Carbanions enamine

The carbanion/enamine is subsequently protonated via aspartic acid. 

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... 

These differences in the control of the product stereochemistry have recently been investigated by molecular modeling techniques [60,154], From these studies, the relevance of the side-chain of isoleucine 476 (PDCS.c.) (Table 2) for the stereo-control during the formation of aromatic a-hydroxy ketones became obvious, since this side-chain may protect one site of the ot-carbanion/enamine 6 (Scheme 3) against the bulky aromatic cosubstrate. Nevertheless, the smaller methyl group of acetaldehyde can bind to both sites of the a-carbanion/en-amine. The preference for one of the two acetoin enantiomers has been interpre-tated in terms of different Boltzmann distributions between the two binding modes of the bound acetaldehyde [155],... 

Although the reactivity of the C2-carbanion is crucial to initiating the reaction, the chemistry of the second C2a-carbanion/enamine is much richer in biochemical systems, as there are known three distinct enzymatic pathways that emanate from the enamine protonation12, oxidation13-15 and condensation16, each known to occur both on enzymes and in appropriate model reactions. 

If the nucleophile is very reactive, the preferred agent for trifluoromethylation is the dimethyl derivative 229b, which is not very powerful. Less reactive substrates were trifluoromethylated in reasonably good yields by the more powerful dinitro salt 230. The unsubstituted salt 229a is intermediate in its trifluoromethylating power. Carbanions, enamines, enol trimethylsilyl ethers, aniline, phenol, and pyrrole have all been successfully trifluoromethylated at their respective nucleophilic carbon atoms (Equations 112-117). 

In these reactions, the C2-atom of ThDP must be deprotonated to allo v this atom to attack the carbonyl carbon of the different substrates. In all ThDP-dependent enzymes this nucleophilic attack of the deprotonated C2-atom of the coenzyme on the substrates results in the formation of a covalent adduct at the C2-atom of the thiazolium ring of the cofactor (Ila and Ilb in Scheme 16.1). This reaction requires protonation of the carbonyl oxygen of the substrate and sterical orientation of the substituents. In the next step during catalysis either CO2, as in the case of decarboxylating enzymes, or an aldo sugar, as in the case of transketo-lase, is eliminated, accompanied by the formation of an a-carbanion/enamine intermediate (Ilia and Illb in Scheme 16.1). Dependent on the enzyme this intermediate reacts either by elimination of an aldehyde, such as in pyruvate decarboxylase, or with a second substrate, such as in transketolase and acetohydroxyacid synthase. In these reaction steps proton transfer reactions are involved. Furthermore, the a-carbanion/enamine intermediate (Ilia in Scheme 16.1) can be oxidized in enzymes containing a second cofactor, such as in the a-ketoacid dehydrogenases and pyruvate oxidases. In principal, this oxidation reaction corresponds to a hydride transfer reaction. 

In the next section, the mechanism of the C2-H deprotonation of ThDP in enzymes is considered, followed by a discussion of the proton transfer reactions during catalysis. Finally, the oxidation mechanism of the a-carbanion/enamine intermediate in pyruvate oxidase is discussed. 

A mechamsm that involves protonation of the a-carbanion/enamine of HEThDP and subsequent hydride transfer, which has been proposed for several flavine-dependent enzymes (Pollegioni et al., 1997), is unlikely since no kinetic solvent isotope effect is evident for this catalytic step (Pig. 16.7). In accordance, after replacement of PAD by 5-carba-5-deaza-FAD, a PAD analog not catalyzing a transfer of single electrons but functioning as hydride acceptor, no reduction is observed by the HEThDP intermediate in pyruvate oxidase from Lactobacillus plantarum (Tittmann et al., 1998). 

Mesecar, A. D., Stoddard, B. L., Koshland Jr., D. E. (1997). Orbital steering in the catalytic power of enzymes small structural changes with large catalytic consequences, Science, 277, 202-206. Recent example Meyer, D., Neumann, R, Parthier, C., Friedemann, R., Nemeria, N., Jordan, F., Tittmann, K. (2010). Double Duty for a Conserved Glutamate in Pyruvate Decarboxylase Evidence of the Participation in Stereoelectronically Controlled Decarboxylation and in Protonation of the Nascent Carbanion/Enamine Intermediate. Biochemistry, 49, 8197-8212. 

Transketolase (TK) is involved in anaerobic carbohydrate metabolisms such as the nonoxidative phase of the pentose phosphate pathway. In plants and photosynthetic bacteria, TK is involved in the Calvin-Benson cycle. TK catalyses the transfer of a 2-carbon dihydroxyethyl group from a ketose phosphate (donor substrate such as D-xylulose 5-phosphate) to the Cl position of an aldose phosphate (acceptor substrate such as o-ribose 5-phosphate) (Figure 4.3) (Schneider and Lindqvist 1998). The first product is an aldose phosphate released from the donor (such as glyceraldehyde 3-phosphate) and the second is a ketose phosphate (such as sedoheptulose 7-phosphate), in which the 2-carbon fragment is attached to the acceptor. Examples of the substrates and the products mentioned above are for the first reaction of the pentose phosphate pathway. In the second reaction of the same pathway, the acceptor is D-ery-throse 4-phosphate and the second product is o-fructose 6-phosphate. A snapshot X-ray crystallographic study revealed that an ot-carbanion/enamine a,p-dihydroxyethyl ThDP is formed as a key intermediate (Fiedler et al. 2002). Then, a nucleophilic attack of the a-carbanion intermediate on the acceptor substrate occurs. 

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