Pyruvate decarboxylase decarboxylation reaction

In the first step, pyruvate is decarboxylated in an irreversible reaction catalyzed by pyruvate decarboxylase. This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate. Pyruvate decarboxylase requires Mg24" and has a tightly bound coenzyme, thiamine pyrophosphate, discussed below. In the second step, acetaldehyde is reduced to ethanol through the action of alcohol dehydrogenase, with... 

The reducing power of NADH, produced by glycolysis, must be transferred to an electron acceptor to regenerate NAD" ". In alcoholic fermentation, it is not pyruvate but rather acetaldehyde, its decarboxylation product, that serves as the terminal electron acceptor. With respect to glycolysis, alcoholic fermentation contains two additional enzymatic reactions, the first of which (catalyzed by pyruvate decarboxylase), decarboxylates pyruvic acid. 

In general, pyruvate decarboxylase (EC 4.1.1.1) catalyzes the decarboxylation of a 2-oxocar-boxylic acid to give the corresponding aldehyde6. Using pyruvic acid, the intermediately formed enzyme-substrate complex can add an acetyl unit to acetaldehyde already present in the reaction mixture, to give optically active acetoin (l-hydroxy-2-butanone)4 26. Although the formation of... 

Kluger and Brandi (1986b) also studied the decarboxylation and base-catalysed elimination reactions of lactylthiamin, the adduct of pyruvate and thiamin (Scheme 2). These reactions are nonenzymic models for reactions of the intermediates formed during the reaction catalysed by the enzyme pyruvate decarboxylase. The secondary j3-deuterium KIE for the decarboxylation was found to be 1.09 at pH 3.8 in 0.5 mol dm-3 sodium acetate at 25°C. In the less polar medium, 38% ethanolic aqueous sodium acetate, chosen to mimic the nonpolar reactive site in the enzyme, the reaction is significantly faster but the KIE was, within experimental error, identical to the KIE found in water. This clearly demonstrates that the stabilization of the transition state by hyperconjugation is unaffected by the change in solvent. 

The project encompassed the comparative characterization of pyruvate decarboxylase from Z. mohilis (PDC) and benzoylformate decarboxylase from P. putida (BED) as well as their optimization for bioorganic synthesis. Both enzymes require thiamine diphosphate (ThDP) and magnesium ions as cofactors. Apart from the decarboxylation of 2-ketoacids, which is the main physiological reaction of these 2-ketoacid decarboxylases, both enzymes show a carboligase site reaction leading to chiral 2-hydroxy ketones (Scheme 2.2.3.1). A well-known example is... 

Decarboxylase reaction Kinetic constants The optimum pH of the decarboxylase reaction was determined with the natural substrates of both enzymes, pyruvate (PDC) and benzoylformate (BFD). Both enzymes show a pH optimum at pH 6.0-6.5 for the decarboxylation reaction [4, 5] and investigation of the kinetic parameters gave hyperbolic v/[S] plots. The kinetic constants are given in Table 2.2.3.1. The catalytic activity of both enzymes increases with the temperature up to about 60 °C. From these data activation energies of 34 kj moT (PDC) and 38 kJ mol (BFD) were calculated using the Arrhenius equation [4, 6-8]. 

Figure 16-6 shows schematically how the pyruvate dehydrogenase complex carries out the five consecutive reactions in the decarboxylation and dehydrogenation of pyruvate. Step CD is essentially identical to the reaction catalyzed by pyruvate decarboxylase (see Fig. 14-13c) C-l of pyruvate is released as C02, and C-2, which in pyruvate has the oxidation state of an aldehyde, is attached to TPP as a hydroxyethyl group. This first step is the slowest and therefore limits the rate of the overall reaction. It is also the point at which the PDH complex exercises its substrate specificity. In step (2) the hydroxyethyl group is oxidized to the level of a car-...

The conversion of pyruvate to ethanol occurs by the two reactions summarized in Figure 8.24. The decarboxylation of pyruvate by pyruvate decarboxylase occurs in yeast and certain microorganisms, but not in humans. The enzyme requires thiamine pyrophosphate as a coenzyme, and catalyzes a reaction similar to that described for pyruvate dehydrogenase (see p. 108). 

