Thiamin diphosphate, decarboxylations

Step 4 of Figure 29.12 Oxidative Decarboxylation The transformation of cr-ketoglutarate to succinyl CoA in step 4 is a multistep process just like the transformation of pyruvate to acetyl CoA that we saw in Figure 29.11. In both cases, an -keto acid loses C02 and is oxidized to a thioester in a series of steps catalyzed by a multienzynie dehydrogenase complex. As in the conversion of pyruvate to acetyl CoA, the reaction involves an initial nucleophilic addition reaction to a-ketoglutarate by thiamin diphosphate vlide, followed by decarboxylation, reaction with lipoamide, elimination of TPP vlide, and finally a transesterification of the dihydrolipoamide thioester with coenzyme A. 

Figure 17-5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (NAD nicotinamide adenine dinucleotide FAD, flavin adenine dinucleotide TDP, thiamin diphosphate.)...

Thiamin has a central role in energy-yielding metabo-hsm, and especially the metabohsm of carbohydrate (Figure 45-9). Thiamin diphosphate is the coenzyme for three multi-enzyme complexes that catalyze oxidative decarboxylation reactions pymvate dehydrogenase in carbohydrate metabolism a-ketoglutarate dehydro-... 

Thiamine diphosphate (TDP) is an essential coenzyme in carbohydrate metabolism. TDP-dependent enzymes catalyze carbon-carbon bond-breaking and -forming reactions such as a-keto acid decarboxylations (oxidative and non-oxidative) and condensations, as well as ketol transfers (trans- and phospho-ketolation). Some of these processes are illustrated in Fig. 12. 

Other organisms are equipped to produce ethanol, by employing a thiamine diphosphate-dependent decarboxylation of pyruvate to acetaldehyde (see Section 15.8) and NAD+ is regenerated by reducing the acetaldehyde to ethanol. This is a characteristic of baker s yeast, and forms the essential process for both bread making (production of CO2) and the brewing industry (formation of ethanol). 

Now this reaction is effectively a repeat of the pyruvate acetyl-CoA oxidative decarboxylation we saw at the beginning of the Krebs cycle. It similarly requires thiamine diphosphate, lipoic acid, coenzyme A and NAD+. A further feature in common with that reaction is that 2-oxoglutarate dehydrogenase is also an enzyme complex comprised of three separate enzyme activities. 2-Oxoglutarate is thus transformed into succinyl-CoA, with the loss of... 

Thiamine diphosphate (TPP, 3), in cooperation with enzymes, is able to activate aldehydes or ketones as hydroxyalkyl groups and then to pass them on to other molecules. This type of transfer is important in the transketo-lase reaction, for example (see p. 152). Hydroxyalkyl residues also arise in the decarboxylation of 0x0 acids. In this case, they are released as aldehydes or transferred to lipoamide residues of 2-oxoacid dehydrogenases (see p. 134). The functional component of TPP is the sulfur- and nitrogen-containing thiazole ring. 

The intermediary metabolism has multienzyme complexes which, in a complex reaction, catalyze the oxidative decarboxylation of 2-oxoacids and the transfer to coenzyme A of the acyl residue produced. NAD" acts as the electron acceptor. In addition, thiamine diphosphate, lipoamide, and FAD are also involved in the reaction. The oxoacid dehydrogenases include a) the pyruvate dehydrogenase complex (PDH, pyruvate acetyl CoA), b) the 2-oxoglutarate dehydrogenase complex of the tricarboxylic acid cycle (ODH, 2-oxoglutarate succinyl CoA), and c) the branched chain dehydrogenase complex, which is involved in the catabolism of valine, leucine, and isoleucine (see p. 414). 

Initially, pyruvate dehydrogenase [El] catalyzes the decarboxylation of pyruvate and the transfer of the resulting hydroxyethyl residue to thiamine diphosphate (TPP, la). The same enzyme then catalyzes oxidation of the TPP-bound hydroxyethyl group to yield an acetyl residue. This residue and the reducing equivalents obtained are then transferred to lipoamide (1b). 

