Thiamine pyrophosphate reactions involving

Transketolase (EC 2.2.1.1) an enzyme that catalyses transketolation, an important process of carbohydrate metabolism, especially in the Pentose phosphate cycle (see) and Calvin cycle (see). T. has been found in a wide variety of cells and tissues, including mammalian liver, green plants and many bacterial species. The enzyme contains divalent metal cations and the coenzyme, thiamin pyrophosphate. Transketolation involves transfer of a C2-unit (often called active glycolaldehyde or a ketol moiety) from a ketose to Cl of an aldose. Only ketoses with L-configuration at C3 and preferably irons configuration on the next carbon (i.e. Cl, 2, 3 and preferably 4 as in fructose) can serve as donors of the C2-unit. The acceptor is always an aldose. Thins-ketolation is reversible. Details of the reaction in which xylulose S-phosphate serves as the donor of... 

As shown in Eigure 18.17, thiamine is composed of a substituted thiazole ring joined to a substituted pyrimidine by a methylene bridge. It is the precursor of thiamine pyrophosphate (TPP), a coenzyme involved in reactions of carbo-... 

The pyruvate dehydrogenase complex (PDC) is a noncovalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA. The active sites of ail three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme and so on, without diffusion of substrates and products through the solution. The overall reaction (see A Deeper Look Reaction Mechanism of the Pyruvate Dehydrogenase Complex ) involves a total of five coenzymes thiamine pyrophosphate, coenzyme A, lipoic acid, NAD+, and FAD. 

The mechanism of the pyruvate dehydrogenase reaction is a tour de force of mechanistic chemistry, involving as it does a total of three enzymes (a) and five different coenzymes—thiamine pyrophosphate, lipoic acid, coenzyme A, FAD, and NAD (b). 

Acyloins (a-hydroxy ketones) are formed enzymatically by a mechanism similar to the classical benzoin condensation. The enzymes that can catalyze reactions of this type arc thiamine dependent. In this sense, the cofactor thiamine pyrophosphate may be regarded as a natural- equivalent of the cyanide catalyst needed for the umpolung step in benzoin condensations. Thus, a suitable carbonyl compound (a -synthon) reacts with thiamine pyrophosphate to form an enzyme-substrate complex that subsequently cleaves to the corresponding a-carbanion (d1-synthon). The latter adds to a carbonyl group resulting in an a-hydroxy ketone after elimination of thiamine pyrophosphate. Stereoselectivity of the addition step (i.e., addition to the Stand Re-face of the carbonyl group, respectively) is achieved by adjustment of a preferred active center conformation. A detailed discussion of the mechanisms involved in thiamine-dependent enzymes, as well as a comparison of the structural similarities, is found in references 1 -4. 

Group-transfer reactions often involve vitamins3, which humans need to have in then-diet, since we are incapable of realizing their synthesis. These include nicotinamide (derived from the vitamin nicotinic acid) and riboflavin (vitamin B2) derivatives, required for electron transfer reactions, biotin for the transfer of C02, pantothenate for acyl group transfer, thiamine (vitamin as thiamine pyrophosphate) for transfer of aldehyde groups and folic acid (as tetrahydrofolate) for exchange of one-carbon fragments. Lipoic acid (not a vitamin) is both an acyl and an electron carrier. In addition, vitamins such as pyridoxine (vitamin B6, as pyridoxal phosphate), vitamin B12 and vitamin C (ascorbic acid) participate as cofactors in an important number of metabolic reactions. 

Thiamine pyrophosphate is a coenzyme for several enzymes involved in carbohydrate metabolism. These enzymes either catalyze the decarboxylation of oi-keto acids or the rearrangement of the carbon skeletons of certain sugars. A particularly important example is provided by the conversion of pyruvic acid, an oi-keto acid, to acetic acid. The pyruvate dehydrogenase complex catalyzes this reaction. This is the key reaction that links the degradation of sugars to the citric acid cycle and fatty acid synthesis (chapters 16 and 18) ... 

The reaction has been shown to be carried out by a pyravate decarboxylase and involves thiamine pyrophosphate in the formation of activated acetaldehyde from pyravate, which then condenses with benzaldehyde. Evidently, pyravate decarboxylase, a crucial enzyme for ethanol biosynthesis, is nsed in an urmatural way... 

In the first step, pyruvate is decarboxylated in an irreversible reaction catalyzed by pyruvate decarboxylase. This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate. Pyruvate decarboxylase requires Mg24" and has a tightly bound coenzyme, thiamine pyrophosphate, discussed below. In the second step, acetaldehyde is reduced to ethanol through the action of alcohol dehydrogenase, with... 

