Hydroxyethylthiamin

Decarboxylation occurs in a step analgous to the loss of CO2 from a /3-keto acid, yielding the enamine hydroxyethylthiamin diphosphate (HETPP). 

Step 3 of Figure 29.11 Reaction with Lipoamide Hydroxyethylthiamin diphosphate is an enamine (R2N—C=C), which, like all enamines, is nucleophilic (Section 23.11). It therefore reacts with the enzyme-bound disulfide lipoamide by nucleophilic attack on a sulfur atom, displacing the second sulfur in an SN2-like process. 

Hydroxyethylthiamine pyrophosphate is also nucleophilic toward a thiol of oxidized lipoic acid. A hemithioacetal is formed, and this decomposes to give a thioester ... 

There are two 2-oxoacid dehydrogenase multienzyme complexes in E. coli. One is specific for pyruvate, the other for 2-oxoglutarate. Each complex is about the size of a ribosome, about 300 A across. The pyruvate dehydrogenase is composed of three types of polypeptide chains El, the pyruvate decarboxylase (an a2 dimer of Mr — 2 X 100 000) E2, lipoate acetyltransferase (Mr = 80 000) and E3, lipoamide dehydrogenase (an a2 dimer of Mr = 2 X 56 000). These catalyze the oxidative decarboxylation of pyruvate via reactions 1.6, 1.7, and 1.8. (The relevant chemistry of the reactions of thiamine pyrophosphate [TPP], hydroxyethylthiamine pyrophosphate [HETPPJ, and lipoic acid [lip-S2] is discussed in detail in Chapter 2, section C3.)... 

The hydroxyethylthiamine pyrophosphates are potent nucleophiles and may add to carbonyl compounds to form carbon-carbon bonds. A good illustration of carbon-carbon bond making and breaking occurs in the reactions of transketo-lase. The enzyme contains tightly bound thiamine pyrophosphate and shuttles a dihydroxyethyl group between D-xylulose 5-phosphate and D-ribose 5-phosphate to form D-sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate (equations 2.55 and 2.56). 

Menon, S., and Ragsdale, S. W., 1997, Mechanism of the Clostridium thernmaceticum pyru-vate ferredoxin oxidoreductase Evidence for the common catalytic intermediacy of the hydroxyethylthiamine pyropyrosphate radical, Biochem. 36 848498494. 

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 half-reaction 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 h followed by fl 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 tetramer and one with an (aP)2 quaternary structure. The isolation of a-hydroxyethylthiamin diphosphate from reaction mixtures of this enzyme with pyruvate provided important verification of the mechanisms of Eqs. 14-14,14-15. Other decarboxylases produce aldehydes in specialized metabolic pathways indolepyruvate decarboxylase in the biosynthesis of the plant hormone indole-3-acetate and ben-zoylformate decarboxylase in the mandelate pathway of bacterial metabolism (Chapter 25). Formation of a-ketols from a-oxo acids also starts with step h of Fig. 14-3 but is followed by condensation with another carbonyl compound in step c, in reverse. An example is decarboxylation of pyruvate and condensation of the resulting active acetaldehyde with a second pyruvate molecule to give l -a-acetolactate, a reaction catalyzed by acetohydroxy acid synthase (acetolactate synthase). Acetolactate is the precursor to valine and leucine. A similar ketol condensation, which is catalyzed by the same S5mthase, is... 

To investigate thiamine metabolism in mammals, thiamine (Rf values 0.16, 0.04, and 0.03), urinary excretion of thiamine metabolites [thiochirome (Rf values 0.31, 0.28, and 0.33), thiazole (Rf values 0.85, 0.79, and 0.81), and 2-methyl-4-amino-pyrimidinecarboxylic acid (Rf values 0.42, 0.21, and 0.26)], and related compounds [pyrimidinesulfonic acid (Rf values 0.48, 0.39, and 0.46), a-hydroxyethylthiamine (Rf values 0.23, 0.09, and 0.06), A -methylnicotinamide (Rf values 0.31, 0.06, and 0.05)] were analyzed and identified by TLC on silica gel with acetonitrile-water (40 10 vol/vol) adjusted to a pH of 2.54, 4.03, and 7.85 with formic acid as solvents, respectively.Although A -methylnicotinamide and thio-chrome could not be separated in single-phase chromatography at pH 2.54, a second phase at right angle, with a pH 4.03 solvent, separated these quite clearly without affecting the resolution of the other compounds. 

Hydroxyethylthiamine (R = MeCH(OH) reacts similarly. When the sodium salt of thiamine was treated with phosgene at -20 C in aqueous ethanol, S. -carbodithiamine was formed [1462] ... 

Although pyruvate and 2-oxobutyrate are substrates of acetohydroxyacid synthase, measurements of the activity of this enzyme have been almost exclusively based on the production of acetolactate from pyruvate. This reaction product is readily decarboxylated under acidic conditions and the acetoin produced can be measured spectrophotometrically. However, ace-toin can be formed during reactions which need not be related to amino acid biosynthesis. Therefore it is unclear whether the enzyme activity characterized by Saytanarayana and Radhakrishnan (1963) can be completely ascribed to acetohydroxyacid synthase. Only a portion of the acetolactate forming activity measured in pea extracts was considered to represent the activity of this enzyme (Davies, 1964). However, the enzyme(s) isolated from barley was shown to facilitate formation of acetohydroxy derivatives of 2-oxobutyrate and pyruvate (Miflin, 1971). Mg or Mn " " as well as the substrate, hydroxyethylthiamine-pyrophosphate, was required for maximum enzyme activity. The fact that the acetolactate forming activity of the barley... 

McNelis, 1959). This subject has been reviewed recently by Metzler (1960) and will not be discussed in detail here. In brief, Breslow postulated that thiamine pyropho.sphate ionizes at the 2-position of the thiazole ring, and that the thiazolium dipolar ion (IV) reacts with pyruvate to form an intermediate (2-laetylthiamine pyrophosphate) (V) which undergoes decarboxylation to produce 2-hydroxyethylthiamine pyrophosphate (VI, VII) (Fig. 2). Species (VI) is regarded as active acetaldehyde, and can... 

Acetylthiamine pjrrophosphate appears to be yet another form of active acetate. It has been assigned a key role in the lipoic acid-Unked oxidative decarboxylation of pyruvate as the primary product of the oxidation of active acetaldehyde, i.e., 2-hydroxyethylthiamine pyrophosphate. It has been proposed that 2-acetylthiamine pyrophosphate is an intermediate in all oxidative transformations of pyruvate and that 2-succinylthiamine pyrophosphate plays a similar role in oxidation of a-ketoglutarate. Further evaluation of this proposal is anticipated in the near future. 

HETPP acronym of hydroxyethylthiamin pyrophosphate. see Thiamin pyrophosphate. 

Ziporin and Waring (1970) used two-dimensional TLC to separate thiamine and its metabolites and precursor compounds, including hydroxyethylthiamine, pyrimidine, thiazole fragments of thiamine, carboxylic acid, and sulfonic deriva-... 

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