Glycolysis aldolase mechanism

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). 

In addition to serving as structural motifs, enols and enolates are involved in diverse biological processes. Several enol/enolate intermediates have been proposed to be involved in glycolysis (Section IV.A), wherein c/ -enediol 21 is proposed to be an intermediate in the catalytic mechanism of phosphohexose isomerase and an enol-containing enamine intermediate (22) has been proposed in the catalytic pathway of class I aldolase. In the case of glucose-fructose (aldose-ketose) isomerization, removal of the proton on Cl-OH produces the aldose while deprotonation of C2-OH yields the ketose, which is accompanied by protonation at the C2 and Cl positions, respectively. There are several cofactors that are involved in various biological reactions, such as NAD(H)/NADP(H) in redox reaction and coenzyme A in group transfer reactions. Pyridoxal phosphate (PLP, 23) is a widely distributed enzyme cofactor involved in the formation of a-keto acids, L/D-amino... 

We will consider the mechanism of transketolase when we meet it again in the pentose phosphate pathway (Section 20.3.2). Aldolase, which we have already encountered in glycolysis (Section 16.1.3). catalyzes an aldol condensation between dihydroxyacetone phosphate and an aldehyde. This enzyme is highly specific for dihydroxyacetone phosphate, but it accepts a wide variety of aldehydes. 

In the actual cleavage reaction of glycolysis, D-fructose< 1,6-diphosphate is converted into D-glyceraldehyde-3-phosphate and dihydroxyacetone, CH2OHCOCH2OH. What kind of reaction is this, basically Sketch out a possible mechanism, neglecting, of course, the all-important role of the enzyme. (Hints The enzyme required is called aldolase. See Problem 21.14, p. 711.)... 

Transaldolase has many features in common with the enzyme aldolase, which we met in the glycolytic pathway. Both an aldol cleavage and an aldol condensation occur at different stages of the reaction. We already saw the mechanism of aldol cleavage, involving the formation of a Schiff hase, when we discussed the aldolase reaction in glycolysis, and we need not discuss this point further. 

A number of studies on the metabolism of 3FG and 4FG in Locusta miaratoria have been undertaken. Both 3FG and 4FG are toxic to locust with LD50 s of 4.8 mg/g and 0.6 mg/g respectively. In vitro studies showed that 3FG is metabolized in the fat body, via the NADP-linked aldose reductase, to 3-deoxy-3-fluoro-D-glucitol (3FGL). This metabolite was detected in the hemolymph of the insect and shown to be both a competitive inhibitor and a substrate for NAD-linked sorbitol dehydrogenase, thereby generating 3-deoxy-3-fluoro-D-fructose (3FF) (541. Subsequently, it was shown by in vivo radio-respirometric analysis of C02 and appropriate chase experiments, that 3FG metabolism irreversibly inhibits glycolysis and not the hexose monophosphate shunt or tricarboxylic acid cycle (55). In addition, the release of fluoride ion and H20 from D-[3- H]-3FG was also observed. Based on the mechanism of aldolase (55) and triosephosphate isomerase... 

The primary amino acid sequence of rabbit muscle aldolase has been determined [27]. The molecular weight of the protein is 160000, and it has 4 equivalent subunits each bearing an active site. Some aldolases are found as tetramers of a and subunits which differ only by the conversion of Asn 358 to an aspartate residue. Because this enzyme is essential to glycolysis, because it is readily available, and because it is mechanistically interesting it has been the subject of a large number of studies. These include determinations of active-site residues, order of substrate addition, stereochemistry, and chemical mechanism which will be integrated below in an attempt to generate a picture of the active-site structure. 

Lyases are enzymes which catalyse the removal of groups from substrates by mechanisms other than hydrolysis, leaving double bonds. An example is provided by one of the steps in glycolysis whereby frnctose-l,6-diphosphate is split into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by the enzyme aldolase. 

The alkaline process of cleavage of o-fructose to give dihydroxy acetone and o-glyceraldehyde is similar to the important glycolysis reaction catalyzed by the enzyme aldolase (see Chapter 11) which reacts with o-fructose-1,6-bisphosphate at 37°C to give 3-phospho-o-glyceraldehyde and dihydroxy acetone phosphate. The comparison of the alkaline and enzyme cleavage and mechanisms of reaction are shown in Fig. 3.7. 

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