Pyruvic acid pyruvate decarboxylase

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... 

The yeast pyruvate decarboxylase is rather specific with respect to the acyl moiety that is added to the aldehyde. Only a few 2-oxo acids can be used as acyl donors besides pyruvic-acid39. For example, treatment of benzaldehyde with 2-oxobutanoic acid and 2-oxopentanoic acid, respectively, and prewashed Saccharomyces cerevisiae gave the corresponding (/ )-acyloin derivatives in 15 25% yield with an enantiomeric excess >95%. 

Figure 5 Model of phosphorus (P) deficiency-induced physiological changes associated with the release of P-mobilizing root exudates in cluster roots of white lupin. Solid lines indicate stimulation and dotted lines inhibition of biochemical reaction sequences or mclaholic pathways in response to P deliciency. For a detailed description see Sec. 4.1. Abbreviations SS = sucrose synthase FK = fructokinase PGM = phosphoglueomutase PEP = phosphoenol pyruvate PE PC = PEP-carboxylase MDH = malate dehydrogenase ME = malic enzyme CS = citrate synthase PDC = pyruvate decarboxylase ALDH — alcohol dehydrogenase E-4-P = erythrosc-4-phosphate DAMP = dihydraxyaceConephos-phate APase = acid phosphatase.

These enzymes catalyse the non-hydrolytic cleavage of bonds in a substrate to remove specific functional groups. Examples include decarboxylases, which remove carboxylic acid groups as carbon dioxide, dehydrases, which remove water, and aldolases. The decarboxylation of pyruvic acid (10.60) to form acetaldehyde (10.61) takes place in the presence of pyruvic decarboxylase (Scheme 10.13), which requires the presence of thiamine pyrophosphate and magnesium ions for activity. 

The requirement for NAD+ is to reoxidize the lipoic acid carrier. It is worth mentioning that the pyruvate acetaldehyde conversion we considered at the end of the glycolytic pathway involves the same initial sequence, and pyruvate decarboxylase is another thiamine diphosphate-dependent enzyme. 

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. 

Various thiamine diphosphate (ThDP)-dependent a-keto acid decarboxylases have been described as catalyzing C-C bond formation and/or cleavage [48]. Extensive work has already been conducted on transketolase (TK) and pyruvate decarboxylase (PDC) from different sources [49]. Here attention should be drawn to some concepts based on the investigation of reactions catalyzed by the enzymes... 

Figure B.l. Mechanism for the conversion of pyruvic acid to acetaldehyde and CO2 by pyruvate decarboxylase.

Alternate fates of pyruvate Compounds other than lactate to which pyruvate can be converted ALTERNATE FATES OF PYRUVATE (p. 103) Pyruvate can be oxidatively decarboxylated by pyruvate dehydrogenase, producing acetyl CoA—a major fuel for the tricarboxylic acid cycle (TCA cycle) and the building block for fatty acid synthesis. Pyruvate can be carboxylated to oxaloacetate (a TCA cycle intermediate) by pyruvate carboxylase. Pyruvate can be reduced by microorganisms to ethanol by pyruvate decarboxylase. 

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>. 

Figure 13.8 Pathway for metabolism of citrate by Leuconostoc spp. and S. lactis subsp. diacetylactis. (1) Citrate permease, (2) citrate lyase, (3) oxaloacetic acid decarboxylase, (4) pyruvate decarboxylase, (5) a-acetolactate synthetase, (6) a-acetolactate carboxylase, (7) diacetyl synthetase, (8) diacetyl reductase, and (9) acetoin reductase.

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... 

Ketols can also be formed enzymatically by cleavage of an aldehyde (step a, Fig. 14-3) followed by condensation with a second aldehyde (step c, in reverse). An enzyme utilizing these steps is transketolase (Eq. 17-15),132b which is essential in the pentose phosphate pathways of metabolism and in photosynthesis. a-Diketones can be cleaved (step d) to a carboxylic acid plus active aldehyde, which can react either via a or c in reverse. These and other combinations of steps are often observed as side reactions of such enzymes as pyruvate decarboxylase. A related thiamin-dependent reaction is that of pyruvate and acetyl-CoA to give the a-diketone, diacetyl, CH3COCOCH3.133 The reaction can be viewed as a displacement of the CoA anion from acetyl-CoA by attack of thiamin-bound active acetaldehyde derived from pyruvate (reverse of step d, Fig. 14-3 with release of CoA). 

