Decarboxylase pyruvate

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 ... 

To what classes do the following enzymes belong (a) Pyruvate decarboxylase (b) Chymotrypsin... 

Of the many enzymes capable of catalyzing the formation of acyloins only transketolases5 (EC 2.2.1.1) and those of the pyruvate decarboxylase complex6 (EC 4.1.1.1) have, as yet, gained considerable importance for preparative purposes. Therefore, applications of both of these enzymes will be discussed here,... 

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 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

Other thiamine diphosphate-dependent enzymes have recently been scrutinized for their preparative value [166]. Although pyruvate decarboxylase (PDC) (EC 4.1.1.1)... 

An enzymatic process using partially purified pyruvate decarboxylase (PDC) with added pyruvate overcomes the problems of benzyl alcohol formation and limiting availability of pyruvate [3]. As a result increased concentrations, yields and productivities of PAC were achieved with concentrations of PAC in excess of 50 g f (330 mM) in 28 h and yields on benzaldehyde above 95% theoretical [4-6]. Screening of a wide range of bacteria, yeasts and other fungi as potential sources of stable, high activity PDC for production of PAC confirmed a strain of the yeast Candida utilis as the most suitable source of PDC [7]. 

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. 

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. 

Figure 1.8 Enzyme pyruvate decarboxylase b. Oxidative deamination NH3 released...

PDH is a multi-enzyme complex consisting of three separate enzyme units pyruvate decarboxylase, transacetylase and dihydrolipoyl dehydrogenase. Serine residues within the decarboxylase subunit are the target for a kinase which causes inhibition of the PDH the inhibition can be rescued by a phosphatase. The PDH kinase (PDH-K) is itself activated, and the phosphatase reciprocally inhibited, by NADH and acetyl-CoA. Figure 3.12(a and b) show the role and control of PDH. 

Pyruvate dehydrogenase is a MEC consisting of three separate catalytic proteins (i) a component with combined pyruvate decarboxylase/dehydrogenase activity (ii) a dihydrolipoyl transacetylase (also called acetyl transferase) unit and (iii) linked dihy-drolipoyl dehydrogenase. This is clearly a very big protein the pyruvate decarboxylase/... 

Control of pymvate dehydrogenase activity is via covalent modification a specific kinase causes inactivation of the PDH by phosphorylation of three serine residues located in the pyruvate decarboxylase/dehydrogenase component whilst a phosphatase activates PDH by removing the phosphates. The kinase and phosphatase enzymes are non-covalently associated with the transacetylase unit of the complex. Here again we have an example of simultaneous but opposite control of enzyme activity, that is, reciprocal regulation. 

The hypE proteins are 302-376 residues long and appear to consist of three domains. Domain 1 shows sequence identity to a domain from phosphoribosyl-aminoimida-zole synthetase which is involved in the fifth step in de novo purine biosynthesis and to a domain in thiamine phosphate kinase which is involved in the synthesis of the cofactor thiamine diphosphate (TDP). TDP is required by enzymes which cleave the bond adjacent to carbonyl groups, e.g. phosphoketolase, transketolase or pyruvate decarboxylase. Domain 2 also shows identity to a domain found in thiamine phosphate kinase. Domain 3 appears to be unique to the HypF proteins. 

Figure 6.1 Pathways involved in glucose oxidation by plant cells (a) glycolysis, (b) Krebs cycle, (c) mitochondrial cytochrome chain. Under anoxic conditions. Reactions 1, 2 and 3 of glycolysis are catalysed by lactate dehydrogenase, pyruvate decarboxylase and alcohol dehydrogenase, respectively. ATP and ADP, adenosine tri- and diphosphate NAD and NADHa, oxidized and reduced forms of nicotinamide adenine dinucleotide PGA, phosphoglyceraldehyde PEP, phosphoenolpyruvate Acetyl-CoA, acetyl coenzyme A FP, flavoprotein cyt, cytochrome e, electron. (Modified from Fitter and Hay, 2002). Reprinted with permission from Elsevier...

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. 

OXALYL-CoA DECARBOXYLASE PHOSPHOKETOLASE PYRUVATE DECARBOXYLASE PYRUVATE DEHYDROGENASE TRANSKETOLASE... 

The second example was the pyruvate decarboxylase catalyzed formation of (ll )-l-hydroxy-l-phenyl-2-propanone (PAC) with benzaldehyde as substrate (Fig. 5 a) [64]. This second reaction shows one potential limitation of this method. Some compounds are too volatile for direct measurement by MALDl mass spectrometry or they do not ionize directly due to their nonpolar character. In this case, these compounds have to be derivatized prior to their measurement in order to reduce their volatihty and to introduce ionizable functions. This is, however, often very easy using well estabhshed quantitative reactions, e.g., formation of oximes from aldehydes and sugars (Fig. 5b). 

Fig. 5. a Pyruvate decarboxylase catalyzed formation of (lJl)-l-hydroxy-l-phenyl-2-pro-panone (PAG) using benzaldehyde (BzA) as substrate, b Derivatization of benzaldehyde by formation of an oxime... 

Pinhero, R. G., Copp, L. J., Amaya, C. -L., Marangoni, A. G., Yada, R. Y. (2007). Roles of Alcohol dehydrogenase. Lactate dehydrogenase and Pyruvate decarboxylase in Low Temperature Sweetening in a tolerant and susceptible varieties of Potato (Solanum tuberosum). Physiologia Plant, 130(2), 230-239. 

Benzoylformate decarboxylase (BFD EC 4.1.1.7) belongs to the class of thiamine diphosphate (ThDP)-dependent enzymes. ThDP is the cofactor for a large number of enzymes, including pyruvate decarboxylase (PDC), benzaldehyde lyase (BAL), cyclohexane-1,2-dione hydrolase (CDH), acetohydroxyacid synthase (AHAS), and (lR,6] )-2-succinyl-6-hydroxy-2,4-cyclohexadiene-l-carboxylate synthase (SHCHC), which all catalyze the cleavage and formation of C-C bonds [1]. The underlying catalytic mechanism is summarized elsewhere [2] (see also Chapter 2.2.3). 

In the framework of SFB380, two projects dealt extensively with acyloin-con-densing ThDP-dependent enzymes such as pyruvate decarboxylase (PDC), ben-zoylformate decarboxylase (BFD), or benzaldehyde lyase (BAL) (see Chapters 2.2.3 and 2.2.7). Another ThDP-dependent decarboxylase, phosphonopyruvate decarboxylase (PPD) from Streptomyces viridochromogenes, became available only recently and was studied in project B21. We wanted to find out whether this PDC-related enzyme could be a valuable tool in the provision of acyloin condensations involving C-P bonds (see Section 2.2.2.23). 

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. 

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... 

M. Pohl, Protein design on pyruvate decarboxylase (PDC) by site-directed mutagenesis. Application to mechanistical investigations, and tailoring PDC for the use in organic synthesis. Adv. Biochem. Eng. BioUchnol. 1997, 58, 15-43. 

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