Thiamine, coenzyme forms

Figure 1 shows the chemical structure ofvitamin B1 or thiamin (3-(4-amino-2-methyl-pyrimidin-5-ylmethyl)-5-(2-hydroxyethyl)-4-methylthiazolium) and its coenzyme form thiaminpyrophosphate (TPP). 

Vitamin B1. Figure 1 Structure of thiamin and its coenzyme form thiaminpyrophosphate (TPP). 

Vitamin deficiency of Bj leads to the disease known as Beriberi. However, nowadays in the Western hemisphere, vitamin Bj deficiency is mainly found as a consequence of extreme alcoholism. In fact, the vitamin absorption by the gut is decreased and its excretion is increased by alcohol. Alcohol also inhibits the activation of vitamin Bj to its coenzyme form, thiamine pyrophosphate ester (TPP). There is no evidence of adverse effects of oral intake of thiamine [417]. The main food sources of vitamin Bj are lean pork, legumes, and cereal grains (germ fraction). It is soluble in water and stable at higher temperature and at pH lower than 5.0, but it is destroyed rapidly by boiling at pH 7.0 or above. 

The weakly basic portion of thiamin or of its coenzyme forms is protonated at low pH, largely on N-l of the pyrimidine ring. 86 88 The pKa value is 4.9. In basic solution, thiamin reacts in two steps with an opening of the thiazole ring (Eq. 14-15) to give the anion of a thiol form which may be crystallized as the sodium salt.79 84 This reaction, like the competing reaction described in Eq. 7-19, and which leads to a yellow... 

Thiamin is synthesized in bacteria, fungi, and plants from 1-deoxyxylulose 5-phosphate (Eq. 25-21), which is also an intermediate in the nonmevalonate pathway of polyprenyl synthesis. However, thiamin diphosphate is a coenzyme for synthesis of this intermediate (p. 736), suggesting that an alternative pathway must also exist. Each of the two rings of thiamin is formed separately as the esters 4-amino-5-hydroxy-methylpyrimidine diphosphate and 4-methyl-5-((i-hydroxyethyl) thiazole monophosphate. These precursors are joined with displacement of pyrophosphate to form thiamin monophosphate.92b In eukaryotes this is hydrolyzed to thiamin, then converted to thiamin diphosphate by transfer of a diphospho group from ATP.92b c In bacteria thiamin monophosphate is converted to the diphosphate by ATP and thiamin monophosphate kinase.92b... 

In practice, donor substituents make it possible actually to isolate a range of carbenes 4.105. With somewhat less stabilisation, the carbene 4.106, although it is only found as a reactive intermediate, is exceptionally easy to form. It is the key intermediate in all the metabolic steps catalysed by thiamine coenzymes, and its reactions are characterised by its nucleophilicity. Similarly, dimethoxycarbene 4.107 reacts as a nucleophile with electrophiles like dimethyl maleate to give the intermediate 4.108, and hence the cyclopropane 4.109, but it does not insert into unactivated alkenes. 

In the tissues of animals, most thiamine is found as its phosphorylated esters (4—6) and is predominantly bound to enzymes as the pyrophosphate (5), the active coenzyme form. As expected for a factor involved in carbohydrate metaboHsm, the highest concentrations are generally found in oigans with high activity, such as the heart, kidney, Hver, and brain. In humans this typically amounts to 1—8 lg/g of wet tissue, with lesser amounts in the skeletal muscles (35). A typical healthy human body may contain about 30 mg of thiamine in all forms, about 40—50% of this being in the muscles owing to their bulk. Almost no excess is stored. Normal human blood contains about 90 ng/mL, mosdy in the red cells and leukocytes. A value below 40 ng/mL is considered indicative of a possible deficiency. Amounts and proportions in the tissues of other animal species vary widely (31,35). 

