Thiamine pyrophosphate catalysis

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 ... 

The next coenzyme for which a mechanism was established was thiamin pyrophosphate [3]. Ronald Breslow used nmr spectroscopy to show that the hydrogen atom at C-2 of a thiazolium salt rapidly exchanges with deuterium in even slightly alkaline solutions (6), so that the coenzyme offers an anionic centre for catalysis (Breslow, 1957). With this established, Breslow could confidently offer the pathway shown in Scheme 2 for the action of the... 

Nucleophilic catalysis is a specific example of covalent catalysis the substrate is transiently modified by formation of a covalent bond with the catalyst to give a reactive intermediate. There are also many examples of electrophilic catalysis by covalent modification. It will be seen later that in the reactions of pyridoxal phosphate, Schiff base formation, and thiamine pyrophosphate, electrons are stabilized by delocalization. 

Effective concentration 65-72 entropy and 68-72 in general-acid-base catalysis 66 in nucleophilic catalysis 66 Elastase 26-30, 40 acylenzyme 27, 40 binding energies of subsites 356, 357 binding site 26-30 kinetic constants for peptide hydrolysis 357 specificity 27 Electrophiles 276 Electrophilic catalysis 61 metal ions 74-77 pyridoxal phosphate 79-82 Schiff bases 77-82 thiamine pyrophosphate 82-84 Electrostatic catalysis 61, 73, 74,498 Electrostatic effects on enzyme-substrate association rates 159-161... 

Decarboxylases (PDC, ADC) decarboxylase carboligation thiamine pyrophosphate (TPP) catalysis Bruhn, 1995... 

The ALS isolated as described in Table III displayed typical Michaelis-Menten kinetics with respect to pyruvate with a Km of 2.44 mM. Substrate concentrations as high as 50x Km had no effect on the rate of the reaction. Thiamine pyrophosphate, FAD and Mg(2+) were an absolute requirement for catalysis by the purified enzyme. These properties are consistent with observations made by others (30). Optimum activity was obtained at pH 7.1 and 37C, which were also the best conditions for inhibition by TP. There was no significant difference in the 1(50) value of TP whether ALS was taken after step 2 or 5, indicating low potential for non-specific binding of the herbicide to other proteins. 

G3P) and D-sedoheptulose 7-P as illustrated in Scheme 5.53. In addition D-erythrose 4-phosphate can function as the ketol acceptor thus producing D-fructose-6-P and G3P (Scheme 5.53). The enzyme relies on two cofactors for activity — thiamin pyrophosphate (TPP) and Mg2+—and utilizes the nucleophilic catalysis mechanism outlined in (Scheme 5.54).83 When TPP is used as a cofactor for nucleophilic catalysis, an activated aldehyde intermediate is formed. This intermediate functions as a nucleophile, and thus TK employs a strategy that is similar to the umpolung strategy exploited in synthetic organic chemistry. 

Several coenzymes are involved in the biosynthesis of their own precursors. Thus, thiamine is the cofactor of the enzyme that converts 1-deoxy-D-xylulose 5-phosphate (43) (the precursor of thiamine pyrophosphate, pyridoxal 5 -phosphate and of iso-prenoids via the nomnevalonate pathway) into 2 C-methyl-D-erythritol 4-phosphate (90, Fig. 11). Similarly, two enzymes required for the biosynthesis of GTP, which is the precursor of tetrahydrofolate, require tetrahydrofolate derivatives as cofactors (Fig. 3). When a given coenzyme is involved in its own biosynthesis, we are faced with a hen and egg problem, namely how the biosynthesis could have evolved in the absence of the cmcially required final product. The answers to that question must remain speculative. The final product may have been formed via an alternative biosynthetic pathway that has been abandoned in later phases of evolution or that may persist in certain organisms but remains to be discovered. Alternatively, the coenzyme under study may have been accessible by a prebiotic sequence of spontaneous reactions. An interesting example in this respect is the biosynthesis of flavin coenzymes, in which several reaction steps can proceed without enzyme catalysis despite their mechanistic complexity. 