TPP-dependent enzymes catalyze either simple decarboxylation of a-keto acids to yield aldehydes (i.e. replacement of C02 with H+), or oxidative decarboxylation to yield acids or thioesters. The latter type of reaction requires a redox coenzyme as well (see below). The best known example of the former non-oxidative type of decarboxylation is the pyruvate decarboxylase-mediated conversion of pyruvate to acetaldehyde and C02. The accepted pathway for this reaction is shown in Scheme 10 (69MI11002, B-70MI11003, B-77MI11001>. 

Most known thiamin diphosphate-dependent reactions (Table 14-2) can be derived from the five halfreactions, a through e, shown in Fig. 14-3. Each halfreaction is an a cleavage which leads to a thiamin- bound enamine (center, Fig. 14-3) The decarboxylation of an a-oxo acid to an aldehyde is represented by step b followed by a in reverse. The most studied enzyme catalyzing a reaction of this type is yeast pyruvate decarboxylase, an enzyme essential to alcoholic fermentation (Fig. 10-3). There are two 250-kDa isoenzyme forms, one an a4 tetramer and one with an ( P)2 quaternary structure. The isolation of ohydroxyethylthiamin diphosphate from reaction mixtures of this enzyme with pyruvate52 provided important verification of the mechanisms of Eqs. 14-14,14-15. Other decarboxylases produce aldehydes in specialized metabolic pathways indolepyruvate decarboxylase126 in the biosynthesis of the plant hormone indoIe-3-acetate and ben-zoylformate decarboxylase in the mandelate pathway of bacterial metabolism (Chapter 25).1243/127... 

There are two 2-oxoacid dehydrogenase multienzyme complexes in E. coli. One is specific for pyruvate, the other for 2-oxoglutarate. Each complex is about the size of a ribosome, about 300 A across. The pyruvate dehydrogenase is composed of three types of polypeptide chains El, the pyruvate decarboxylase (an a2 dimer of Mr — 2 X 100 000) E2, lipoate acetyltransferase (Mr = 80 000) and E3, lipoamide dehydrogenase (an a2 dimer of Mr = 2 X 56 000). These catalyze the oxidative decarboxylation of pyruvate via reactions 1.6, 1.7, and 1.8. (The relevant chemistry of the reactions of thiamine pyrophosphate [TPP], hydroxyethylthiamine pyrophosphate [HETPPJ, and lipoic acid [lip-S2] is discussed in detail in Chapter 2, section C3.)... 

In the first step of the conversion catalyzed by pyruvate decarboxylase, a carbon atom from thiamine pyrophosphate adds to the carbonyl carbon of pyruvate. Decarboxylation produces the key reactive intermediate, hydroxyethyl thiamine pyrophosphate (HETPP). As shown in figure 13.5, the ionized ylid form of HETPP is resonance-stabilized by the existence of a form without charge separation. The next enzyme, dihydrolipoyltransacetylase, catalyzes the transfer of the two-carbon moiety to lipoic acid. A nucleophilic attack by HETPP on the sulfur atom attached to carbon 8 of oxidized lipoic acid displaces the electrons of the disulfide bond to the sulfur atom attached to carbon 6. The sulfur then picks up a proton from the environment as shown in figure 13.5. This simple displacement reaction is also an oxidation-reduction reaction, in which the attacking carbon atom is oxidized from the aldehyde level in HETPP to the carboxyl level in the lipoic acid derivative. The oxidized (disulfide) form of lipoic acid is converted to the reduced (mer-capto) form. The fact that the two-carbon moiety has become an acyl group is shown more clearly after dissocia-... 

There are significant differences in the reactivity of synthetic intermediate analogues for these reactions and the corresponding intermediates in the enzymic system. Lienhard and coworkers42,43 reported that the rate of decarboxylation of 2-(l-carboxy-l-hydroxyethyl)-3,4-dimethylthiazolium chloride is very fast relative to pyruvate (whose reaction is too slow to observe) but slower than the enzymic decarboxylation of pyruvate decarboxylase (PDC) by a factor of 105. Similar observations of a catalytic gap were seen for the rate of decarboxylation of lactylthiamin compared to PDC and the... 

As a side activity, many decarboxylases catalyze the formation of C-C bonds. In the reaction of two pyruvate molecules, catalyzed by pyruvate decarboxylase (PDC, E.C. 4.1.1.1), a-acetolactate is formed, an important intermediate of valine biosynthesis. In turn, a-acetolactale can be oxidatively decarboxylated by oxygen to diacetyl or enzymatically decarboxylated by acetolactate decarboxylase (ADC, E.C. 4.1.1.5) to (] )-acetoin (Figure 7.29). 

