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Results for “Pyruvate Decarboxylase”

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PubMed1992

The catalytic power of pyruvate decarboxylase. A stochastic model for the molecular evolution of enzymes.

Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate anion by a factor of around 10(12), compared with the non-enzymic decarboxylation by thiamine, under standard state conditions of 1 mM pyruvate and thiamine diphosphate (TDP), pH 6.2. Free-energy diagrams constructed on the basis of earlier measurements for the enzymic and non-enzymic reactions give some information on catalysis by PDC. PDC stabilizes the reactant state preceding TDP addition to pyruvate by 76 kJ mol-1 and the transition state for the addition by 83 kJ mol-1. PDC stabilizes the reactant state preceding decarboxylation (presumably alpha-lactyl-TDP) by 27 kJ mol-1 and the transition state for decarboxylation by 68 kJ mol-1. In addition, the free-energy diagrams reveal a leveling of reactant-state free energies in the enzymic reaction compared with the non-enzymic reaction, in that the former are nearly equal to each other. The enzyme-bound transition-state energies are similarly leveled. The energetic leveling of reactant states has been noted by Albery, Knowles and their coworkers in many enzymic reactions and termed 'matched internal thermodynamics.' They showed that the result would arise naturally (and inevitably) in the 'evolution to perfection' of enzymes, when the evolutionary process was treated by a deterministic model. The critical assumption of this model was the validity of a Marcus-type or Brønsted-type linear free-energy relationship between rate and equilibrium constants for reactions occurring wholly within enzyme complexes. Here a completely stochastic simulation of molecular evolution, with no deterministic assumptions, is shown to reproduce both 'matched internal thermodynamics' and the 'matched internal kinetics' or leveling of transition-state energies noted here. The Albery-Knowles result is thus more general than might have been supposed.

Biological Evolution↗

PubMed2005

Functional expression of bacterial Zymobacter palmae pyruvate decarboxylase gene in Lactococcus lactis.

A pyruvate decarboxylase (PDC) gene from bacterial Zymobacter palmae (Zymopdc) was cloned, characterized, and introduced into Lactococcus lactis via a shuttle vector pAK80 as part of a research strategy to develop an efficient ethanol-producing lactic acid bacteria (LAB). The expression levels of Zymopdc gene in the host, as measured by a colorimetric assay based on PDC catalyzed formation of (R)-phenylacetylcarbinol ((R)-PAC), appeared to be dependent on the strength of corresponding Gram-positive promoters. A constitutive, highly expressed promoter conferred the greatest PDC activity, and an acid-inducible promoter demonstrated acid-inducible expression. The metabolic production of ethanol and other products was examined in flask fermentations. More than eightfold increases in acetaldehyde concentrations were detected in two recombinant strains. However, no detectable differences for ethanol fermentation in these engineered strains were observed compared with that of the strain carrying lacZ reporter.

Base Sequence↗

PubMed1980

Hydroxyl-ion-induced subunit dissociation of east cytoplasmic pyruvate decarboxylase. A circular dichroism study.

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250--320-nm range a multiple two-signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are delta G = 87.7 kJ mol-1, delta H = + 56.0 kJ mol-1, delta S = - 108 J mol-1 K-1, very characteristic for the deprotonation of an amino-acid side chain. The second class of the protons has the following thermodynamic data: delta G = 88.3 kJ mol-1, delta H = - 64.3 kJ mol-1, delta S = - 520 J mol-1 K-1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. Th enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate-determining for the overall process. The data are kp = 1.4 . 10(-3), delta H not equal to = + 128.3 kJ mol-1, delta S not equal = + 136 J mol-1 K-1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor beta = kc/(1 + Kc) (0 < beta less than or equal to 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo-enzyme are derived from the data.

Apoenzymes↗

PubMed1996

RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis.

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3-1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.

Amino Acid Sequence↗

PubMed1983

A rapid procedure for the preparation of highly purified pyruvate decarboxylase from brewer's yeast.

