Transketolase mechanism

Figure 20.21. Transketolase Mechanism. The carbanion of thiamine pyrophosphate (TPP) attacks the ketose substrate. Cleavage of a carbon-carbon bond frees the aldose product and leaves a two-carbon fragment joined to TPP. This activated glycoaldehyde intermediate attacks the aldose substrate to form a new carbon-carbon bond. The ketose product is released, freeing the TPP for the next reaction cycle.

The mechanistic chemistry of the acetolactate synthase and phosphoketolase reactions (shown below) is similar to that of the transketolase reaction (Figure 23.34). Write suitable mechanisms for these reactions. 

TPP-dependent enzymes are involved in oxidative decarboxylation of a-keto acids, making them available for energy metabolism. Transketolase is involved in the formation of NADPH and pentose in the pentose phosphate pathway. This reaction is important for several other synthetic pathways. It is furthermore assumed that the above-mentioned enzymes are involved in the function of neurotransmitters and nerve conduction, though the exact mechanisms remain unclear. 

Figure 18 Reaction mechanism of transketolase and the stereochemical course.

An example of an a-ketol formation that does not involve decarboxylation is provided by the reaction catalyzed by transketolase, an enzyme that plays an essential role in the pentose phosphate pathway and in photosynthesis (equation 21) (B-77MI11001). The mechanism of the reaction of equation (21) is similar to that of acetolactate synthesis (equation 20). The addition of (39) to the carbonyl group of (44) is followed by aldol cleavage to give a TPP-stabilized carbanion (analogous to (41)). The condensation of this carbanionic intermediate with the second substrate, followed by the elimination of (39), accounts for the observed products (B-7IMIHOO1). 

A reaction that is related to that of transketolase but is likely to function via acetyl-TDP is phosphoketolase, whose action is required in the energy metabolism of some bacteria (Eq. 14-23). A product of phosphoketolase is acetyl phosphate, whose cleavage can be coupled to synthesis of ATP. Phosphoketolase presumably catalyzes an a cleavage to the thiamin-containing enamine shown in Fig. 14-3. A possible mechanism of formation of acetyl phosphate is elimination of HzO from this enamine, tautomerization to 2-acetylthiamin, and reaction of the latter with inorganic phosphate. 

The reactions enclosed within the shaded box of Fig. 17-14 do not give the whole story about the coupling mechanism. A phospho group was transferred from ATP in step a and to complete the hydrolysis it must be removed in some future step. This is indicated in a general way in Fig. 17-14 by the reaction steps d, e, and/. Step/represents the action of specific phosphatases that remove phospho groups from the seven-carbon sedoheptulose bisphosphate and from fructose bisphosphate. In either case the resulting ketose monophosphate reacts with an aldose (via transketolase, step g) to regenerate ribulose 5-phosphate, the C02 acceptor. The overall reductive pentose phosphate cycle (Fig. 17-14B) is easy to understand as a reversal of the oxidative pentose phosphate pathway in which the oxidative decarboxylation system of Eq. 17-12 is... 

The transketolase-catalyzed conversion of xylulose-5-phosphate and ribose-5-phosphate to glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate. Although the aldolase and ketolase reactions superficially resemble each other, they proceed by very different mechanisms. This is because in the aldolase reaction the carbon adjacent to a carbonyl... 

A number of lyases are known which, unlike the aldolases, require thiamine diphosphate (TDP) as a cofactor in the transfer of acyl anion equivalents [389-391], but proceed via enolate-type intermediates by a mechanism that resembles the classical benzoin addition. The most important representative is the transketolase (EC 2,2.1.1) [392] which stems from the oxidative pentose... 

Figure 9.13 Catalytic mechanism of transketolase (taken from Stryer, 1995).

Related systems were later developed for transaldolases 10-12 (Scheme 1.3) [12]. The fructo/tagato stereoselectivity of various transaldolases was determined by fluorescence for the stereoisomeric substrate pair 11/12. However, the reactivity of the substrates towards transaldolases is much lower than with the natural substrate due to the replacement of the phosphate group at position 6 of the natural fructose-6-phosphate substrate with the neutral, aromatic coumarin ether, which is not well recognized by the enzyme. Sevestre et al. [13] reported substrate 13 as a fluorogenic substrate for transketolases, based on a similar fluorescence release mechanism. 

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. 

Part of the dark reactions of photosynthesis is interconversion of sugars with an enzyme called transketolase using thiamine pyrophosphate, TPP, as a catalyst (Section 8.12.8). Provide a reasonable mechanism for this enzymatic reaction. In addition to water, there are weak general acids and general bases present in the active site at pH 7. 

Mechanism Transketolase and Transaldolase Stabilize Carbanionic Intermediates by Different Mechanisms... 

Transketolase Reaction. Transketolase contains a tightly bound thiamine pyrophosphate as its prosthetic group. The enzyme transfers a two-carbon glycoaldehyde from a ketose donor to an aldose acceptor. The site of the addition of the two-carbon unit is the thiazole ring of TPR Transketolase is homologous to the Ej subunit of the pyruvate dehydrogenase complex (p. 478) and the reaction mechanism is similar (Figure 20.21). 

The same coenzyme binding pattern and no structural changes in the protein component were detectable for the mutant enzymes of transketolase from Saccha-romyces cerevisiae and their complexes with coenzyme analogs studied by X-ray crystallography (Konig et ah, 1994 Wikner et ah, 1994). Summarizing, it can be ruled out that the differences in the H/D exchange rate constants of transketolase from Saccharomyces cerevisiae are a result of a different solvent accessibility of a base involved in the proton abstraction mechanism of ThDP. 

TPP is a coenzyme for transketolase, the enzyme that catalyzes the conversion of a ke-topentose (xylulose-5-phosphate) and an aldopentose (ribose-5-phosphate) into an al-dotriose (glyceraldehyde-3-phosphate) and a ketoheptose (sedoheptulose-7-phosphate). Notice that the total number of carbon atoms in the reactants and products does not change (5+5 = 3+ 7). Propose a mechanism for this reaction. 

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