DHAP

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 lysiae S-amino group of the enzyme and a carbonyl group of the substrate. Class II aldolases are found primarily ia microorganisms and utilize a divalent ziac to activate the electrophilic component of the reaction. The most studied aldolases are fmctose-1,6-diphosphate (FDP) enzymes from rabbit muscle, rabbit muscle adolase (RAMA), and a Zn " -containing aldolase from E. coli. In vivo these enzymes catalyze the reversible reaction of D-glyceraldehyde-3-phosphate [591-57-1] (G-3-P) and dihydroxyacetone phosphate [57-04-5] (DHAP). 

Fig. 6. FDP-aldolase-catalyzed addition of electrophiles (94) with DHAP (139—146). Representative R groups ia (94) are given as (a—j) (a) methyl, CH (b)...

The chemical reaction catalyzed by triosephosphate isomerase (TIM) was the first application of the QM-MM method in CHARMM to the smdy of enzyme catalysis [26]. The study calculated an energy pathway for the reaction in the enzyme and decomposed the energetics into specific contributions from each of the residues of the enzyme. TIM catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) as part of the glycolytic pathway. Extensive experimental studies have been performed on TIM, and it has been proposed that Glu-165 acts as a base for deprotonation of DHAP and that His-95 acts as an acid to protonate the carbonyl oxygen of DHAP, forming an enediolate (see Fig. 3) [58]. 

Figure 3 A possible mechanism for the isomerization of dihydroxyacetone phosphate (DHAP) to D glyceraldehyde 3 phosphate (GAP) by the enzyme triosephosphate isomerase (TIM). The general acid (Glu 165) and general base (His 95) are shown.

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 fatty acids released on triacylglycerol hydrolysis are transported to mitochondria and degraded to acetyl CoA, while the glycerol is carried to the liver for further metabolism. In the liver, glycerol is first phosphorylated by reaction with ATP. Oxidation by NAD+ then yields dihydroxyacetone phosphate (DHAP), which enters the carbohydrate metabolic pathway. We ll discuss this carbohydrate pathway in more detail in Section 29.5. 

Fructose 1,6-bisphosphate undergoes ring opening and is cleaved by a retro-aldol reaction into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). 

Step 4 of Figure 29.7 Cleavage Fructose 1,6-bisphosphate is cleaved in step 4 into two 3-carbon pieces, dihydroxyacetone phosphate (DHAP) and glyceraldehvde 3-phosphate (GAP). The bond between C3 and C4 of fructose 1,6-bisphosphate... 

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. 

Four DHAP converting aldolases are known, these can synthesize different diastereomers with complementary configurations D-fructose (FruA EC 4.1.2.13) and D-tagatose 1,6-bisphos-phate (TagA, F.C 4.1.2.-), L-fuculose (FucA EC 4.1.2.17) and L-rhamnulose 1-phosphate aldolase (RhuA EC 4.1.2.19)3. The synthetic application of the first (class 1 or 2) and the latter two types (class 2) has been examined. 

A mixture of dihydroxyacetone and inorganic arsenate can replace DHAP due to the transient formation of a monoarsenate ester which is recognized by the aldolase as a DHAP mimic21. This approach suffers from the high toxicity of arsenate, especially at the relatively high levels (>0.5 M) needed for efficient conversion, and from problems in product isolation. 

Similar to DHAP aldolases, the 3-hexulose 6-phosphate aldolase found in Methylomonas Ml 5 is highly specific for the aldol donor component D-ribulose 5-phosphate, but accepts a wide variety of aldehydes as replacement for formaldehyde as the acceptor. With propanal,... 

Functionally related to FruA is the novel class I fructose 6-phosphate aldolase (FSA) from E. coli, which catalyzes the reversible cleavage of fructose 6-phosphate (30) to give dihydroxyacetone (31) and d-(18) [90]. It is the only known enzyme that does not require the expensive phosphorylated nucleophile DHAP for synthetic purpose. 

Figure 10.17 Kinetic enantiopreference of class II DHAP aldolases useful for racemic resolution of a-hydroxyaldehydes.

Apparently, all DHAP aldolases are highly specific for (25) as the donor component for mechanistic reason [30-33], a fact which requires an economical access to this compound for synthetic applications. Owing to the limited stability of (25) in solution, particularly at alkaline pH, it is preferentially generated in situ to avoid high stationary concentrations. 

Figure 10.19 Oxidative enzymatic generation of dihydroxyacetone phosphate in situ for stereoselective aldol reactions using DHAP aldolases (a), and extension by pH-controlled, integrated precursor preparation and product liberation (b).

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