A rapid purification procedure for pyruvate decarboxylase (E.C. 4.1.1.1.) from fresh cells of brewer's yeast (Saccharomyces carlsb.) is reported. The preparation of a crude enzyme (30-45 U/mg) by the use of fractionation steps with protamine sulfate, acetone, and ammonium sulfate takes about 6-7 h. A stable pyruvate decarboxylase (70-85 U/mg) was obtained from such preparations after purification on CM Sephadex C 50 after another 2-3 h. Stability and structural properties are compared for enzymes prepared from fresh and dried yeast.

Carboxy-Lyases↗

PubMed2005

Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde.

Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.

Acetone↗

PubMed1994

A pyruvate decarboxylase gene from Aspergillus parasiticus.

A gene encoding a putative pyruvate decarboxylase (EC 4.1.1.1) was isolated from a genomic library of the filamentous fungus Aspergillus parasiticus strain SU-1. The deduced amino acid sequence showed 37% homology to PDC1 from Saccharomyces cerevisiae. Although A. parasiticus has an obligate growth requirement for oxygen, it produced ethanol in shake flask cultures indicating a response to anoxic conditions mediated by pyruvate decarboxylase.

Amino Acid Sequence↗

PubMed1989

A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations.

We deleted most of the pyruvate decarboxylase structural gene PDC1 from the genome of Saccharomyces cerevisiae. Surprisingly, mutants carrying this deletion allele showed a completely different phenotype than previously described point mutations. They were able to ferment glucose and their specific pyruvate decarboxylase activity was only reduced to 45% of the wild type level. Northern blot analysis revealed that a sequence in the yeast genome homologous to PDC1 and formerly designated as a possible pseudogene is expressed and may code for a different but closely related pyruvate decarboxylase. The products of the two PDC genes seem to form hybrid oligomers, however both homooligomers have enzyme activity. Thus, the product of the PDC1 gene is not absolutely necessary for glucose fermentation in yeast.

Blotting, Southern↗

PubMed1975

Conversion of nitrosobenzene to N-phenylacetohydroxamic acid by yeast pyruvate decarboxylase.

In the presence of yeast enzyme concentrate or purified yeast pyruvate decarboxylase, nitrosobenzene was converted in part to N-phenylacetohydroxamic acid. This transformation had to be catalyzed by the enzyme, since the incubation of nitrosobenzene with the cofactor of pyruvate decarboxylase did not produce the hydroxamic acid. Similar incubations conducted with phenylhydroxylamine did not yield any detectable amounts of N-phenylacetohydroxamic acid.

Carboxy-Lyases↗

PubMed1970

A defect in pyruvate decarboxylase in a child with an intermittent movement disorder.

A patient with an intermittent movement disorder has been found to have an inherited defect in pyruvate decarboxylase ((2-oxo-acid carboxy-lyase, E.C. 4.1.1.1.). The patient is a 9 yr old boy who since infancy has had repeated episodes of a combined cerebellar and choreoathetoid movement disorder. He has an elevated level of pyruvic acid in his blood, an elevated urinary alanine content, and less marked elevations in blood alanine and lactate. Methods were developed to study his metabolic abnormality in dilute suspensions of white blood cells and cultured skin fibroblasts, as well as in cell-free sonicates of fibroblasts. Oxidation of pyruvic acid-1-(14)C and pyruvic acid-2-(14)C by his cells and pyruvate decarboxylase activity in sonicates of his cells were less than 20% of those in cells from control subjects. Oxidation of glutamic acid-U-(14)C, acetate-1-(14)C, and palmitate-1-(14)C was normal, as was incorporation of alanine-U-(14)C into protein. The rate of oxidation of pyruvic acid by the father's cells and the activity of pyruvate decarboxylase in the father's sonicated fibroblasts were intermediate between those of the patient and those of controls. Values for the mother were at or just below the lower limits of the ranges in controls. Kinetic data suggested the posibility of several forms of pyruvate decarboxylase in this family. Possible mechanisms relating the chemical abnormality and the clinical symptoms in this patient are discussed.

Alanine↗

PubMed1982

Enzymologic studies and therapy of Leigh's disease associated with pyruvate decarboxylase deficiency.

