Aldolase enzyme mechanism

Nature eontinues to excel at facile synthesis of asymmetric molecules under mild eonditions, and has constantly served as a wellspring of knowledge and strategies. Aldolase enzymes are merely another example of Nature s prowess, in their ability to perform asymmetric aldol reactions with unactivated and unprotected highly functionalised carbonyl groups under mild eonditions with impressive efficieneies and ehemoselectivities. In light of sueh enzymatie reactions, the mechanism for the Hajos-Parrish-Eder-Sauer-Wieehert reaction was postulated to oeeur via a similar pathway to elass I aldolase-eatalysed reactions. 

An example of a lysine lyase is the aldolase enzyme isolated from rabbit muscle. The intermediary product formed with dihydroxyacetone phosphate (cf. mechanism in Fig. 2.19) is detected as follows ... 

Wagner, J., Lerner, R. A., and Barbas, C. F., Ill, 1995. Efficient adolase catalytic antibodies that use tlie enamine mechanism of natural enzymes. Science 270 1797-1800. See also tlie discussion entitled Aldolase antibody in Science 270 1737. 

These observations are explained by the mechanism shown in the figure. NaBH4 inactivates Class I aldolases by transfer of a hydride ion (H ) to the imine carbon atom of the enzyme-substrate adduct. The resulting secondary amine is stable to hydrolysis, and the active-site lysine is thus permanently modified and inactivated. NaBH4 inactivates Class I aldolases in the presence of either dihydroxyacetone-P or fructose-1,6-bisP, but inhibition doesn t occur in the presence of glyceraldehyde-3-P. 

Due to mechanistic requirements, most of these enzymes are quite specific for the nucleophilic component, which most often is dihydroxyacetone phosphate (DHAP, 3-hydroxy-2-ox-opropyl phosphate) or pyruvate (2-oxopropanoate), while they allow a reasonable variation of the electrophile, which usually is an aldehyde. Activation of the donor substrate by stereospecific deprotonation is either achieved via imine/enamine formation (type 1 aldolases) or via transition metal ion induced enolization (type 2 aldolases mostly Zn2 )2. The approach of the aldol acceptor occurs stereospecifically following an overall retention mechanism, while facial differentiation of the aldehyde is responsible for the relative stereoselectivity. 

Like many other antibodies, the activity of antibody 14D9 is sufficient for preparative application, yet it remains modest when compared to that of enzymes. The protein is relatively difficult to produce, although a recombinant format as a fusion vdth the NusA protein was found to provide the antibody in soluble form with good activity [20]. It should be mentioned that aldolase catalytic antibodies operating by an enamine mechanism, obtained by the principle of reactive immunization mentioned above [15], represent another example of enantioselective antibodies, which have proven to be preparatively useful in organic synthesis [21]. One such aldolase antibody, antibody 38C2, is commercially available and provides a useful alternative to natural aldolases to prepare a variety of enantiomerically pure aldol products, which are otherwise difficult to prepare, allovdng applications in natural product synthesis [22]. 

An interesting case in the perspective of artificial enzymes for enantioselective synthesis is the recently described peptide dendrimer aldolases [36]. These dendrimers utilize the enamine type I aldolase mechanism, which is found in natural aldolases [37] and antibodies [21].These aldolase dendrimers, for example, L2Dl,have multiple N-terminal proline residues as found in catalytic aldolase peptides [38], and display catalytic activity in aqueous medium under conditions where the small molecule catalysts are inactive (Figure 3.8). As most enzyme models, these dendrimers remain very far from natural enzymes in terms ofboth activity and selectivity, and at present should only be considered in the perspective of fundamental studies. 

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).

Fig. 6 Enzymatic splitting of the oxidised polymer backbone proceeds by two different enzymes depending on the target moiety in the oxidised polymer backbone. Above, the [1-OH-ketone can be opened by an aldolase activity (apoenzyme of PVADH). Below, the diketone element is cleaved by a specific [l-diketone hydrolase (BDH). A non-enzymatic mechanism is also possible...

Figure 2.20 The two mechanisms of aldolases. Group 1 enzymes from animals and higher plants use an amino group in the enzyme to form a Schiff s base intermediate to activate the aldol donors. Group II enzymes from lower organisms, use a metal ion, usually Zn " in the active site to form an enolate intermediate. The two mechanisms are examplified by fiuctose-1,6-diphosphate aldolase, a very important aldolase in synthesis and breakdown of sugars.

