Acetyl-thiamine pyrophosphate

Intermediates of this type have the necessary chemical reactivity for cleaving the bonds indicated in figure 10.1b and c. The decarboxylated product of the pyruvate adduct shown in equation (2) is resonance-stabilized by the thiazolium ring (fig. 10.2a). This intermediate may be protonated to a-hydroxyethyl thiamine pyrophosphate (fig. I0.2d) alternatively, it may react with other electrophiles, such as the carbonyl groups of acetaldehyde or pyruvate, to form the species in figure 10.2b and c or it may be oxidized to acetyl-thiamine pyrophosphate (fig. 10.2e). The fate of the intermediate depends on the reaction specificity of the enzyme with which the coenzyme is associated. 

Mechanism of thiamine pyrophosphate action. Intermediate (a) is represented as a resonance-stabilized species. It arises from the decarboxylation of the pyruvate-thiamine pyrophosphate addition compound shown at the left of (a) and in equation (2). It can react as a carbanion with acetaldehyde, pyruvate, or H+ to form (b), (c), or (d), depending on the specificity of the enzyme. It can also be oxidized to acetyl-thiamine pyrophosphate (TPP) (e) by other enzymes, such as pyruvate oxidase. The intermediates (b) through (e) are further transformed to the products shown by the actions of specific enzymes. 

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 first step of this reaction, decarboxylation of pyruvate and transfer of the acetyl group to lipoic acid, depends on accumulation of negative charge on the carbonyl carbon of pyruvate. This is facilitated by the quaternary nitrogen on the thiazolium group of thiamine pyrophosphate. As shown in (c), this cationic... 

Stepl of Figure 29.11 Addition of Thiamin Diphosphate The conversion of pyruvate to acetyl CoA begins by reaction of pyruvate with thiamin diphosphate, a derivative of vitamin B(. Formerly called thiamin pyrophosphate, thiamin diphosphate is usually abbreviated as TPP. The spelling thiamine is also correct and frequently used. 

In this reaction, pyruvic acid is oxidized to carbon dioxide with formation of acetyl-SCoA and NAD+ is reduced to NADH. As noted in chapter 15, this reaction requires the participation of thiamine pyrophosphate as coenzyme. Here too the NADH formed is converted back to NAD+ by the electron transport chain. As noted above, the acetyl-SCoA is consumed by the citric acid cycle and CoASH is regenerated. 

This thiamin pyrophosphate-dependent enzyme [EC 4.1.2.9] catalyzes the reaction of D-xylulose 5-phosphate with orthophosphate to produce acetyl phosphate, d-glyceraldehyde 3-phosphate, and water. 

This enzyme [EC 1.2.3.3], which requires thiamin pyrophosphate and FAD, catalyzes the reaction of pyruvate with orthophosphate, dioxygen, and water to produce acetyl phosphate, carbon dioxide, and hydrogen peroxide. 

PDH removes CO2 and transfers the remaining acetyl group to the enzyme-bound coenzyme thiamine pyrophosphate,... 

Figure 7-1. Conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase complex. The three enzymes, pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase, exist in a complex associated with the mitochondrial matrix. Each enzyme requires at least one coenzyme that participates in the reaction. TPP, thiamine pyrophosphate Lip, lipoic acid CoA, coenzyme A.

Fig. 9. A schematic drawing of a possible mechanism for the reaction catalyzed by the pyruvate dehydrogenase complex. The three enzymes Elf E2, and E3 are located so that lipoic acid covalently linked to E2 can rotate between the active sites containing thiamine pyrophosphate (TPP) and pyruvate (Pyr) on Elt CoA on E2, and FAD on E3. Acetyl-CoA and GTP are allosteric effectors of E, and NAD+ is an inhibitor of the overall reaction.

The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA (Fig. 16-2) requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups—thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the —SH group), nicotinamide adenine dinucleotide (NAD), and lipoate. Four different vitamins required in human nutrition are vital components of this system thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA). We have already described the roles of FAD and NAD as electron carriers (Chapter 13), and we have encountered TPP as the coenzyme of pyruvate decarboxylase (see Fig. 14-13). 

Vitamin Bt Thiamine Thiamine pyrophosphate Y Cofactor of enzymes catalyzing Pyruvate -> acetyl CoA a-Ketoglutarate -> Succinyl CoA RiboseS-P xylulose S-P -> Sedoheptulose 7-P + Glyceraldehyde 3-P... 

Reactions of the TCA cycle Enzyme that oxidatively decarboxylates pyruvate, its coenzymes, activators, and inhibitors REACTIONS OF THE TRICARBOXYLIC ACID CYCLE (p. 107) Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase complex producing acetyl CoA, which is the major fuel for the tricarboxylic acid cycle (TCA cycle). The irreversible set of reactions catalyzed by this enzyme complex requires five coenzymes thiamine pyrophosphate, lipoic acid, coenzyme A (which contains the vitamin pantothenic acid), FAD, and NAD. The reaction is activated by NAD, coenzyme A, and pyruvate, and inhibited by ATP, acetyl CoA, and NADH. 

