Hydroxyethylthiamine pyrophosphate

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). 

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. 

The properties of pyruvate decarboxylase have been extensively studied particularly with respect to its catal3dic mechanism involving the enzyme-bound reaction intermediate 2a-hydroxyethylthiamine pyrophosphate (HETPP) [24-26]. The ability of the coenzyme (TPP) and more particularly its thiazolium ring to bind to the carbonyl group and act as an electronic sink makes the decarboxylation of an a-keto acid possible. This is because decarboxylation of an a-keto acid requires the buildup of negative charge on the carbonyl atom in the transition state. The reaction mechanism of pyruvate decarboxylase is described by Voet and Voet [27] and is based on isolation and identification of die HETPP intermediate. 

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