Some of the vitamins in the coenzyme form associate tightly with specific enzymes, but not via a covalent linkage. Immediately after biosynthesis on the ribosome, enzymes do not contain their cofactor, and these are called apoenzymes. An enzyme containing its required cofactor is called a holoenzyme. With removal of the cofactor, the enzyme is also called an apoenzyme. The enzymes that exist in apoenzyme and holoenzyme forms include those that use vitamin B12/ vitamin B, thiamin, and riboflavin-based cofactors. Enz)mies that use niacin-based cofactors, folate, ascorbate, and vitamin K are not said to exist in apoenzyme and holoenzyme forms. These enzymes bind their cofactors relatively weakly, and the cofactors behave in a manner similar to substrates. 

This vitamin, also called aneurin, is the antiberiberi factor. The active coenzyme form is thiamine pyrophosphate (TPP), or coearboxylase. Thiamine triphosphate (TTP) may be an active form in the central nervous system. Of the thiamine in the body, 10% occurs as TTP, 80% as TPP, and 10% as TMP (thiamine monophosphate) (Figure 38-12). 

The answer is b. (Murray, pp 627-661. Scriver, pp 3897-3964. Sack, pp 121-138. Wilson, pp 287-320.) Nicotinamide adenine dinucleotide (NAD+) is the functional coenzyme derivative of niacin. It is the major electron acceptor in the oxidation of molecules, generating NADH, which is the major electron donor for reduction reactions. Thiamine (also known as vitamin Bi) occurs functionally as thiamine pyrophosphate and is a coenzyme for enzymes such as pyruvate dehydrogenase. Riboflavin (vitamin B2) functions in the coenzyme forms of flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD). When concentrated, both have a yellow color due to the riboflavin they contain. Both function as prosthetic groups of oxidation-reduction enzymes or flavoproteins. Flavoproteins are active in selected oxidation reactions and in electron transport, but they do not have the ubiquitous role of NAD+. 

Thiamin contains a substituted thiazole group which is involved in the decarboxylation of pyruvate by pyruvate decarboxylase. Thiamin occurs in cells largely as its active coenzyme form thiamin pyrophosphate (TPP) (Fig. 7.14). 

The cardiomyopathy is directly related to a reduction in the normal biochemical function of the vitamin thiamine in heart muscle. Inhibition of the a-keto acid dehydrogenase complexes causes accumulation of a-keto acids in heart muscle (and in blood), resulting in a chemically-induced cardiomyopathy. Impairment of two other functions of thiamine may also contribute to the cardiomyopathy. Thiamine pyrophosphate serves as the coenzyme for transketolase in the pentose phosphate pathway, and pentose phosphates accumulate in thiamine deficiency. In addition, thiamine triphosphate (a different coenzyme form) may function in Na conductance channels. 

Immediate treatment with large doses (50-KX) mg) of intravenous thiamine may produce a measurable decrease in cardiac output and increase in peripheral vascular resistance as early as 30 minutes after the initial injection. Dietary supplementation of thiamine is not as effective because ethanol consumption interferes with thiamine absorption. Because ethanol also affects the absorption of most water-soluble vitamins, or their conversion to the coenzyme form, Al Martini was also given a bolus containing a multivitamin supplement. 

Vitamin B, is thiamine (ll.lOSd), which is often encountered in coenzyme form as thiamine pyrophosphate. It can be made by phosphorylation of thiamine with ATP (11.105). This anti-beri-beri vitamin was first isolated and synthesised in 1936. Thiamine is found as free B, mostly in plants. It usually occurs in pyrophosphate form in yeast and in tissue. 

Biosynthesis of vitamin B, and its coenzyme form, thiamin pyrophosphate. HMP = 2-methyl-6-8mino-S-hydroxymethylpyrimidine. HET = 4-methyl-5-hydroxyethylthiazole. 

Figure 1.1 Thiamin (Vitamin Bi) and thiamin diphosphate. The diphosphate ester of thiamin is the coenzyme form of thiamin.

Reed and De Busk have suggested that the true coenzyme form of lipoic acid is a conjugate of a-lipoic acid and thiamine pyrophosphate, given the name lipothiamide. The bond between the two is presumed to be a peptide bond between the amino group on Ci of the pyrimidine moiety of thiamine and the carboxyl group of a-lipoic acid. The true coenzyme is reported to be the lipothiamide pyrophosphate. 

