly,substrates with dual 2- or 3-hydroxyaldehyde termini seemed to be a logical choice for potential tandem aldolizations. 

Bromopyruvic acid inhibits S-deoxy-D-erj t ro-hexulosonate 6-phosphate aldolase " and 3-deoxy-D-ar-a6mo-heptulosonate 7-phosphate synthetase " in a similar way. The enzymes are protected from inhibition by pyruvate and phospho-enol pyruvate respectively. Since aldolases must possess a nucleophilic group to initiate the reaction, this type of inhibition should be a general property of aldolases. 

FDP aldolase catalyzes the reversible aldol addition reaction of DHAP and d-glyceraldehyde 3-phosphate (D-Gly 3-P) to form d-FDP (Fig. 14.1-1). The equilibrium constant for this reaction has a value of -104 m-1 in favor of FDP formation. The enzyme has been isolated from a variety of eukaryotic and prokaryotic sources, both in type I and type II forms[7 21). Generally, the type I FDP aldolases exist as tetramers (M.W. 160 KDa), while the type II enzymes are dimers (M. W. 80 KDa). For the... 

An exhaustive review of all of the types of reactions that are catalyzed by metal-requiring enzymes and the specific functions of these metals, as currently understood, is beyond the scope of this chapter. To complicate this general area of investigation, even within a single group of enzymes, the metal ions may play different roles in the catalytic processes for reaction-related enzymes. Not all enzymes of a specific class necessarily require a cation for activity. In some cases, the roles of the cation may be substituted by specific amino acid residues in the protein. A classic example of such a case is the muscle and yeast fmctose-bisphosphate aldolases. The muscle enzyme catalyzes the aldol condensation using a Schiff base intermediate to activate the substrate, whereas the yeast enzyme is a Zn +-metalloenzyme (1). The cation appears to serve as the electrophile in the activation of the substrate for the same reaction. 

Enzymes catalyze the formation of carbon-carbon bonds between allylic and homoallylic pyrophosphate species by mechanisms that are very different from those for carbonyl compounds. Here, carbonium ions, stabilized as ion pairs and generated from allylic pyrophosphates, are likely to be the intermediates that add to the TT-electron density of carbon-carbon double bonds to form new carbon-carbon single bonds. Reaction patterns are consistent with model systems and the mechanisms are based on analogies with the models, stereochemical information (which is subject to interpretation), and the structural requirements for inhibitors. Detailed kinetic studies, including isotope effects, which provide probes in the aldolase and Claisen enzymes discussed in Section II, have not yet been performed in these systems. The possibility for surprising discoveries remains and further work is needed to confirm the proposed mechanisms and to generalize them. 

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