Pyruvate decarboxylase

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Pyruvate decarboxylase
Pyruvate decarboxylase
Ribbon model (from above, to the side) of the PDC tetramer of the baker's yeast , with pyruvate / TPP / Mg ++ as balls, according to PDB 2VK1
Secondary to quaternary structure Homotetramer
Cofactor Thiamine pyrophosphate, magnesium
Enzyme classification
EC, category 4.1.1.1 ,  lyase
Response type Decarboxylation
Substrate Pyruvate
Products Acetaldehyde, carbon dioxide
Occurrence
Parent taxon Bacteria , fungi , plants

Figure 1: Ribbon / surface model of the PDC dimer of baker's yeast , according to PDB 1PVD

Pyruvate decarboxylase is a homotetrameric enzyme (EC 4.1.1.1) that catalyzes the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide in the cytoplasm . Under anaerobic conditions, this enzyme is part of the fermentation that takes place in yeasts, particularly in the genus Saccharomyces , to produce ethanol through fermentation . The conversion of pyruvate to acetaldehyde and carbon dioxide by pyruvate decarboxylase is at the beginning of this process. Pyruvate decarboxylase is dependent on the cofactors thiamine pyrophosphate and magnesium. The enzyme should not be confused with pyruvate dehydrogenase , an oxidoreductase (EC 1.2.4.1) that catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA .

yeast

In yeast, pyruvate decarboxylase works independently during anaerobic fermentation and releases acetaldehyde and carbon dioxide as the C2 body. Pyruvate decarboxylase is a route to CO 2 elimination, which the cell releases. The ethanol produced by the enzyme acts as an antibiotic that is used to eliminate competing organisms. The enzyme is necessary to aid in the decarboxylation of alpha-keto acids because more negative charges are accumulated on the carbonyl carbon atom in the transition state; the enzyme thus offers the optimal environment for thiamine pyrophosphate and the alpha-keto acids ( pyruvate ) to meet.

structure

Pyruvate decarboxylase is built up as a dimer of dimers, with two active centers, formed from two monomers of each dimer. The enzyme contains a beta-alpha-beta structure resulting in parallel beta sheets and 563 residual subunits in each dimer. The monomers are assembled into dimers through strong interactions, but the dimers only interact loosely with each other to form a loose tetramer.

The active center

Pyruvate decarboxylase is a homotetramer and therefore has four active centers. The active centers are located in a cavity in the core area of ​​the enzyme, where hydrogen bonds can be formed and where pyruvate reacts with thiamine pyrophosphate (TPP, see Figure 2). Each active site is made up of 20 amino acids, including Glu-477 (helps maintain the stability of the TPP ring) and Glu-51 (helps with cofactor binding). These glutamates also help to form the TPP ylide by acting as a proton donor towards the substituted TPP aminopyrimidine ring. The microenvironment around Glu 477 is very non-polar, with a higher than usual pKa (typically the pKA of Glu and Asp in small proteins is around 4.6).

The lipophilic residues Ile-476, Ile-480 and Pro-26 contribute to the non-polarity of the area around Glu-477. The only other negatively charged residue aside from the TPP coenzyme is Asp-28, which also helps increase the pKa of Glu-477. The environment of the enzyme must allow the γ-carboxy group of Glu-477 to be protonated at a pH around 6.

The aminopyrimidine ring on the TPP, as soon as it is present as an imine, deprotonates the C2 atom of TPP to form the nucleophilic ylide. This has to be done in this way, as the enzyme does not have any basic side chains to deprotonate the C2 of TPP. A mutation in the active site associated with Glu can lead to the inefficiency or inactivity of the enzyme. This inactivity has been demonstrated in experiments in which either the N1 'and / or 4' amino groups were absent. In NMR analyzes it was found that when TPP is bound to the enzyme together with the pyruvamide analogous to the substrate, the rate of ylid formation is greater than that of the normal enzyme. In addition, the mutation rate from Glu 51 to Gln significantly reduces this rate.

Figure 2: Structural formula of the cofactor thiamine pyrophosphate.

Also included are Asp-444 and Asp-28, which stabilize the active center. They act as stabilizers for the Mg 2+ ion that is in every active center. To ensure that only pyruvate binds, two Cys-221 (more than 20 Angstroms away from each center) and His-92 trigger a conformational change that inhibits or activates the enzyme, depending on the substrate it interacts with. If the substrate that is bound in the active site is pyruvate, then the enzyme is activated by a conformational change in this regulatory site. The conformational change involves a 1,2 nucleophilic addition. This reaction, the formation of a thioketal, converts the enzyme from the inactive to the active form.