Coenzymes - Many enzymes require nonprotein coenzymes for catalytic activity.8 These are cosubstrates, and must be constantly reconverted into their active form for catalysis to continue. This is not a problem for growing microorganisms since the normal metabolic processes ensure an adequate supply of coenzymes. However, with purified, or immobilized enzymes, maintaining a sufficient concentration of coenzyme can pose a major problem. Coenzymes are expensive and it is seldom economically feasible to add them in stoichiometric amounts. This is often undesirable for chemical reasons, e.g., the coenzyme may be unstable, or the eventual build-up of high concentrations of its inactive form may Induce displacement of an equilibrium reaction in the opposite direction to that desired.3 It is therefore necessary to use catalytic amounts of coenzymes and to ensure that the active forms are continuously regenerated. Some coenzymes present little or no problem in this regard since they are automatically reformed under the normal aqueous reaction conditions or in the presence of oxygen. These include biotin, pyrldoxal phosphate (PLP), thiamine pyrophosphate, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).1 ... 

Kluger R, Gish G (1988) Stereochemical aspects of thiamin catalysis. In Schellenberg A, Schowen RL (eds) Thiamin pyrophosphate biochemistry. CRC Press, Boca Raton, Florida, chap 1... 

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. 

Fig. 8.11. The role of the functional group of thiamine pyrophosphate (the reactive carbon shown in blue) in formation of a covalent intermediate. A. A base on the enzyme (B) abstracts a proton from thiamine, creating a carbanion (general acid-base catalysis). B. The carbanion is a strong nucleophile and attacks the partially positively charged keto group on the substrate. C. A covalent intermediate is formed, which is stabilized by resonance forms. The uncharged intermediate is the stabilized transition state complex.

Thiamine pyrophosphate is a coenzyme in the transfer of two-carbon units. It is required for catalysis by pyruvate decarboxylase in alcoholic fermentation. 

A variety of models have been developed to study TD. TD in rodents can be produced in multiple ways. Simple deprivation of thiamine will deplete thiamine and thiamine-dependent processes. However, this expands the time until the symptoms occur, and increases the variability for time of the onset of the symptoms. Injection of inhibitors of thiamine utilization in conjunction with the thiamine deficient diet shortens the time until onset of symptoms and provides a remarkably reproducible model. Pyrithiamine, which is structurally similar to thiamine, blocks the thiamine pyrophospho kinase, which catalysis the phosphorylation of thiamine to thiamine pyrophosphate (TPP) so that the production of the metabolically active form of thiamine, TPP, is impaired. Pyrithiamine readily crosses the blood brain barrier so that TD is produced in the brain and in the periphery. On the other hand, oxythiamine does not cross the BBB and only produces TD in the periphery. The precise timing of the acute and chronic changes in TD varies with the model. All the models lead to diminished food intake, so, often paired fed controls are used. These have never shown that pathology is related to TD. 

In addition to the preparatively useful aldolases, several mechanistically distinct enzymes can be employed for synthesis of product structures identical w ith those accessible from aldolase catalysis. Such alternative enzymes (e.g. transketolase), w hich are actually categorized as transferases but also catalyze aldol-related additions w ith the aid of cofactors (Eigure 5.4) such as pyridoxal 5-phosphate (PEP), thiamine pyrophosphate (TPP), or tetra-hydrofolate (THE), are emerging as useful catalysts in organic synthesis. Because these operations often extend and/or complement the synthetic strategies open to aldolases, a selection of such enzymes and examples of their synthetic utility are included also in this overview . 

Real Life 18-1 described an aldol-based biosynthesis of carbohydrates, and Real Life 23-2 introduced thiamine pyrophosphate (TPP) in its role in the metabolism of glucose to produce biochemical energy. Here we illustrate another biochemical sequence that uses thiamine catalysis to interconvert ketoses. 

Very different prosthetic groups that are involved in enzymatic catalysis and that are usually covalently linked or very tightly bound to the enzyme can be found. However, all of those carrying phosphorus are part of the class of the B vitamins (B, B2, B, and 6,2). For example, vitamin B (thiamin) is the precursor for synthesis of the prosthetic group thiamin pyrophosphate. It functions as a coenzyme, usually in conjunction with Mg2+, which is thought to complex to the pyrophosphate group (Chauvet-Monges et ai, 1981) in enzyme-catalyzed reactions of aldehyde removal or transfer. 

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