An 11-month-old boy with muscle hypotonia and neurologic deteriorations had lactic acidosis, pyruvic acidemia and alaninemia due to deficiency of the pyruvate dehydrogenase complex in his platelets and of pyruvate decarboxylase in his muscle. When placed on a low carbohydrate-high fat diet for 6 months, his blood levels of lactate and pyruvate became nearly normal, but his cerebrospinal fluid levels of lactate and pyruvate remained high. Despite this dietary therapy, neurologic deterioration progressed slowly. He died of pneumonia after artificial respiration for 3 wk. At autopsy, extensive symmetric lesions were found in the brain including proliferation of capillaries and gliosis in the brain stem and diffuse demyelination in the white matter. These lesions were consistent with those observed in Leigh's disease. The activities of the pyruvate dehydrogenase complex and pyruvate decarboxylase in various tissues obtained at autopsy were less than 10% of control values; however, the activities of pyruvate carboxylase and alpha-ketoglutarate decarboxylase were within the normal limits. This patient with Leigh's disease had an isolated deficiency of pyruvate decarboxylase in various tissues.

Blood Platelets↗

PubMed2002

Folding and stability of different oligomeric states of thiamin diphosphate dependent homomeric pyruvate decarboxylase.

The folding and stability of recombinant homomeric (alpha-only) pyruvate decarboxylase from yeast was investigated. Different oligomeric states (tetramers, dimers and monomers) of the enzyme occur under defined conditions. The enzymatic activity is used as a sensitive probe for structural differences between the active and inactive form (mis-assembled forms, aggregates) of the folded protein. Unfolding kinetics starting from the native protein comprise both the dissociation of the oligomers into monomers and their subsequent denaturation, which could be monitored by stopped-flow kinetics. In the course of unfolding, the tetramers do not directly dissociate into monomers, but via a stable dimeric state. Starting from the unfolded state, a reactivation of homomeric pyruvate decarboxylase requires both refolding to monomers and their correct association to enzymatically active dimers or tetramers. The reactivation yield under the in vitro conditions used follows an optimum behavior.

Circular Dichroism↗

PubMed1983

Carbon dioxide effects on ethanol production, pyruvate decarboxylase, and alcohol dehydrogenase activities in anaerobic sweet potato roots.

The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O(2) and N(2) gases disappeared quickly and were replaced by CO(2). There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO(2) environment, followed by submergence and a N(2) environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO(2)-treated and 100% N(2)-treated roots. CO(2) atmospheres also resulted in higher pyruvate decarboxylase activity than did N(2) atmospheres. Concentrations of CO(2) were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO(2) concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase.

Journal Article↗

PubMed1998

Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures.

The crystal structures of pyruvate decarboxylase from the yeast Saccharomyces uvarum and Saccharomyces cerevisiae have been determined at 2.4 and 2.3 A resolution, respectively. These structures provide details about the protein fold and domain assembly within subunits, about subunit assembly to form dimers and about dimer assembly to form tetramers. They also provide a clear picture of the active site centered on the thiamin diphosphate cofactor, and have allowed amino acids critical for catalysis and involved in stabilization of the unusual cofactor conformation to be identified. The structural information has enabled identification of the site of allosteric activation to be centered on Cys-221, and suggests that a six residue segment leading from the regulatory site to the catalytic site may be involved in transmission of a binding signal. The importance of several amino acids within this segment in the regulatory process, as well as some involved in stabilizing and activating the cofactor has been confirmed by analyzing the behavior of recombinant enzymes with single point mutations introduced at these sites. Additional structures have been determined for pyruvate decarboxylase in multiple crystal forms, some of which were obtained from crystals grown with known allosteric activators present in the media. Currently four distinct types of tetramers have been observed, with each showing a different mode of association of dimers to form the tetramers. In some of the cases involving the presence of allosteric activators drastic changes in the mode of dimer assembly to form tetramers is seen.

Allosteric Regulation↗

PubMedDate not supplied

[Paracatalytic interaction of pyruvate decarboxylase with quinones in the presence of an organic solvent. Formation of an active site which hinders proton transfer].

