Hydroxyethyl thiamine pyrophosphate

The CPPase substrate DMAPP (15) is formed from isopentenyl pyrophosphate (IPP) (14) via the IPP isomerase reaction. It had been assumed that IPP was generated only via mevalonic acid (12) (Fig. 2), but Rohmer discovered another route, 2-C-methyl-D-erythritol 4-phosphate (13) (MEP) pathway (Fig. 2) [22, 23]. A key step in the MEP pathway is the reaction catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which combines hydroxyethyl thiamine pyrophosphate (hydroxyethyl TPP) generated from pyruvic acid (17) and TPP with glyceral-dehyde 3-phosphate (18) to yield 1-deoxy-D-xylulose 5-phosphate (19) containing five carbons. The mevalonate pathway operates in the cytosol of plants and animals, whereas the MEP pathway is present in the plastid of plants or in eubacteria [24-27]. 

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. 

In the first step of the conversion catalyzed by pyruvate decarboxylase, a carbon atom from thiamine pyrophosphate adds to the carbonyl carbon of pyruvate. Decarboxylation produces the key reactive intermediate, hydroxyethyl thiamine pyrophosphate (HETPP). As shown in figure 13.5, the ionized ylid form of HETPP is resonance-stabilized by the existence of a form without charge separation. The next enzyme, dihydrolipoyltransacetylase, catalyzes the transfer of the two-carbon moiety to lipoic acid. A nucleophilic attack by HETPP on the sulfur atom attached to carbon 8 of oxidized lipoic acid displaces the electrons of the disulfide bond to the sulfur atom attached to carbon 6. The sulfur then picks up a proton from the environment as shown in figure 13.5. This simple displacement reaction is also an oxidation-reduction reaction, in which the attacking carbon atom is oxidized from the aldehyde level in HETPP to the carboxyl level in the lipoic acid derivative. The oxidized (disulfide) form of lipoic acid is converted to the reduced (mer-capto) form. The fact that the two-carbon moiety has become an acyl group is shown more clearly after dissocia-... 

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. 

That 2-acetylthiamine pyrophosphate is an intermediate in the oxidative decarboxylation of pyruvate may also be. inferred from (a) the demonstration by Goedde et al. (1961) that the hydroxyethyl group of 2-hydroxyethyl-thiamine pyrophosphate can be converted to acetyl CoA in the presence of a pyruvate oxidation system from yeast mitochondria and (b) the demonstration by Krampitz et al. (1961) that the hydroxyethyl group of 2-hydrox-yethylthiamine pyrophosphate is oxidized to acetate in the presence of fcrricyanide and the acetoin-forming system from A. aerogenes. 

Of the pyruvic acid formed during glycolysis, a proportion is used for biosynthetic reactions (see Fig. 17.8). Pyruvate is converted to a-acetolactate by the enzyme acetohydroxy acid synthetase. The substrates for this reaction are pyruvate and hydroxyethyl thiamine pyrophosphate. In a similar reaction involving hydroxyethyl TPP and a-oxobutyrate, a-acetohydroxybutyrate is formed (Fig. 17.13). Both acetohydroxy acids are excreted by yeast and are non-enzymically converted, in the medium, to vicinal diketones. 

Thiamine pyrophosphate is the essential coenzyme in the enzymatic decarboxylation of pyruvate to acetaldehyde. It has been proposed that the decarboxylation proceeds by way of 2-(l-carboxy-l-hydroxyethyl)-thiamine pyrophosphate [(134a) arising from pyruvic acid and thecoenzyme]. 

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. 

Hydroxyethyl-thiamine pyrophosphate is also nucleophilic toward lipoic acid. In nature lipoic acid operates in tandem with thiamine pyrophosphate. 

The pathways for thiamine biosynthesis have been elucidated only partiy. Thiamine pyrophosphate is made universally from the precursors 4-amino-5-hydroxymethyl-2-methylpytimidinepyrophosphate [841-01-0] (47) and 4-methyl-5-(2-hydroxyethyl)thiazolephosphate [3269-79-2] (48), but there appear to be different pathways ia the eadier steps. In bacteria, the early steps of the pyrimidine biosynthesis are same as those of purine nucleotide biosynthesis, 5-Aminoimidazole ribotide [41535-66-4] (AIR) (49) appears to be the sole and last common iatermediate ultimately the elements are suppHed by glycine, formate, and ribose. AIR is rearranged in a complex manner to the pyrimidine by an as-yet undetermined mechanism. In yeasts, the pathway to the pyrimidine is less well understood and maybe different (74—83) (Fig. 9). 

This resonance-stabilized intermediate can be protonated to give hydroxyethyl-TPP. This well-characterized intermediate was once thought to be so unstable that it could not be synthesized or isolated. However, its synthesis and isolation are actually routine. (In fact, a substantial amount of the thiamine pyrophosphate in living things exists as the hydroxyethyl form.)... 

Pyruvate is decarboxylated to form a hydroxyethyl derivative bound to the reactive carbon of thiamine pyrophosphate, the coenzyme of pyruvate dehydrogenase. 

Decarboxylation of an a-keto acid like pyruvate is a difficult reaction for the same reason as are the ketol condensations (see fig. 12.33) Both kinds of reactions require the participation of an intermediate in which the carbonyl carbon carries a negative charge. In all such reactions that occur in metabolism, the intermediate is stabilized by prior condensation of the carbonyl group with thiamine pyrophosphate. In figure 13.5 thiamine pyrophosphate and its hydroxyethyl derivative are written in the doubly ionized ylid form rather than the neutral form because this is the form that actually participates in the reaction even though it is present in much smaller amounts. 

