Aldolase and

Fructose bisphosphate aldolase of animal muscle is a Class I aldolase, which forms a Schiff base or imme intermediate between the substrate (fructose-1,6-bisP or dihydroxyacetone-P) and a lysine amino group at the enzyme active site. The chemical evidence for this intermediate comes from studies with the aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of fructose bisphosphate aldolase with dihydroxyacetone-P and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. 

Figure 10.10 Natural substrates of the 2-keto-3-deoxy-monno-octosonic acid aldolase, and nonnatural sialic acids obtained by KdoA catalysis.

Furthermore, the GPO procedure can also be used for a preparative synthesis of the corresponding phosphorothioate (37), phosphoramidate (38), and methylene phosphonate (39) analogs of (25) (Figure 10.20) from suitable diol precursors [106] to be used as aldolase substrates [102]. In fact, such isosteric replacements of the phosphate ester oxygen were found to be tolerable by a number of class I and class II aldolases, and only some specific enzymes failed to accept the less polar phosphonate (39) [107]. Thus, sugar phosphonates (e.g. (71)/(72)) that mimic metabolic intermediates but are hydrolytically stable to phosphatase degradation can be rapidly synthesized (Figure 10.28). 

Hake, S., Kelley, P.M., Taylor, W.C. Freeling, M. (1985). Coordinate induction of alcohol dehydrogenase 1, aldolase, and other anaerobic RNAs in maize. Journal of Biological Chemistry, 260, 5050-4. 

Dihydroxyacetone phosphate (82) is a substrate for a-glycero-phosphate dehydrogenase, aldolase, and triose phosphate isomerase, and its O-alkyl ethers are intermediates in the biosynthesis of phospholipids. In neutral aqueous solution at 20 °C, dihydroxyacetone phosphate exists as an equilibrium mixture of the keto (82), gem-d o (83), and enol (84) forms, as shown by n.m.r. spectroscopy. The proportion of (82) to (83)... 

Fessner, W.D. andHelaine, V. (2001) Biocatalytic synthesis of hydroxy lated natural products using aldolases and related enzymes. Current Opinion in Biotechnology, 12, 574-586. 

Schurmann, M., Schurmann, M. and Sprenger, G.A. (2002) Fructose 6-phosphate aldolase and 1-deoxy-D-xy lulose 5-phosphate synthase from Escherichia coli as tools in enzymatic synthesis of 1-deoxysugars. Journal of Molecular Catalysis B, Enzymatic, 19, 247-252. 

In contrast to the above two examples, for which applications were developed long before the responsible biocatalyst was discovered, aldolase applications are more recently developed. Indeed, aldolases and their natural function were extensively studied between the end of the 1960s and the beginning of the 1970s. The first patents about their applications in organic synthesis appear in the 1990s [67-69] and the first ton-scale applications were reported in 1997... 

One of the most important metals with regard to its role in enzyme chemistry is zinc. There are several significant enzymes that contain the metal, among which are carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, aldolase, and carbonic anhydrase. Although most of these enzymes are involved in catalyzing biochemical reactions, carbonic anhydrase is involved in a process that is inorganic in nature. That reaction can be shown as... 

In further studies, Amstein and Bentley5 demonstrated the presence of aldolase and triosephosphate isomerase in fungi producing kojic acid. They also found that both production and destruction of kojic acid were rapid in media with high phosphate levels, and slow at lower phosphate levels. They preferred to consider kojic acid as a normal metabolite of the fungi, rather than as an end product. 

Jack-bean aldolase and liver aldolase catalyze the conversion of one mole of D-fructose 1-phosphate into one mole each of dihydroxyacetone phosphate and D-glycerose, and the reaction is reversible.73-77... 

The aldol reaction is of fundamental importance in organic chemistry and has been used as a key reaction in the synthesis of many complex natural products. There are biocatalysts for this reaction (aldolases) and one (rabbit muscle... 

Genetically-determined deficiency of G6PD is the most common cause of haemolysis arising from enzyme defects. Mutated glycolytic enzymes such as hexokinase, phosphofructokinase, aldolase and pyruvate kinase can also bring about haemolysis but the occurrence of these defects are much rarer than for G6PD deficiency (see Case N otes at the end of this chapter). 

Small molecules that act as collisional quenchers may penetrate into the internal structure of proteins, diffuse, and cause quenching upon collision with the aromatic groups. Lakowicz and Weber(53) have shown that the interaction of oxygen molecules with buried tryptophan residues in proteins leads to quenching with unexpectedly high rate constants—from 2 x 109 to 7 x 109 M l s 1. Acrylamide is also capable of quenching the fluorescence of buried tryptophan residues, as was shown for aldolase and ribonuclease 7V(54) A more hydrophobic quencher, trichloroethanol, is a considerably more efficient quencher of internal chromophore groups in proteins.(55)... 

The question of what mechanisms is involved in the case of other quenchers is still unclear. For the quenching of aldolase and ribonuclease Ti by acrylamide, the activation energy is rather high, 40-45 kJ/mol,(54) but the value in the case of cod parvalbumin(60) is lower (27 kJ/mol), being similar to that for oxygen quenching. According to Bushueva et al., the efficiency of... 

Kai and Imakubo(76) found that the temperature at which emission from the exposed tryptophan is no longer observed appears to be characteristic of the protein, having values of 180 K for trypsin, 200 K for aldolase, and 230 K for alkaline phosphatase. 

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... 

Figure 1. Synthesis of usual and unusual sugars using FDP aldolase and glucose isomerase as catalysts.

This enzyme [EC 4.1.2.16] (also known as phospho-2-dehydro-3-deoxyoctonate aldolase, phospho-2-keto-3-deoxyoctonate aldolase, and 3-deoxy-D-manno-octulo-sonic acid 8-phosphate synthetase) catalyzes the reaction of 2-dehydro-3-deoxy-D-octonate 8-phosphate and orthophosphate to produce phosphoenolpyruvate, D-arabinose 5-phosphate, and water. 

This enzyme [EC 4.1.3.16], also known as 2-keto-4-hy-droxyglutarate aldolase and 2-oxo-4-hydroxyglutarate aldolase, catalyzes the reversible conversion of 4-hy-droxy-2-oxoglutarate to pyruvate and glyoxylate. Interestingly, the enzyme is reported to be able to act on both stereoisomers. 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

Scheme 2.35 Combined use of threonine aldolase and L-tyrosine decarboxylase.

Scheme 4.10 General strategy for the chemoenzymatic synthesis of iminocyclitols based in the use of aldolases and palladium-mediated reductive amination.

Scheme 4.12 Synthesis of polyhydroxylated azepanes based in the combined use of aldolases and isomerases.

Combined Use of Aldolases and Isomerases for the Synthesis of Natural and Unnatural Sugars... 

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