Aldolase-isomerase-catalyzed reactions

Phosphotriose Isomerase. Early work on aldolase was made difficult by the presence of phosphotriose isomerase. This enzyme is usually present in much larger effective concentrations than aldolase, and catalyzes the interconversion of phosphoglyceraldehyde and dihydroxyacetone phosphate. Since the equilibrium of this reaction lies to the... 

Further steps m glycolysis use the d glyceraldehyde 3 phosphate formed m the aldolase catalyzed cleavage reaction as a substrate Its coproduct dihydroxyacetone phosphate is not wasted however The enzyme triose phosphate isomerase converts dihydroxyacetone phosphate to d glyceraldehyde 3 phosphate which enters the glycol ysis pathway for further transformations... 

This reaction is followed by another phosphorylation with ATP catalyzed by the enzyme phosphofructoki-nase (phosphofructokinase-1), forming fructose 1,6-bisphosphate. The phosphofructokinase reaction may be considered to be functionally irreversible under physiologic conditions it is both inducible and subject to allosteric regulation and has a major role in regulating the rate of glycolysis. Fructose 1,6-bisphosphate is cleaved by aldolase (fructose 1,6-bisphosphate aldolase) into two triose phosphates, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Glyceraldehyde 3-phosphate and dihydroxyacetone phosphate are inter-converted by the enzyme phosphotriose isomerase. 

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). 

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.

Reactions catalyzed by aldolase (ALD) and triosophosphate isomerase (TPI) are considered rapid, and in equilibrium. Therefore, fructose-1,6-diphosphate (FDP), glyceraldehyde phosphate (GAP) and dihydroxyacetone phosphate (DHAP) change in phase, thus may be described by... 

Fuc 1-P and Rha 1-P aldolases have also been utilized in whole cell systems with DHA and catalytic inorganic aresenate1971. With L-lactaldehyde as the substrate in the Rha 1-P aldolase reaction, the aldol product L-rhamnulose was subsequently iso-merized to L-rhamnose, catalyzed by rhamnose isomerase. No such isomerization was observed with L-xylulose, the corresponding aldol product using glycolaldehyde as the substrate. Recent studies have since shown that both rhamnose and fucose isomerase require fixed stereochemistry only up to C3 for aldohexose substrates ... 

Nuclear relaxation studies of substrates and inhibitors have resulted in the detection of 10 enzyme-Mur-substrate and 4 enzyme-Mn-inhibitor bridge complexes possessing kinetic and thermodynamic properties consistent with their participation in enzyme catalysis. Three cases of a activation, by divalent cations, of enzyme-catalyzed enolization reactions (pyruvate carboxylase, yeast aldolase, v-xylose isomerase), and one case of 8 activation of an enzyme-catalyzed elimination reaction (histidine deaminase) have thereby been established, Thus, in each proven case, the enzyme-bound Mn coordinates an electronegative atom (Z) of the substrate, which is attached to a carbon atom one or two bonds away from the carbon atom which is to be deprotonated ... 

An economically viable alternative to the synthesis of deoxyribonuclosides has been developed as a two stage process involving 2-deoxy-D-ribose 5-phosphate aldolase (DERA) (Fig. 6.5.14) (Tischer et al. 2001). The first step was the aldol addition of G3P to acetaldehyde catalyzed by DERA. G3P was generated in situ by a reverse action of EruA on L-fructose-1,6-diphosphate and triose phosphate isomerase which transformed the DHAP released into G3P. In a second stage, the action of pentose-phosphate mutase (PPM) and purine nucleoside phosphorylase (PNP), in the presence of adenine furnished the desired product. The released phosphate was consumed by sucrose phosphorylase (SP) that converts sucrose to fructose-1-phosphate, shifting the unfavorable equilibrium position of the later reaction. 

Synthesis of DHAP. Both RAMA and Fuc-l-P aldolase require DHAP as the nucleophilic component. Although DHAP is commercially available, it is too expensive for synthetic use, and must be synthesized for preparative-scale enzymatic reactions. DHAP has been prepared via three major routes enzymatically from FDP using a combination of RAMA and triose isomerase (TIM, E.C. 5.3.1.1) (25), chemically, by phosphorylation of dihydroxyacetone dimer (26), and enzymatically by phosphorylation of dihydroxy acetone catalyzed by glycerokinase (Scheme 8) (25). 

Some of the ketoses prepared in the FDP aldolase reactions can be converted to the corresponding aldolases catalyzed by glucose isomerase ( ) (from Miles), the enzyme used for the manufacture of high fructose com syrup. Figure 5 summarizes some of the isomerization reactions. The equilibrium mixture can be separated by Dowex-50-Ba column chromatography using water as the mobile phase. This isomerization step provides a new entry to several interesting aldolases. 

Figure 5. Isomerization of the products from FDP aldolase reactions to aldolases catalyzed by glucose isomerase. The numbers in parenthesis indicate the equilibrium ratio.

An enzyme catalyzes the interconversion of dihydroxyacetone phosphate into D-phosphoglyceraldehyde in presence of NAD. Thus, triose phosphate isomerase breaks a carbon-hydrogen bond in the hydroxymethyl group of the D-phosphoglyceraldehyde to yield dihydroxyacetone phosphate. The equilibrium of that reaction favors the formation of the dihydroxyacetone phosphate. From the description of the glycolytic pathway, it is evident that dihydroxyacetone phosphate is produced in two different enzymic reactions, catalyzed by aldolase or triose phosphate isomerase. The exact mechanism of the reaction is not known, but it has ben suggested that it involves the formation of an enolate anion that is bound to the enzyme. 

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