The a-keto acid decarboxylases such as pyruvate (E.C. 4.1.1.1) and benzoyl formate (E.C. 4.1.1.7) decarboxylases are a thiamine pyrophosphate (TPP)-dependent group of enzymes, which in addition to nonoxidatively decarboxylating their substrates, catalyze a carboligation reaction forming a C-C bond leading to the formation of a-hydroxy ketones.269-270 The hydroxy ketone (R)-phenylacetylcarbinol (55), a precursor to L-ephedrine (56), has been synthesized with pyruvate decarboxylase (Scheme 19.35). BASF scientists have made mutations in the pyruvate decarboxylase from Zymomonas mobilis to make the enzyme more resistant than the wild-type enzyme to inactivation by acetaldehyde for the preparation of chiral phenylacetylcarbinols.271... 

The identity of the enzyme(s) involved in the latter reaction has been debated (13). However, the formation of the above hydro-xyketone, in analogy with acetoin, has been conceptualized as the consequence of the condensation of the "active" form of acetaldehyde, that is formed by decarboxylative addition of pyruvate to thiamine pyrophospate, with benzaldehyde.The role of pyruvate, in fact has been established. The same mechanism can be invoked for the reaction of cinnamaldehyde.lt is known that the pyruvate decarboxylase (E.C. 4.1.1.1) accepts as substrates a-oxoacids... 

Unfortunately diacetyl formation is still not well understood. Acetoin formation occurs either by nonspecific interaction of acetaldehyde with the a-hydroxyethyl thiamine pyrophosphate intermediate in pyruvate decarboxylation (209) or by decarboxylation of a-acetolactate (210), which in turn arises either from interaction of pyruvate with a-hydroxyethyl thiamine pyrophosphate (211) or as a specific intermediate in valine biosynthesis (212, 213). Diacetyl does not appear to be formed directly from acetoin (208, 214). It is formed from a-acetolactate, in absence of cells, by O2 oxidation (215), and even under N2 (216), although an oxidation must occur. It is also formed from acetyl CoA (217, 218), probably by interaction with a-hydroxyethyl thiamine pyrophosphate [cf. stimulation by acetyl CoA addition to a solution of pyruvate and pyruvate decarboxylase (2i5)]. It is not known whether this involves a specific enzyme or is a mere side reaction. 

Ethanol is formed from pyruvate in yeast and several other microorganisms. The first step is the decarboxylation of pyruvate. This reaction is catalyzed by pyruvate decarboxylase, which requires the coenzyme thiamine pyrophosphate. This coenzyme, derived from the vitamin thiamine (Bj), also participates in reactions catalyzed by other enzymes (Section 17.1.1). The second step is the reduction of acetaldehyde to ethanol by NADH, in a reaction catalyzed by... 

Pyruvate decarboxylase catalyzes the nonoxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. When an aldehyde is present with pyruvate, the enzyme promotes an acyloin condensation reaction. The mechanistic reason for this fortuitous reaction is well understood and involves the aldehyde outcompeting a proton for bond formation with a reactive thiamine pyrophosphate-bound intermediate (90,91). When acetaldehyde is present, the product formed is acetoin. Benzalde-hyde results in the production of phenylacetylcarbinol (Fig. 26). Both of these condensations are enantioselective, forming the R enantiomer preferentially in both cases. 

As early as 1930 it was proposed fhat fhe production of 2-hydroxy ketones (here (P)-PAC) is a side reaction of pyruvate decarboxylase (PDC) [18, 19]. Later, it was shown unequivocally that fhiamine diphosphate (ThDP)-dependent PDC catalyzes both the decarboxylation of pyruvate and fhe carboligation of the intermediate activated aldehyde to benzaldehyde (Scheme 4.2 A). 

An alternative method for driving the reaction equilibrium of transaminations was developed by Hohne et al. (Figure 14.44) [65]. Alanine served as the amine donor, and pyruvate decarboxylase was used to remove the pyruvate coproduct by decarboxyl ation to acetaldehyde. An advantage of pyruvate decarboxylase over lactate dehydro genase is that it requires no cofactor recycling system, and the high volatility of the coproducts allows for the desired shift of equilibrium. 

---