Pyruvate decarboxylase 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 ... 

In order to increase the understanding of ThDP-dependent enzymes, the identification of amino acid side chains important for the catalysis of the carboligase reaction in pyruvate decarboxylase from Zymomonas mohilis (E.C. 4.1.1.1) and benzoylformate decarboxylase from Pseudomonasputida (E.C. 4.1.1.7) was a major task. Using site-directed mutagenesis and directed evolution, various enzyme variants were obtained, differing in substrate specificity and enantioselectivity. 

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. 

On the basis of the crystal structure of a Bacillus stearothermophilus pyruvate dehydrogenase subcomplex formed between the heterotetrameric El and the peripheral subunit binding domain of E2 with an evident stmctural dissymmetry of the two active sites, a direct active center communication via an acidic proton tunnel has been proposed (Frank et ak, 2004). According to this, one active site is in a closed state with an activated cofactor even before a substrate molecule is engaged, whereas the activation of the second active site is coupled to decarboxylation in the first site. Our own kinetic NMR studies on human PDH El (unpublished) support the model suggested, but similar studies on related thiamin enzymes, such as pyruvate decarboxylase, transketolase or pyruvate oxidase reveal that half-of-the-sites reactivity is a unique feature of ketoacid dehydrogenases. In line with this. X-ray crystallography studies on intermediates in transketolase catalysis indicated an active site occupancy close to unity in both active sites (Fiedler et al., 2002 and G. Schneider, personal communication). 

Table 16.4. Microscopic rate constants of catalysis in pyruvate decarboxylase wildtype and variants...

Linkage of catalysis and regulation in enzyme action — carbon isotope effects, solvent isotope effects, and proton inventories for the unregulated pyruvate decarboxylase of Zymomonas mohilis, J. Am. Chem. Soc. 117, 7317-7322. 

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

Although the utility of transaminases has been widely examined, one snch limitation is the fact that the equilibrium constant for the reaction is aronnd one. A shift in this eqnilibrinm is necessary, therefore, for the reaction to be synthetically usefnl. " Aspartate, when used as the amino donor, is converted into oxaloacetate (26) upon reaction (Scheme 9.29). Because 26 is unstable, it decomposes to pyrnvate (27) and thus favors product formation. However, because pyruvate is also an a-keto acid, it conld be a substrate and be transaminated into alanine. The enzyme acetolactate synthase, which condenses two moles of pyrnvate to form (5)-acetolactate (28), is, therefore, inclnded in the reaction. The (5)-acetolactate can nndergo decarboxylation either spontaneously or by the enzyme catalysis with acetolactate decarboxylase to give the final by-prodnct, (/ )-acetoin... 

Other examples of electrophilic metal catalysis are given under section 2.3.3.1. Electrophilic reactions are also carried out by enzymes which have an a-keto acid (pyruvic acid or a-keto butyric acid) at the transforming locus of the active site. One example of such an enzyme is histidine decarboxylase in which the N-terminal amino acid residue is bound to pyruvate. Histidine decarboxylation is initiated by the formation of a Schiff base by the reaction mechanism in Fig. 2.20. 

Under the catalysis of ligases of the acid-thiol type, 2-oxoacids are transformed into acyl-CoA esters or by the action of decarboxylases (carboxylyases) into aldehydes having one carbon atom less than the original amino acid. For example, decarboxylation of pyruvic acid yields acetaldehyde and decarboxylation of 2-oxobutanoic acid gives propanal. The same aldehydes also form by Strecker... 

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