Vitamin Influences. The involvement of NAD and NADP in many carbohydrate reactions explains the importance of nicotinamide in carbohydrate melaholism. Thiamine, in the form or thiamine pyrophosphate (cocarboxylase), is the cofaclor necessary in the decarboxylation of pyruvic acid, in the iraru-kelolase-calalyzed reactions of the pentose phosphaie cycle, and in the decarboxylation of alpha-keloglutaric acid in the citric acid cycle, among other reactions. Biotin is a hound cofaclor in the fixation of carbon dioxide to form nxalacetic acid from pyruvic acid. Pantothenic acid is a part of the C oA molecule. There are separate alphabetical entries in this volume on the various specific vitamins as well as a review entry on Vitamin. 

The quinone ring is derived from isochorismic acid, formed by isomerization of chorismic acid, an intermediate in the shikirnic acid pathway for synthesis of the aromatic amino acids. The first intermediate unique to menaquinone formation is o-succinyl benzoate, which is formed by a thiamin pyrophosphate-dependent condensation between 2-oxoglutarate and chorismic acid. The reaction catalyzed by o-succinylbenzoate synthetase is a complex one, involving initially the formation of the succinic semialdehyde-thiamin diphosphate complex by decarboxylation of 2-oxoglutarate, then addition of the succinyl moiety to isochorismate, followed by removal of the pyruvoyl side chain and the hydroxyl group of isochorismate. 

Unfortunately diacetyl formation is still not well understood. Acetoin formation occurs either by nonspecific interaction of acetaldehyde with the a-hydroxyethyl thiamine pyrophosphate intermediate in pyruvate decarboxylation (209) or by decarboxylation of a-acetolactate (210), which in turn arises either from interaction of pyruvate with a-hydroxyethyl thiamine pyrophosphate (211) or as a specific intermediate in valine biosynthesis (212, 213). Diacetyl does not appear to be formed directly from acetoin (208, 214). It is formed from a-acetolactate, in absence of cells, by O2 oxidation (215), and even under N2 (216), although an oxidation must occur. It is also formed from acetyl CoA (217, 218), probably by interaction with a-hydroxyethyl thiamine pyrophosphate [cf. stimulation by acetyl CoA addition to a solution of pyruvate and pyruvate decarboxylase (2i5)]. It is not known whether this involves a specific enzyme or is a mere side reaction. 

Several coenzymes are involved in the biosynthesis of their own precursors. Thus, thiamine is the cofactor of the enzyme that converts 1-deoxy-D-xylulose 5-phosphate (43) (the precursor of thiamine pyrophosphate, pyridoxal 5 -phosphate and of iso-prenoids via the nomnevalonate pathway) into 2 C-methyl-D-erythritol 4-phosphate (90, Fig. 11). Similarly, two enzymes required for the biosynthesis of GTP, which is the precursor of tetrahydrofolate, require tetrahydrofolate derivatives as cofactors (Fig. 3). When a given coenzyme is involved in its own biosynthesis, we are faced with a hen and egg problem, namely how the biosynthesis could have evolved in the absence of the cmcially required final product. The answers to that question must remain speculative. The final product may have been formed via an alternative biosynthetic pathway that has been abandoned in later phases of evolution or that may persist in certain organisms but remains to be discovered. Alternatively, the coenzyme under study may have been accessible by a prebiotic sequence of spontaneous reactions. An interesting example in this respect is the biosynthesis of flavin coenzymes, in which several reaction steps can proceed without enzyme catalysis despite their mechanistic complexity. 

Figure 1 Thiamine, vitamin B1, is the cofactor, as its pyrophosphate ester, for many important biologic reactions. These reactions involve the formation of an anion 2 that is stabilized by resonance with a carbene form 3. The related species 4 and derivatives have been developed as important ligands for metal ions in chemical synthesis.

Pyruvate decarboxylase catalyzes the nonoxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. When an aldehyde is present with pyruvate, the enzyme promotes an acyloin condensation reaction. The mechanistic reason for this fortuitous reaction is well understood and involves the aldehyde outcompeting a proton for bond formation with a reactive thiamine pyrophosphate-bound intermediate (90,91). When acetaldehyde is present, the product formed is acetoin. Benzalde-hyde results in the production of phenylacetylcarbinol (Fig. 26). Both of these condensations are enantioselective, forming the R enantiomer preferentially in both cases. 

This looks like a simple reaction based on very small molecules. But look again. It Is a very strange reaction indeed. The molecule of CO2 clearly comes from the carboxyl group of pyruvate, but how is the C-C bond cleaved, and how does acetyl CoA Join on If you try to draw a mechanism you will see that there must be more to this reaction than meets the eye. The extra features are two new cofactors, thiamine pyrophosphate and lipoic acid, and the reaction takes place in several stages with some interesting chemistry involved. 