In concert, structure determinations and enzymological studies for catalytic rates and product distributions with structurally varied aldehydes of native enzymes and numerous active-site mutants have allowed us to derive a conclusive blueprint for the catalytic cycle of FucA (Fig. 2.2.5.2). The proposed mechanism, which has general implications for other metal-dependent aldolases, is able to rationalize all key stereochemical issues successfully ]15]. Independent work by other groups has recently provided further insight into related proteins with Fru A and TagA specificity [16]. 

In the catalysis of the lyase from C. perfringens, the participation of lysine residues forming intennediary Schiff bases between enzyme and substrate molecules, and of histidine residues, has been demonstrated with the aid of photooxidation, reagents for histidine modification, and borohydride reduction in the presence of substrate.408-418 Thus, according to Frazi and coworkers,414 the lyase belongs to the class I lyases (aldolases). The catalytic mechanism proposed is outlined in Scheme 3. Evidence has been educed for the existence of a similar mechanism of cleavage of sialic acid by the lyase enriched from pig kidney.411... 

The enzyme that hydrolyzes phosphonoacetaldehyde (Fig. 7) is bacterial and provides a pathway for breaking the C—P bond. It has a mechanism like that of aldolase (69-71) in that an imine forms between the carbonyl group of substrate and an amino group of the enzyme since this imine is hydronated at neutral pH, the electron attraction is increased, and this facilitates the breakage of the C—P bond. When arsonoacetaldehyde was tried in this reaction (63), it proved not to be a substrate, and it did not inhibit the enzyme appreciably. 

There are two classes of aldolases. Class I aldolases, found in animals and plants, use the mechanism shown in Figure 14-5. Class II enzymes, in fungi and bacteria, do not form the Schiff base intermediate. Instead, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2 the Zn2+ polarizes the carbonyl group... 

Type I aldolases, which include the most studied mammalian enzymes, have a more complex mechanism involving intermediate Schiff base forms (Eq. 13-36, steps a, V, c, d ).m The best known members of this group are the fructose bisphosphate aldolases (often referred to simply as aldolases), which cleave fructose-1,6-P2 during glycolysis (Fig. 10-2, step e). 

Closely related to aldolases is transaldolase, an important enzyme in the pentose phosphate pathways of sugar metabolism and in photosynthesis. The mechanism of the transaldolase reaction (Eq. 17-15) is similar to that used by fructose-1,6-bisphosphate aldolase with a lysine side chain forming a Schiff base and catalytic aspartate and glutamate side chains.198... 

Write a step-by-step sequence showing the chemical mechanisms involved in the action of a type I aldolase that catalyzes cleavage of fructose 1,6-bisphosphate. The enzyme is inactivated by sodium borohydride in the presence of the substrate. Explain this inactivation. 

In 2008 Resmini et al. [76] presented their work on the synthesis of novel molecularly imprinted nanogels with Aldolase type I activity in the cross-aldol reaction between 4-nitrobenzaldehyde and acetone. A polymerisable proline derivative was used as the functional monomer to mimic the enamine-based mechanism of aldolase type I enzymes. A 1,3-diketone template, used to create the cavity, was... 

The mechanism similarities to enzymatic processes In principle, L-proline acts as an enzyme mimic of type I metal-free aldolases. Similar to this enzyme, L-proline catalyzes the direct aldol reaction according to an enamine mechanism. Thus, for the first time a mimic of type I aldolases has been found. The close similarity of... 

In principle, L-proline acts as an enzyme mimic of the metal-free aldolase of type I. Similar to this enzyme L-proline catalyzes the direct aldol reaction according to an enamine mechanism. Thus, for the first time a mimic of the aldolase of type I was found. The close relation of the reaction mechanisms of the aldolase of type 1 [5b] and L-proline [4] is shown in a graphical comparison of both reaction cycles in Scheme 3. In both cases the formation of the enamines Ila and lib, respectively, represents the initial step. These reactions are carried out starting from the corresponding ketone and the amino functionality of the enzyme or L-proline. The conversion of the enamine intermediates Ha and lib, respectively, with an aldehyde, and the subsequent release of the catalytic system (aldolase of type I or L-proline) furnishes the aldol product. 

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