Inactivation kinetics of pyruvate decarboxylase under joint action of substrate and substituted quinones in aqueous solutions which contain 1.0-13.5 vol.% of methyl alcohol has been investigated. The observed inactivation rate constant of pyruvate decarboxylase sharply decreases with the increase of methanol concentration from 4 up to 7 vol.% at pH 5.8-6.4. The decrease of the rate constant is independent of quinone order in the kinetic inactivation equation. The result is that the decrease of microscopic dielectric permeability by interaction of methanol with hydrophobic cities of enzyme active surface hinders the transfer of proton at the stage which is limiting in the inactivation process. It is assumed that the organization of active centre of pyruvate decarboxylase may depend on hydrophobic contact.

Benzoquinones↗

PubMed1980

Leigh's disease with decreased activities of pyruvate carboxylase and pyruvate decarboxylase.

In a patient with the clinical symptoms of Leigh's disease a partial deficiency of hepatic pyruvate carboxylase and pyruvate decarboxylase was found at necropsy. Cerebral pyruvate decarboxylase activity was also diminished. All enzyme activities were measured in total homogenates. The finding of typical necrotic lesions in the brain stem was consistent with the clinical diagnosis. During life moderate lactic acidaemia and no hypoglycaemia were observed, but an abnormal organic acid excretion pattern was present. The contribution of the enzyme defects to the aetiology of the disease is discussed.

Brain↗

PubMed2000

Generation of odorous acyloins by yeast pyruvate decarboxylases and their occurrence in sherry and soy sauce.

Volatile acyloins (alpha-hydroxy ketones) were obtained by condensing either aldehydes with pyruvate or 2-keto acids with acetaldehyde in a reaction catalyzed by yeast pyruvate decarboxylases (EC 4.1.1.1). Odor qualities and threshold values of 34 acyloins were evaluated, and 23 of them possessed distinct flavor properties. Sherry and soy sauce flavors were analyzed: 2-hydroxy-3-pentanone and 3-hydroxy-2-pentanone were identified in soy sauce for the first time; these and 2-hydroxy-5-methyl-3-hexanone and 3-hydroxy-1-phenyl-2-butanone were isolated from sherry for the first time. The biocatalytic efficiencies of crude pyruvate decarboxylase preparations from Zygosaccharomyces bisporus, Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxianus were compared. Product yields comparable to those of conversions with purified pyruvate decarboxylase demonstrated the suitability of crude enzyme extracts as cost-effective biocatalysts in acyloin formation. Conversion rates of >50% showed that the potential of this type of enzyme to catalyze the formation of aliphatic acyloins has been underestimated before.

Alcoholic Beverages↗

PubMed1991

PDC6, a weakly expressed pyruvate decarboxylase gene from yeast, is activated when fused spontaneously under the control of the PDC1 promoter.

Three structural genes encode the pyruvate decarboxylase isoenzymes in the yeast Saccharomyces cerevisiae. PDC1 and PDC5 are active during glucose fermentation where PDC1 is expressed about six times more strongly than PDC5. Expression of PDC6 is weak and seems to be induced in ethanol medium. Consequently, pdc1 delta pdc5 delta double mutants do not ferment glucose and do not grow on glucose medium. Spontaneous mutants, derived from such a pdc1 pdc5 strain, were isolated which could again ferment glucose. They showed pyruvate decarboxylase activity due to a duplication of PDC6. The second copy of PDC6 was expressed under the control of the PDC1 promoter, which was still present in the pdc1 strain. However, the resulting PDC1-PDC6 fusion gene could only partially substitute for PDC1: to achieve normal growth and high pyruvate decarboxylase activity strains carrying PDC1-PDC6 required a functional PDC5 gene which is dispensable in a PDC1 wild-type background. Thus, expression of PDC5 depends on the state of the PDC1 locus: low in the PDC1 wild-type background and high in PDC1-PDC6 fusion strains and, as shown previously, in pdc1 mutants. The activation of PDC5 expression in PDC1-PDC6 strains may be due to particular properties of the PDC1-PDC6 fusion protein or simply to the weaker expression of PDC1-PDC6 in comparison to the wild-type PDC1 gene.

Alleles↗