The metabolic functions of pantothenic acid in human biochemistry are mediated through the synthesis of CoA. Pantothenic acid is a structural component of CoA. which is necessary for many important metabolic processes. Pantothenic acid is incorporated into CoA by a. series of five enzyme-catalyzed reactions. CoA is involved in the activation of fatty acids before oxidation, which requires ATP to form the respective fatty ocyl-CoA derivatives. Pantothenic acid aI.so participates in fatty acid oxidation in the final step, forming acetyl-CoA. Acetyl-CoA is also formed from pyruvate decarboxylation, in which CoA participates with thiamine pyrophosphate and lipoic acid, two other important coenzymes. Thiamine pyrophosphate is the actual decarboxylating coenzyme that functions with lipoic acid to form acetyidihydrolipoic acid from pyruvate decarboxylation. CoA then accepts the acetyl group from acetyidihydrolipoic acid to form acetyl-CoA. Acetyl-CoA is an acetyl donor in many processes and is the precursor in important biosyntheses (e.g.. those of fatty acids, steroids, porphyrins, and acetylcholine). 

Thiamine (vitamin Bj) is an important water-soluble vitamin that, in its active form of thiamine pyrophosphate, is used as a cofactor in enzymatic reactions that involve the transfer of an aldehyde group. Thiamine can be synthesized by plants and some microorganisms, but not usually by animals. Hence, humans must obtain thiamine from the diet, though small amounts may be obtained from synthesis by intestinal bacteria. Because of its importance in metabolic reactions, it is present in large amounts in skeletal muscle, heart, liver, kidney, and brain. Thus, it has a widespread distribution in foods, but there can be a substantial loss of thiamine during cooking above 100°C (212°F). 

Thiamine (vitamin Bj) is an important water-soluble vitamin that, in its active form of thiamine pyrophosphate, is used as a cofactor in enzymatic reactions that involve the transfer of an aldehyde group. 

The syntheses of valine, leucine, and isoleucine from pyruvate are illustrated in Figure 14.9. Valine and isoleucine are synthesized in parallel pathways with the same four enzymes. Valine synthesis begins with the condensation of pyruvate with hydroxyethyl-TPP (a decarboxylation product of a pyruvate-thiamine pyrophosphate intermediate) catalyzed by acetohydroxy acid synthase. The a-acetolactate product is then reduced to form a,/3-dihydroxyisovalerate followed by a dehydration to a-ketoisovalerate. Valine is produced in a subsequent transamination reaction. (a-Ketoisovalerate is also a precursor of leucine.) Isoleucine synthesis also involves hydroxyethyl-TPP, which condenses with a-ketobutyrate to form a-aceto-a-hydroxybutyrate. (a-Ketobutyrate is derived from L-threonine in a deamination reaction catalyzed by threonine deaminase.) a,/3-Dihydroxy-/3-methylvalerate, the reduced product of a-aceto-a-hydroxybutyrate, subsequently loses an HzO molecule, thus forming a-keto-/kmethylvalerate. Isoleucine is then produced during a transamination reaction. In the first step of leucine biosynthesis from a-ketoisovalerate, acetyl-CoA donates a two-carbon unit. Leucine is formed after isomerization, reduction, and transamination. 

TK is one of the enzymes involved in the oxidative pentose phosphate pathway, and requires the cofactors thiamine pyrophosphate (TPP)12191 and Mg2+[218). It reversibly transfers the C1-C2 ketol unit from D-xylulose 5-phosphate to D-ribose 5-phosphate, and generates D-sedoheptulose 7-phosphate and D-Gly 3-P. D-Erythrose 4-phosphate also functions as an acceptor of the ketol unit from D-xylulose 5-phosphate, to produce Fru 6-P and D-Gly 3-P (Fig. 14.2-1). TK from baker s yeast is commercially available, and the enzyme can also be isolated from spinach[220, 2211 TK from E. coli has been overexpressed and prepared on a large scale12221. In ketol transfer reactions,... 

In many decarboxylations, particularly those involving pyridoxal 5 -phos-phate and thiamin pyrophosphate, the starting state for the decarboxylation is a zwitterion in which the positive and negative charges are widely separated. In the decarboxylation step, this zwitterion is converted into a neutral intermediate. Such reactions are faster in less polar environments. Some enzymes appear to take advantage of this fact. 

Thiamine pyrophosphate (TPP). The purple color highlights the parts of this cofactor that are directly involved in enzyme reactions. 

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