From a nutritional standpoint, it is significant that five of the B-complex vitamins (riboflavin, nicotinamide, thiamine, vitamin Be, and pantothenic acid) have been shown to be constituents of the coenzymes. The nutritional requirement of these vitamins is explained on the basis of their coenzyme function. In all cases the coenzyme form appears to be the sole bound form of the vitamin, and this then becomes the only metabolically active form for these particular vitamins. 

Recently Reed and DeBusk have isolated from natural materials a bound form of lipoic acid which seems to be somewhat closer to the coenzyme form. Analysis of this compound as well as its preparation from synthetic materials indicates that it is the amide of thiamine and lipoic acid (lipothiamide). This important discovery may eventually cast some light upon the mechanism of action of lipoic acid. It is significant that lipothiamide can restore pyruvate oxidation to deficient cells, and the authors conclude that lipothiamide may be part of the coenzyme for the oxidative decarboxylation of a-keto acids. 

The structures of the phosphate esters of thiamine are also shown in Figure 1. Thiamine monophosphate (TMP), thiamine pyrophosphate (TPP), and thiamine triphosphate (TTP) are commonly found in organisms. About 80% to 90% of the total thiamine content in cells is TPP, the coenzyme form of thiamine. In some animal tissues, especially pig skeletal muscle (2) and chicken white skeletal muscle (3), TTP is present in an extremely high amount (70% to 80% of total thiamine—i.e., thiamine plus thiamine phosphate esters). However, TTP has no coenzyme activity. Thiamine pyrophosphate in the dried state is stable for several months when stored at a low temperature in the dark. In solution, TPP is unstable and partially decomposes to TMP and/or thiamine when stored for several months at pH 5 and 38°C. However, TPP in solution at pH 2 to 6 and at 0°C is... 

With a mobile phase of 0.2 M ammonium phosphate buffer (pH 5.1) in a reversed-phase system, folate, pyridoxine, nicotinamide, and thiamine could be separated, in this order, within 20 min, followed by vitamin Bn and riboflavin (17). The latter two compounds eluted after a step gradient to 30% aqueous methanol. This procedure has the advantage of completely separating at least nine coenzyme forms of water-soluble vitamins, including TPP. A similar mobile phase, composed of methanol-water (50 50) was used in connection with a LiCh-rosorb RP-18 column to separate thiamine, pyridoxine, vitamin and riboflavin in 3 min, as shown in Figure 4 (23). The detection limit at 254 nm is 5 ng (15 pmol) for thiamine and 10 to 20 ng for the other three vitamins, with a coefficient of variation of <4%. 

FIGURE 2 The vitamin thiamin and its coenzyme form thiamin diphosphate (thiamin pyrophosphate). 

Most coenzymes have aromatic heterocycles as major constituents. While enzymes possess purely protein structures, coenzymes incorporate non-amino acid moieties, most of them aromatic nitrogen het-erocycles. Coenzymes are essential for the redox biochemical transformations, e.g., nicotinamide adenine dinucleotide (NAD, 13) and flavin adenine dinucleotide (FAD, 14) (Scheme 5). Both are hydrogen transporters through their tautomeric forms that allow hydrogen uptake at the termini of the quinon-oid chain. Thiamine pyrophosphate (15) is a coenzyme that assists the decarboxylation of pyruvic acid, a very important biologic reaction (Scheme 6). 

Not all vitamin coenzymes need to be in the form of a nucleotide (base, sugar, phosphate). For example thiamine biotin pyridoxine vitamin B12. 

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. 

So what does riboflavin do As such riboflavin does nothing. Like thiamine, riboflavin must undergo metabolic change to become effective as a coenzyme. It fact, it undergoes two reactions. The first converts riboflavin to riboflavin-5-phosphate (commonly known as flavin adenine mononucleotide, FMN), about which we will say no more, and the second converts it to flavin adenine dinucleotide, FAD. The flavins are a class of redox agents of very general importance in biochemistry. FAD is the oxidized form and FADH2 is the reduced form. ... 

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