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-... 

The conversion of pyruvate to acetyl-CoA. The reactions are catalyzed by the enzymes of the pyruvate dehydrogenase complex. This complex has three enzymes pyruvate decarboxylase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. In addition, five coenzymes are required thiamine pyrophosphate, lipoic acid, CoASH, FAD, and NAD+. Lipoic acid is covalently attached to... 

EC 1.11.1.7) (68) and diphenol oxidase (EC 1.10.3.1) (69) have been identified. The potential role of pyruvic decarboxylase (EC 4.1.1.1) catalyzed reaction as a source of acetaldehyde and other aldehydes in juice was discussed (70). Raymond et al. (71) isolated the decarboxylase from orange juice sections and demonstrated that only 10 to 15% of the enzyme was in an active form. Since the purified enzyme was only active with pyruvic acid and 2-ketobutyric acid of the series of 2-ketoacids examined, they (71) concluded that the direct contribution of orange pyruvic decarboxylase to the orange volatile profile was limited to acetaldehyde and possibly propionaldehyde. 

A carboxylic side-chain of a glutamic acid (which is conserved in pyruvate decarboxylases, pyruvate oxidase and transketolase) forms a hydrogen bond to l N of ThDP, and thus keeps this nitrogen in the protonated form. The positive charge is delocalized to the 4 -NH2-group, making it possible for it to act as a proton donor [35,60]. 

Biological compounds with long chains of carbon atoms are broken down into molecules with shorter chains by the breaking of carbon-carbon bonds. This commonly occurs by the elimination of -C02H groups from carboxylic acids. For example, pyruvic decarboxylase enzyme acts upon pyruvic acid,... 

Cadmium in the body is known to affect several enzymes. It is believed that the renal damage that results in proteinuria is the result of cadmium adversely affecting enzymes responsible for reabsorption of proteins in kidney tubules. Cadmium also reduces the activity of delta-aminolevulinic acid synthetase (Figure 10.3), arylsulfatase, alcohol dehydrogenase, and lipoamide dehydrogenase, whereas it enhances the activity of delta-aminolevulinic acid dehydratase, pyruvate dehydrogenase, and pyruvate decarboxylase. 

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... 

Pyruvate decarboxylase Plants, yeast 2-Oxo-acid Aldehyde + C02... 

Calculate the entropy change for the conversion of pyruvic acid (CH3COCOOH) into acetaldehyde and C02 by the enzyme pyruvate decarboxylase at 25°C and 100 atm. 

The lipoic acid is covalently attached to the transacetylase (E2). It acts as a swinging arm , interacting with pyruvate decarboxylase (E ) to accept the hydroxyethyl derivative of pyruvate, with CoA to produce acetyl-CoA, and with dihydrolipoyl dehydrogenase (E3) so that it can be reoxidized. 

Fig. 12-7 The reactions of the pyruvate dehydrogenase complex. The reactants in the overall reaction are shown in boxes. E, = pyruvate decarboxylase (TPP = thiamine pyrophosphate as prosthetic group), E2 = dihydrolipoyl trans-acetylase (oxidized lipoic acid as prosthetic group). E3 = dihydrolipoyl dehydrogenase (FAD as prosthetic group).

Nichols, N. N., Dien, B. S., and Bothast, R. J. 2003. Engineering lactic acid bacteria with pyruvate decarboxylase and alcohol dehydrogenase genes for ethanol production from Zymomonas mobilis. J. Ind. Microbiol. Biotechnol., 30, 315-321. 

Fig. 3 Metabolic engineering for L-lactic acid production in Saccharomyces cerevisiae. The abbreviation of enzymes is described as follows LDH bovine lactate dehydrogenase, PDC pyruvate decarboxylase complex encoded by the genes PDC1, PDC5, and PDC6, which were deleted in this strategy...

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