Vitamin B1 (thiamine) has the active form, thiamine pyrophosphate. It is a cofactor of enzymes catalyzing the conversion of pyruvate to acetyl CoA, a-ketoglutarate to succinyl CoA, and the transketolase reactions in the pentose phosphate pathway. A deficiency of thiamine causes beriberi, with symptoms of tachycardia, vomiting, and convulsions. In Wernicke-Korsakoff syndrome (most common in alcoholics), individuals suffer from apa thy, loss of memory, and eye movements. There is no known toxicity for this vitamin. 

The conversion of pyruvate to acetyl-CoA. The reactions are catalyzed by the enzymes of the pyruvate dehydrogenase complex. This complex has three enzymes pyruvate decarboxylase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. In addition, five coenzymes are required thiamine pyrophosphate, lipoic acid, CoASH, FAD, and NAD+. Lipoic acid is covalently attached to... 

This reaction was found later in extracts of another bacterium by Wolfe and O Kane (113) who showed requirements for coenzyme A (CoA) and thiamine pyrophosphate (TPP). Further experiments (Mortlock, Valentine, and Wolfe (76)) established acetyl-CoA as the first stable 2-carbon compound formed in this reaction (Eq. 2). 

Pyruvate produced by the glycolytic pathway may be transported into the mitochondria (via an antiport with OH"), where it is converted to acetyl-CoA by the action of the enzyme complex pyruvate dehydrogenase. The pertinent enzyme activities are pyruvate dehydrogenase (PD), lipoic acid acetyltransferase, and dihydrolipoic acid dehydrogenase. In addition, several cofactors are utilized thiamine pyrophosphate (TPP), lipoic acid, NAD+, Co A, and FAD. Only Co A and NAD+ are used in stoichiometric amounts, whereas the others are required in catalytic amounts. Arsenite and Hg2+ are inhibitors of this system. The overall reaction sequence may be represented by Figure 18.5. The NADH generated may enter the oxidative phosphorylation pathway to generate three ATP molecules per NADH molecule reduced. The reaction is practically irreversible its AGq = -9.4 kcal/mol. 

Figure 7-2. Reactions of the pyruvate dehydrogenase (PDU) multienzyme complex (PDC). Pyruvate is decarboxylated by the PDH subunit (I ,) in the presence of thiamine pyrophosphate (TPP). The resulting hydroxyethyl-TPP complex reacts with oxidized lipoamide (LipS3), the prosthetic group of dehydrolipoamide transacetylase (Ii2), to form acetyl lipoamide. In turn, this intermediate reacts with coenzyme A (CoASH) to yield acetyl-CoA and reduced lipoamide [Lip(SH)2]. The cycle of reaction is completed when reduced lipoamide is reoxidized by the flavoprotein, dehydrolipoamide dehydrogenase (E3). Finally, the reduced flavoprotein is oxidized by NAD+ and transfers reducing equivalents to the respiratory chain via reduced NADH. PDC is regulated in part by reversible phosphorylation, in which the phosphorylated enzyme is inactive. Increases in the in-tramitochondrial ratios of NADH/NAD+ and acetyl-CoA/CoASH also stimulate kinase-mediated phosphorylation of PDC. The drug dichloroacetate (DCA) inhibits the kinase responsible for phosphorylating PDC, thus locking the enzyme in its unphosphory-lated, catalytically active state. Reprinted with permission from Stacpoole et al. (2003).

At the centre is enzyme 2 which binds the acetyl group through a lipoic acid-lysine amide. On the one side this acetyl group is delivered from pyruvate by the ministrations of thiamine pyrophosphate and enzyme T and on the other it is delivered to CoA as the free thiol ester. Enzyme 3 recycles... 

The compound formed from thiamine pyrophosphate and pyruvic acid is Nature s nucleophilic acetyl group. This is a d1 reagent like the dithiane anion you met in Chapter 46. 

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. 

Figure 11 Biosynthesis of isoprenoid type cofactors. 18, Heme a 39, pyridoxal 5 -phosphate 43, 1-deoxy-D-xylulose 5-phosphate 46, thiamine pyrophosphate 83, acetyl-CoA 84, (S)-3-hydroxy-3-methylglutaryl-CoA 85, mevalonate 86, isopentenyl diphosphate (IPP) 87, dimethylallyl diphosphate (DMAPP) 88, pyruvate 89, D-glyceraldehyde 3-phosphate 90, 2C-methyl-D-erythritol 4-phosphate 91, 2C-methyl-erythritol 2,4-cyclodiphosphate 92, 1-hydroxy-2-methyl-2-( )-butenyl 4-diphosphate 93, polyprenyl diphosphate 94, cholecalciferol 95, fS-carotene 96, retinol 97, ubiquinone 98, menaquinone 99, a-tocopherol.

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. 

STEP 4 Elimination of thiamine pyrophosphate from the tetrahedral intermediate then yields acetyl dihydrolipoamide. 

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