Catalysis by thiamine

P. A. Frey, Chemical Intermediates In Catalysis by Thiamine Diphosphate. In Thiamine. Cataiytic Mechanisms in Normai and Disease States F. Jordan, M. S. Patel, Eds. Marcel Dekker, Inc. New York, 2004. 

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. 

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. 

Thiamine is synthesized biologically from the pyrimidine derivative 4-amino-5-hydroxymethyl-2-mcthyl pyrimidine methylpyrimidine and 5-( hydtoxycthyl)-4-methylthia-zole. These two precursors are converted to phosphate derivatives under kinase catalysis, which requires ATP. The respective phosphate derivatives then interact to form thiamine phosphate in a reaction catalyzed by thiamine phosphate pyr-ophosphotyla.se. 

Jordan, F., Zhang, Z., Seegienko, E. (2002), Spectroscopic evidence for participation of the l, 4 -imino tautomer of thiamin diphosphate in catalysis by yeast pymvate decarboxylase, Bioorg. Chem. 30, 188-198. 

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

The finding that thiamine, and even simple thiazolium ring derivatives, can perform many reactions in the absence of the host apoenzyme has allowed detailed analyses of its chemistry [33, 34]. In 1958 Breslow first proposed a mechanism for thiamine catalysis to this day, this mechanism remains as the generally accepted model [35]. NMR deuterium exchange experiments were enlisted to show that the thiazolium C2-proton of thiamine was exchangeable, suggesting that a carbanion zwitterion could be formed at that center. This nucleophilic carbanion was proposed to interact with sites in the substrates. The thiazolium thus acts as an electron sink to stabilize a carbonyl carbanion generated by deprotonation of an aldehydic carbon or decarboxylation of an a-keto acid. The nucleophilic carbonyl equivalent could then react with other electro-... 

Figure 14-2 (A) Stereoscopic view of the active site of pyruvate oxidase from the bacterium Lactobacillus plantarium showing the thiamin diphosphate as well as the flavin part of the bound FAD. The planar structure of the part of the intermediate enamine that arises from pyruvate is shown by dotted lines. Only some residues that may be important for catalysis are displayed G35 , S36 , E59 , H89 , F12T, Q122 , R264, F479, and E483. Courtesy of Georg E. Schulz.119 (B) Simplified view with some atoms labeled and some side chains omitted. The atoms of the hypothetical enamine that are formed from pyruvate, by decarboxylation, are shown in green.

First steps to elucidate the reaction mechanism of PDC were achieved by the investigation of model reactions using ThDP or thiamine [36,37], Besides the identification of C2-ThDP as the catalytic center of the cofactor [36], the mechanism of the ThDP-catalyzed decarboxylation of a-keto acids as well as the formation of acyloins was explained by the formation of a common reaction intermediate, active acetaldehyde . This active species was first identified as HEThDP 7 (Scheme 3) [38,39]. Later studies revealed the a-carbanion/enamine 6 as the most likely candidate for the active acetaldehyde [40 47] (for a comprehensive review see [48]). The relevance of different functional groups in the ThDP-molecule for the enzymatic catalysis was elucidated by site-directed substitutions of the cofactor ThDP by chemical means (for a review see... 

It is the purpose of this review to discuss some of the better known biological enamines. Due to the preoccupation of the author with thiamin chemistry and enzymology, many of the properties of enamines will be exemplified by a variety of results obtained on this particular system. Enamines are, however, present in a number of other biochemical processes as well, where their remarkably rich chemistry is required. In most of these reactions the enamines are only present as intermediates, that are the result of covalent catalysis, in which the substrate of the enzymatic reaction forms a covalent bond with either a protein side chain or a required coenzyme. 

Referring to a mechanistic classification of organocatalysts (Seayad and List 2005), currently the two most prominent classes are Brpnsted acid catalysts and Lewis base catalysts. Within the latter class chiral secondary amines (enamine, iminium, dienamine activation for a short review please refer to List 2006) play an important role and can be considered as—by now—already widely extended mimetics of type I aldolases, whereas acylation catalysts, for example, refer to hydrolases or peptidases (Spivey and McDaid 2007). Thiamine-dependent enzymes, a versatile class of C-C bond forming and destructing biocatalysts (Pohl et al. 2002) with their common catalytically active coenzyme thiamine (vitamin Bi), are understood to be the biomimetic roots ofcar-bene catalysis, a further class of nucleophilic, Lewis base catalysis with increasing importance in the last 5 years. 

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. 

---