The enzymatic decarboxylation of hydroxypyruvate associated with purified pyruvate decarboxylase from wheat germ

Highly purified pyruvic decarboxylase (EC 4.1.1.1) from wheat germ catalyses the decarboxylation of hydroxypyruvate. A kinetic analysis of the activity of the enzyme with pyruvate and hydroxypyruvate as substrates suggests that a single enzyme is involved. The kinetics of decarboxylation are autocatalytic. The time lag before maximum activity is reached is affected by the concentration of hydroxypyruvate and the pH. The question whether or not hydroxypyruvate is a natural substrate for the enzyme remains unresolved, but it may be significant that at physiological pH (ca 7.5) the enzyme shows optimum activity with hydroxypyruvate, but negligible activity with pyruvate.     

Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast

Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.     

Sucrose synthase from wheat leaves. Comparison with the wheat germ enzyme

Sucrose synthase (UDP glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) was partially purified from wheat ( Triticum aestivum L. cv. San Agustin INTA) leaves and its properties compared with the wheat germ enzyme. The leaf enzyme moved faster in polyacrylamide gel electrophoresis, was more sensitive to SH reagents and crossreacted more slowly with antibody prepared towards the germ enzyme. Kinetic constants were of the same order for all substrates. UDP was a strong inhibitor of the synthesis reaction. MgCl₂ stimulated this reaction and partially reversed UDP inhibition. Molecular weight determined by gel filtration was 380 and 370 kdalton for the leaf and germ enzymes respectively. Both enzymes presented forms of higher molecular weight estimated to around 800 and 1000 kdalton. Neither sucrose synthase from leaves nor from germ were affected by fructose 6-P, fructose 1,6—P₂, glucose 1—P, glucose 6—P, fructose 2,6—P₂ and cAMP.     

Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity.

In the yeast, Saccharomyces cerevisiae, pyruvate decarboxylase (Pdc) is encoded by the two isogenes PDC1 and PDC5. Deletion of the more strongly expressed PDC1 gene stimulates the promoter activity of both PDC1 and PDC5, a phenomenon called Pdc autoregulation. Hence, pdc1Delta strains have high Pdc specific activity and can grow on glucose medium. In this work we have characterized the mutant alleles pdc1-8 and pdc1-14, which cause strongly diminished Pdc activity and an inability to grow on glucose. Both mutant alleles are expressed as detectable proteins, each of which differs from the wild-type by a single amino acid. The cloned pdc1-8 and pdc1-14 alleles, as well as the in-vitro-generated pdc1-51 (Glu51Ala) allele, repressed expression of PDC5 and diminished Pdc specific activity. Thus, the repressive effect of Pdc1p on PDC5 expression seems to be independent of its catalytic activity. A pdc1-8 mutant was used to isolate spontaneous suppressor mutations, which allowed expression of PDC5. All three mutants characterized had additional mutations within the pdc1-8 allele. Two of these mutations resulted in a premature translational stop conferring phenotypes virtually indistinguishable from those of a pdc1Delta mutation. The third mutation, pdc1-803, led to a deletion of two amino acids adjacent to the pdc1-8 mutation. The alleles pdc1-8 and pdc1-803 were expressed in Escherichia coli and purified to homogeneity. In the crude extract, both proteins had 10% residual activity, which was lost during purification, probably due to dissociation of the cofactor thiamin diphosphate (ThDP). The defect in pdc1-8 (Asp291Asn) and the two amino acids deleted in pdc1-803 (Ser296 and Phe297) are located within a flexible loop in the beta domain. This domain appears to determine the relative orientation of the alpha and gamma domains, which bind ThDP. Alterations in this loop may also affect the conformational change upon substrate binding. The mutation in pdc1-14 (Ser455Phe) is located within the ThDP fold and is likely to affect binding and/or orientation of the cofactor in the protein. We suggest that autoregulation is triggered by a certain conformation of Pdc1p and that the mutations in pdc1-8 and pdc1-14 may lock Pdc1p in vivo in a conformational state which leads to repression of PDC5.     

Partial purification of actin from wheat germ

A protein, one of two major components in a preparation obtained from wheat germ, has been identified as actin by its solubility properties, mobility in SDS-acrylamide gel electrophoresis, ability to react with an antiserum against turkey-gizzard actin, and ability to form needle-shaped fibrils.     