Thiamine diphosphate (ThDP) is an important cofactor in many enzymes which either transfer carbon units between molecules or decarboxylate organic acids. 

The project encompassed the comparative characterization of pyruvate decarboxylase from Z. mohilis (PDC) and benzoylformate decarboxylase from P. putida (BED) as well as their optimization for bioorganic synthesis. Both enzymes require thiamine diphosphate (ThDP) and magnesium ions as cofactors. Apart from the decarboxylation of 2-ketoacids, which is the main physiological reaction of these 2-ketoacid decarboxylases, both enzymes show a carboligase site reaction leading to chiral 2-hydroxy ketones (Scheme 2.2.3.1). A well-known example is... 

In most organisms undergoing aerobic metabolism, pyruvate is oxidized to acetyl-CoA in a complex process involving its decarboxylation (Eq. 10-6). This oxidative decarboxylation, like the decarboxylation of pyruvate to acetaldehyde, requires thiamin diphosphate. In addition, an array of other catalysts participate in the process (see Fig. 15-15). Among these are the electron carrier flavin adenine diphosphate (FAD), which is derived from the vitamin riboflavin. Like NAD+, this... 

Why do we need vitamins Early clues came in 1935 when nicotinamide was found in NAD+ by H. von Euler and associates and in NADP+ by Warburg and Christian. Two years later, K. Lohman and P. Schuster isolated pure cocarboxylase, a dialyz-able material required for decarboxylation of pyruvate by an enzyme from yeast. It was shown to be thiamin diphosphate (Fig. 15-3). Most of the water-soluble vitamins are converted into coenzymes or are covalently bound into active sites of enzymes. Some lipid-soluble vitamins have similar functions but others, such as vitamin D and some metabolites of vitamin A, act more like hormones, binding to receptors that control gene expression or other aspects of metabolism. 

We see that the essence of the action of thiamin diphosphate as a coenzyme is to convert the substrate into a form in which electron flow can occur from the bond to be broken into the structure of the coenzyme. Because of this alteration in structure, a bond breaking reaction that would not otherwise have been possible occurs readily. To complete the catalytic cycle, the electron flow has to be reversed again. The thiamin-bound cleavage product (an enamine) from either of the adducts in Eq. 14-20 can be reconverted to the thiazolium dipolar ion and an aldehyde as shown in step b of Eq. 14-21 for decarboxylation of pyruvate to acetaldehyde. 

Most known thiamin diphosphate-dependent reactions (Table 14-2) can be derived from the five halfreactions, a through e, shown in Fig. 14-3. Each halfreaction is an a cleavage which leads to a thiamin- bound enamine (center, Fig. 14-3) The decarboxylation of an a-oxo acid to an aldehyde is represented by step b followed by a in reverse. The most studied enzyme catalyzing a reaction of this type is yeast pyruvate decarboxylase, an enzyme essential to alcoholic fermentation (Fig. 10-3). There are two 250-kDa isoenzyme forms, one an a4 tetramer and one with an ( P)2 quaternary structure. The isolation of ohydroxyethylthiamin diphosphate from reaction mixtures of this enzyme with pyruvate52 provided important verification of the mechanisms of Eqs. 14-14,14-15. Other decarboxylases produce aldehydes in specialized metabolic pathways indolepyruvate decarboxylase126 in the biosynthesis of the plant hormone indoIe-3-acetate and ben-zoylformate decarboxylase in the mandelate pathway of bacterial metabolism (Chapter 25).1243/127... 