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).

Consider, for example, the biosynthesis of the amino acids valine, leucine, and isoleucine. A common intermediate, hydroxy ethyl thiamine pyrophosphate (hydroxy ethyl-TPP Section 17.1.1). initiates the pathways leading to all three of these amino acids. Hydroxyethyl-TPP can react with a-ketobutyrate in the initial step for the synthesis of isoleucine. Alternatively, hydroxyethyl-TPP can react with pyruvate in the committed step for the pathways leading to valine and leucine. Thus, the relative concentrations of a-ketobutyrate and pyruvate determine how much isoleucine is produced compared with valine and leucine. Threonine deaminase, the PLP enzyme that catalyzes the formation of a-ketobutyrate, is allosterically inhibited by isoleucine (Figure 24.22). This enzyme is also allosterically activated by valine. Thus, this enzyme is inhibited by the product of the pathway that it initiates and is activated by the end product of a competitive pathway. This mechanism balances the amounts of different amino acids that are synthesized. 

A. form a hydroxyethyl derivative of the thiazole ring of enzyme-bound thiamine pyrophosphate... 

D. decarboxylate the hydroxyethyl group bound to the thiamine pyrophosphate coenzyme... 

In the first step, pyruvate dehydrogenase catalyzes the decarboxylation of pyruvate. A nucleophile is formed when a basic residue of the enzyme extracts a proton from the thiazole ring of thiamine pyrophosphate (TPP). The intermediate, hydroxyethyl-TPP (HETPP), forms after the nucleophilic thiazole ring attacks the carbonyl group of pyruvate with the resulting loss of COz (Figure 9.8). 

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. 

The C-2-exchange of azolium salts via an ylide mechanism was discussed in Section 24.1.2.1. Thiamin pyrophosphate acts as a coenzyme in several biochemical processes and in these, its mode of action depends on the intermediacy of a 2-deprotonated species (32.2.4). In the laboratory, thiazolium salts (3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride is commercially available) will act as catalysts for the benzoin condensation, and in contrast to cyanide, the classical catalyst, allow such reactions to proceed with alkanals, as opposed to araldehydes the key steps in thiazolium ion catalysis for the synthesis of 2-hydroxy-ketones are shown below and depend on the formation and nucleophilic reactivity of the C-2-ylide. Such catalysis provides acyl-anion equivalents. 

Vitamin Bi is an essential co-factor for several enzymes of carbohydrate metabolism such as transketolase, pyruvate dehydrogenase (PDH), pyruvate decarboxylase and a-ketoglutarate dehydrogenase. To become the active co-factor thiamin pyrophosphate (TPP), thiamin has to be salvaged by thiamin pyrophosphokinase or synthesized de novo. In Escherichia coli and Saccharomyces cerevisiae thiamin biosynthesis proceeds via two branches that have to be combined. In the pyrimidine branch, 4-amino-5-hydroxymethy-2-methylpyrimidine (PIMP) is phosphorylated to 4-amino-2-methyl-5-hydroxymethyl pyrimidine diphosphate (PIMP-PP) by the enzyme HMP/HMP-P kinase (ThiD) however, the step can also be catalyzed by pyridoxine kinase (PdxK), an enzyme also responsible for the activation of vitamin B6 (see below). The second precursor of thiamin biosynthesis, 5-(2-hydroxyethyl)-4-methylthiazole (THZ), is activated by THZ kinase (ThiM) to 4-methyl-5-(2-phosphoethyl)-thiazole (THZ-P), and then the thia-zole and pyrimidine moieties, HMP-PP and THZ-P, are combined to form thiamin phosphate (ThiP) by thiamin phosphate synthase (ThiE). The final step, pyrophosphorylation, yields TPP and is carried out by thiamin pyrophosphorylase (TPK). 

Fig. 11-15 The pyruvate dehydrogenase reaction takes piace via three enzymes in a complex Pyruvate is decarboxyiated by the pyruvate decarboxylase) component of the enzyme complex a key cofactor is thiamine pyrophosphate (TPP) that transfers the hydroxyethyl moiety to one of the sulfur atoms on oxidized lipoamide that is covalently bound to Ej(c//hyc/ro//poy/ transacetylase). When this transfer takes place, the 2-carbon moiety is oxidized to an acetyl moiety and then the acetyl moiety is transferred to CoA to yield acetyl-CoA which is then released from the active site of Ej. The reduced lipoamide moiety is recycled back to its oxidized form by donating hydrogen atoms to FAD in E3 lipoamide dehydrogenase), and the reaction cycle begins over again.

Figure 7.8 Thiamine pyrophosphate (TPP) biosynthesis in 8. subtilis. in addition to the de novo route, salvage reactions to recruit nonphosphorylated 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), 5-(2-hydroxyethyl)-4-methylthiazole (HET), and thiamine are shown.

According to Breslow, the active aldehyde intermediate in the decarboxylation of pyruvate could be an a-hydroxyethyl derivative of thiamine pyrophosphate, the substituent being attached in position 2 to the thiazole ring . His starting point was the observation that thiazolium salts easily lose a proton at C-2. Thus a stable and reactive zwitterion results that could be capable of forming an acyl carbanion derivative. The near-by amino-pyrimidine ring would have an inductive effect upon electron withdrawal at C-2. Breslow pictures the formation of acetoin from pyruvate and acetaldehyde as follows ... 

A 8-hydroxyethyl group in position 5 of the thiazolium ring. Activity is lost upon replacing this group by hydrogen. The need for a free hydroxyl group is obvious, for here phosphorylation takes place when thiamine is converted into thiamine pyrophosphate. 

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