The inhibition of the site is triggered by an inhibitor or a substrate analog of the form XC6H4CH = CHCOCOOH, as well as by products of the decarboxylation of substances such as cinnamaldehyde. Other potential targets for nucleophilic inhibitors are Cys-152, Asp-28, His-114, His-115 and Gln-477.

The normal catalytic rate of pyruvate decarboxylase is kcat = 10 s −1 . In contrast, with a Glu-51 mutation to Gln, it is 1.7 s −1 .

mechanism

The decarboxylation of pyruvate poses the challenge that a negative charge on a carbonyl carbon, which would result from the elimination of CO 2 , is very unstable.

Decarboxylation of Pyruvate

In the pyruvate decarboxylase reaction, the cofactor TPP absorbs this high electron density by delocalization (the same "strategy" is found in the pyruvate dehydrogenase reaction). The corresponding carbonyl carbon is first transformed into alcohol. This reaction procedure is also found in synthetic chemistry, so-called umpolung reactions , such as the Stetter reaction or the benzoin addition .

The proton at C2 of the substituted thiazolium ring (see Figure 2) is sufficiently acidic to be deprotonated by the neighboring aminopyrimidine ring. The reaction mechanism of deprotonation is shown in the figure below. A base deprotonates the amino group of the aminopyridine ring, which then leads to the formation of the imine ( imine-enamine tautomerism ). This is followed by an internal proton transfer from the thiazolium ring to the amino group.

Deprotonation of thiamine pyrophosphate using a base

The negative charge is stabilized by the resonance structures 2A and 2B (see Figure 3). The carbene or carbanion of the thiazolium ring attacks the keto group of pyruvate 1 nucleophilically. The resulting alcoholate 3 is protonated to form alcohol 4 . The subsequent decarboxylation takes place easily through the electron pull of the ammonium ion . The remaining electrons are stabilized by resonance, 5A and 5B . After protonation to 6 , the cofactor 2A and acetaldehyde 7 are released.

Figure 3: Reaction mechanism of the decarboxylation of pyruvate with TPP.

Individual evidence

  1. ↑ Entry at BRENDA
  2. ↑ John McMurry, Tadhg P. Begley: The organic chemistry of biological pathways . Roberts and Co. Publishers, 2005, ISBN 0-9747077-1-6 , pp. 179 (English).
  3. ↑ a b c d e f PDB  1pyd ; F. Dyda, W. Furey, S. Swaminathan, M. Sax, B. Farrenkopf, F. Jordan: Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution . In: Biochemistry . Vol. 32, No. June 24 , 1993, pp. 6165-6170 , doi : 10.1021 / bi00075a008 , PMID 8512926 (English).
  4. ↑ a b M. Lobell, DHG Crout: Pyruvate Decarboxylase: A Molecular Modeling Study of Pyruvate Decarboxylation and Acyloin Formation . In: J. Am. Chem. Soc. Vol. 118, No. 8 , 1996, pp. 1867–1873 , doi : 10.1021 / ja951830t (English).
  5. ↑ a b I. Baburina, G. Dikdan, F. Guo, GI Tous, B. Root, F. Jordan: Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions . In: Biochemistry . Vol. 37, No. 5 , February 1998, p. 1245-1255 , doi : 10.1021 / bi9709912 , PMID 9477950 (English).
  6. ^ Donald Voet, Judith G. Voet, Charlotte W. Pratt: Textbook of Biochemistry . 3. Edition. Wiley-VCH Verlag, Weinheim 2019, ISBN 978-3-527-34286-0 , pp. 605 .
  7. ↑ Georg Fuchs (Ed.): General Microbiology . 10th edition. Thieme, 2017, ISBN 978-3-13-241885-1 , p. 271 .
  8. ↑ John McMurry, Tadhg P. Begley: The Organic Chemistry of Biological Pathways . Roberts and Company Publishers, 2005, ISBN 0-9747077-1-6 , pp. 132 ( limited preview in Google Book search).
  9. ^ TDH Bugg: Introduction to Enzyme and Coenzyme Chemistry . John Wiley & Sons, 2012, ISBN 978-1-118-34899-4 , pp. 155 ( limited preview in Google Book search).