Figure 14-2 (A) Stereoscopic view of the active site of pyruvate oxidase from the bacterium Lactobacillus plantarium showing the thiamin diphosphate as well as the flavin part of the bound FAD. The planar structure of the part of the intermediate enamine that arises from pyruvate is shown by dotted lines. Only some residues that may be important for catalysis are displayed G35 , S36 , E59 , H89 , F12T, Q122 , R264, F479, and E483. Courtesy of Georg E. Schulz.119 (B) Simplified view with some atoms labeled and some side chains omitted. The atoms of the hypothetical enamine that are formed from pyruvate, by decarboxylation, are shown in green.

Oxidative decarboxylation of an a-oxoacid with thiamin diphosphate... 

The third type of carbon-branched unit is 2-oxoisovalerate, from which valine is formed by transamination. The starting units are two molecules of pyruvate which combine in a thiamin diphosphate-dependent a condensation with decarboxylation. The resulting a-acetolactate contains a branched chain but is quite unsuitable for formation of an a amino acid. A rearrangement moves the methyl group to the (3 position (Fig. 24-17), and elimination of water from the diol forms the enol of the desired a-oxo acid (Fig. 17-19). The precursor of isoleucine is formed in an analogous way by condensation, with decarboxylation of one molecule of pyruvate with one of 2-oxobutyrate. 

The pathway also operates in some bacteria and apparently is the sole source of isoprenoid compounds for the unicellular alga Scenedesmus.28 The pathway is outlined in Fig. 22-2. Pyruvate is decarboxylated by a thiamin diphosphate-dependent enzyme,29 and the resulting enamine is condensed with D-glyceraldehyde 3-phosphate to form 1-deoxyxylulose 5-phosphate.28, i0 31a The latter undergoes an isomeroreductase rearrange-... 

In a rare autosomal recessive condition (discovered in 1954) the urine and perspiration has a maple syrup odor/ High concentrations of the branched-chain 2-oxoacids formed by transamination of valine, leucine, and isoleucine are present, and the odor arises from decomposition products of these acids. The branched-chain amino acids as well as the related alcohols also accumulate in the blood and are found in the urine. The biochemical defect lies in the enzyme catalyzing oxidative decarboxylation of the oxoacids, as is indicated in Fig. 24-18. Insertions, deletions, and substitutions may be present in any of the subunits (Figs. 15-14,15-15). The disease which may affect one person in 200,000, is usually fatal in early childhood if untreated. Children suffer seizures, mental retardation, and coma. They may survive on a low-protein (gelatin) diet supplemented with essential amino acids, but treatment is difficult and a sudden relapse is apt to prove fatal. Some patients respond to administration of thiamin at 20 times the normal daily requirement. The branched-chain oxoacid dehydrogenase from some of these children shows a reduced affinity for the essential coenzyme thiamin diphosphate.d... 

Polled hereford calves in Australia develop maple syrup urine disease relatively often/ 6 One cause was established as a mutation that introduces a stop codon that causes premature termination within the leader peptide during synthesis of the thiamin diphosphate-dependent El subunit. A similar biochemical defect in a mutant of Bacillus subtilis causes difficulties for this bacterium, which requires branched-chain fatty acids in its membranes. Branched acyl-CoA derivatives are needed as starter pieces for their synthesis (Chapter 29). With the oxidative decarboxylation of the necessary oxoacids blocked, the mutant is unable to grow unless supplemented with branched-chain fatty acids. 

The vitamins K and other naphthoquinones arise from O-succinylbenzoate84 86 whose synthesis from chorismate and 2-oxoglutarate depends upon a thiamine diphosphate-bound intermediate, as indicated in Fig. 25-4. Elimination of pyruvate yields O-succinylbenzoate. The remaining reactions of decarboxylation, methylation, and prenylation (Fig. 25-4) resemble those of ubiquinone synthesis. 

Scheme 21. Thiamine-diphosphate-dependent decarboxylation of hydroxypyruvate by transketolase...

Scheme 3 Thiamin diphosphate-dependent decarboxylation of benzoylfonnic acid to benzaldehyde. Reprinted with permission from Reference 50. Copyright 2006 American Chemical Society.

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