Purification, characterisation and cDNA sequencing of pyruvate decarboxylase from Zygosaccharomyces bisporus

Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the non-oxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha4) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent Km value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.     

Aspartate aminotransferase from wheat germ: purification and kinetic properties.

A convenient method for the purification of aspartate aminotransferase [L-aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1)] from wheat germ is described. An overall purification of 150 fold was achieved. On polyacrylamide gel electrophoresis at pH 8.9 the purified enzyme revealed two protein bands both provided with enzymatic activity. The holoenzyme is readily resolved on conversion to the aminic form and gel-filtration. The apoenzyme is reactivated by pyridoxal-5-phosphate. Kinetic data indicate that a Ping-Pong mechanism is operative similar to that found for the tyrosine aminotransferase by Litwack and Cleland (1968). Phosphate ion behaves as a competitive inhibitor towards the coenzyme. The relatively low affinity between coenzyme and apoenzyme from wheat germ allowed the determination of the dissociation constants for coenzymes (pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate) and of the inhibition constant for phosphate.     

Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis.

Six different pyruvate decarboxylase mutants of Saccharomyces cerevisiae were isolated. They belong to two unlinked complementation groups. Evidence is presented that one group is affected in a structural gene. The fact that five of the six mutants had residual pyruvate decarboxylase activity provided the opportunity for an intensive physiological characterization. It was shown that the loss of enzyme activity in vitro is reflected in a lower fermentation rate, an increased pyruvate secretion, and slower growth on a 2% glucose medium. The different effects of antimycin A on leaky mutants grown on ethanol versus the same mutants grown on glucose support the view that glucose induces some of the glycolytic enzymes, especially pyruvate decarboxylase.     

Purification and partial characterization of pyruvate decarboxylase from Oryza sativa L

Pyruvate decarboxylase(PyrDC) was purified from rice bran to a specific activity of 1 mu kat/mg and partially characterized. The holoenzyme is a tetramer of two types of subunits with molecular masses 64 kDa and 62 kDa. Purified rice PyrDC exhibits positive cooperative kinetics with respect to pyruvate and functions with a significant lag phase. When compared to other plant PyrDC, the lag phase was shorter at low pyruvate concentrations and the S0.5 was smaller. The optimum pH (6.25) was also less acidic and the enzyme retained 30% of its maximal activity at neutral pH. In contrast to other plant PyrDC, rice PyrDC could be active at the onset of anoxia and would be activated by small changes in pyruvate concentration.     

Expression of active yeast pyruvate decarboxylase in Escherichia coli.

We have shown by appropriate modification of the translational signals and using the strong T7 RNA polymerase promoter phi 10, that a cloned Saccharomyces cerevisiae pyruvate decarboxylase gene (pdc1) can be expressed in Escherichia coli. This protein, which migrated as a single band on SDS-polyacrylamide gels, was found to have a subunit molecular mass of approximately 62 kDa, similar to that of the enzyme produced by yeast. Polyclonal antibodies raised against purified yeast pyruvate decarboxylase recognized this bacterially produced protein. We found that this recombinant enzyme is active, indicating that the homotetramer encoded by the pdc1 gene is functional.     

Solubilization and partial purification of yeast chitin synthetase. Confirmation of the zymogenic nature of the enzyme.

Chitin synthetase was solubilized with digitonin from a particulate yeast fraction. The solubilized enzyme, which did not sediment at 200,000 X g and emerged after the void volume in a Sepharose 6B column, was active only after treatment with a protease. This confirms that chitin synthetase exists in the plasma membrane as a zymogen and that initiation of the chitin septum occurs by localized activation of the enzyme. By differential extraction with sodium cholate and digitonin, followed by chromatography on Sepharose 6B, a 20-fold purification of the enzyme was achieved with respect to the crude particles. The purified enzyme showed a requirement for a phospholipid; phosphatidylserine and lysophosphatidylserine were the best activators. Unsaturated fatty acids strongly inhibited synthetase activity, whereas their saturated counterparts were inert. The solubilized enzyme catalyzed the formation of insoluble chitin in the absence of added primer. The synthetic polysaccharide was examined by electron microscopy and found to consist of lozenge-shaped particles about 60 nm long and 10 nm wide.