Transketolase mechanism enzyme

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). 

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. 

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. 

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. 

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. 

Transketolase resembles pyruvate decarboxylase, the enzyme that converts pyruvate to acetaldehyde (Section 17.4), in that it also requires Mg and thiamine pyrophosphate (TPP). As in the pyruvate decarboxylase reaction, a carb-anion plays a crucial role in the reaction mechanism, which is similar to that of the conversion of pyruvate to acetaldehyde. 

With such an extensive knowledge base, what is the present state of our understanding of the mechanisms of this disorder Not unexpectedly, initial studies, primarily in experimental animal models, focused on the known metabolic pathways which involve thiamine. Indeed, the classical studies of Peters in 1930 (Peters, 1969) showed lactate accumulation in the brainstem of thiamine deficient birds with normalization of this in vitro when thiamine was added to the tissue. This led to the concept of the biochemical lesion of the brain in thiamine deficiency. The enzymes which depend on thiamine are shown in Fig. 14.1. They are transketolase, pyruvate and a-ketoglutarate dehydrogenase. Transketolase is involved in the pentose phosphate pathway needed to form nucleic acids and membrane lipids, including myelin. The ketoacid dehydrogenases are key enzymes of the Krebs cycle needed for energy (ATP) synthesis and also to form acetylcholine via Acetyl CoA synthesis. Decrease in activity of this cycle would result in anaerobic metabolism and lead to lactate formation (i.e., tissue acidosis) (Fig. 14.1). 

Indeed both -lactylthiamine pyrophosphate (XX) and a-hydroxyethyl-thiamine pyrophosphate (XXI) have been isolated and identified as products after incubation of pyruvate with a purified carboxylase preparation " . When [2- - C]pyruvate is used, the radioactivity is found in the thiazole part of the molecule after sulfite cleavage of XXL Acetaldehyde is formed from pyruvic acid by yeast carboxylase by enzymic cleavage of intermediate XXI, Uberating thiamine pyTophosphate . XXI has also been identified as intermediate in the formation of acetyl-coenzyme A from pyruvic acid by p3u uvic oxidase . The transketolase reaction has been shown to proceed via a gly-colaldehyde-enzyme intermediate here one may expect to find dihydroxy-ethylthiamine pyrophosphate as active glycol-aldehyde . Such experiments strongly support Breslow s concept of the reaction mechanism. 

Evidence from a number of sources indicated that pentose phosphates were metabolized in a series of reactions that resulted in the formation of hexose monophosphates and hexose diphosphates. Several enzyme steps are involved in these transformations. The reaction between D-ribulose 5-phosphate and D-ribose 5-phosphate to form D-sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate is catalyzed by an enzyme known as transketolase (91). This enzyme is found in plant, animal, and bacterial cells. Thiamine pyrophosphate (TPP) and Mg ions are required as cofactors. The mechanism of the reaction was suggested (92) as shown in reaction (28). 

The Transketolase-Transaldolase Pathway. The second, non-oxidative, mechanism involves a series of transfer reactions catalyzed by the enzymes transketolase and transaldolase. 

---