Thiamine dehydrogenase

Until recently, it was not known that flavoproteins contain also covalently bound phosphate. A. vinelandii flavodoxin, L-amino acid oxidase, glucose oxidase, NADPH-cytochrome c reductase, thiamine dehydrogenase possess one mole of covalently bound phosphate/per mole protein function of these phosphate residues... 

Enzymes of Still Undefined Specificity The nature of the specific acceptor is unknown for choline-dehydrogenase, thiamine-dehydrogenase, and succinic dehydrogenase which is a metalloflavoprotein containing iron. 

The same flavin occurs at the active center of bacterial thiamine dehydrogenase (Singer and Kenney, 1974, Kenney et al., 1974b) and in L-gulono-y-lactone oxidase from rat liver (Kenney et al., 1976b). 

A similar type of linkage between coenzyme and protein in which position 8a of the flavin is at the oxidation level of carbonyl was found in thiamine dehydrogenase (from soil bacterial 138) and in P-cyclo-piazonate oxidocyclase from Penicillium cyclopium 88, 155). In these cases, however, it is probable that N(l) of a histidine residue constitutes the bridge to the protein backbone. The oxidation level of position 8a was deduced from decay data similar to those mentioned above for the 8a-thiohemiacetals. Of particular interest is the fluorescence profile of the flavin peptide obtained by trypsin-chymotrypsin hydrolysis of p-cyclopiazonate oxidocyclase. Fluorescence quenching similar to that observed with SD-flavin (9) is observed, but in this case the pK attributed to protonation of the histidine imidazole is shifted from 4.7 to 5.4, and the maximal fluorescence obtained is only 20% of that of FMN 164). Upon performic acid oxidation of the peptide the emission intensity is... 

Kenney, W. C., D. E. Edmondson, and T. P. Singer A Novel Form of Covalently Bound Flavin from Thiamine Dehydrogenase. Biochem. Biophys. Res. Commun. 57, 106 (1974). 

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 mechanism of the pyruvate dehydrogenase reaction is a tour de force of mechanistic chemistry, involving as it does a total of three enzymes (a) and five different coenzymes—thiamine pyrophosphate, lipoic acid, coenzyme A, FAD, and NAD (b). 

Based on the action of thiamine pyrophosphate in catalysis of the pyruvate dehydrogenase reaction, suggest a suitable chemical mechanism for the pyruvate decarboxylase reaction in yeast ... 

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.)...

Pyruvate is oxidized to acetyl-GoA by a multienzyme complex, pyruvate dehydrogenase, that is dependent on the vitamin cofactor thiamin diphosphate. 

B, Thiamin Coenzyme in pyruvate and a-ketoglutarate, dehydrogenases, and transketolase poorly defined function in nerve conduction Peripheral nerve damage (beriberi) or central nervous system lesions (Wernicke-Korsakoff syndrome)... 

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-... 

A somewhat more trivial thing to remember about the HMP pathway is that this is one of the places you ve seen the vitamin thiamin pyrophosphate. This cofactor is necessary for the transketolase reaction that is in the middle of the HMP pathway. The transketolase reaction converts two C-5 sugars to a C-7 and a C-3. The other place you ve seen thiamin pyrophosphate as a cofactor is in the pyruvate dehydrogenase and a-ketoglutarate dehydrogenase reactions. 

Thiamine deficiency results in early decreases in activity of the mitochondrial enzyme a-ketoglutarate dehydrogenase in brain. Wernicke s encephalopathy, also known as the Wernicke-Korsakoff syndrome is a neuropsychiatric disorder characterized by ophthalmoplegia, ataxia and memory loss. Wernicke s encephalopathy is encountered in chronic alcoholism, in patients with HIV-AIDS and in other disorders associated with grossly impaired nutritional status. The condition results from thiamine deficiency. 

In the 1930s, Peters and co-workers showed that thiamine deficiency in pigeons resulted in the accumulation of lactate in the brainstem [ 15]. Furthermore, they showed that the addition of small quantities of crystalline thiamine to the isolated brainstem tissue from thiamine-deficient birds in vitro resulted in normalization of lactate levels. These findings led to the formulation of the concept of the biochemical lesion in thiamine deficiency. Subsequent studies showed that the enzyme defect responsible for the biochemical lesion was a-KGDH rather than pyruvate dehydrogenase (PHDC), as had previously been presumed. a-KGDH and PHDC are major thiamine diphosphate (TDP)-dependent enzymes involved in brain glucose oxidation (Fig. 34-4). 

Rare patients respond to the administration of thiamine in large doses (10-30mg/day). The clinical course is even more mild than that of patients with intermittent disease. Thiamine is a cofactor for the branched-chain ketoacid dehydrogenase, and the presumed mutation involves faulty binding of the apoprotein to this vitamin. 

The PDHC catalyzes the irreversible conversion of pyruvate to acetyl-CoA (Fig. 42-3) and is dependent on thiamine and lipoic acid as cofactors (see Ch. 35). The complex has five enzymes three subserving a catalytic function and two subserving a regulatory role. The catalytic components include PDH, El dihydrolipoyl trans-acetylase, E2 and dihydrolipoyl dehydrogenase, E3. The two regulatory enzymes include PDH-specific kinase and phospho-PDH-specific phosphatase. The multienzyme complex contains nine protein subunits, including... 

The way in which thiamine participated in the oxidation of pyruvate became clearer when Lohmann and Schuster (1937) showed vitamin Bj to be present intracellularly as thiamine pyrophosphate. In yeast, decarboxylation of pyruvate yielded ethanal which was reduced by alcohol dehydrogenase to give ethanol. A cofactor was needed for this decarboxylation, co-carboxylase. Like the cofactor needed in animal cells for the decarboxylation of pyruvate, cocarboxylase was found to be identical to thiamine pyrophosphate. Vitamin Bj thus became the first vitamin whose intracellular function as a coenzyme had been established in vitro. Another aphorism therefore arose about vitamins—B vitamins are (parts of) coenzymes—an idea that was to be completely confirmed. 

Thiamine (Bj) Pyruvate dehydrogenase PDH MCC alcoholism (alcohol interferes... 

Two other enzyme complexes similar to pyruvate dehydrogenase that use thiamine are ... 

---