1887

Microbiology Society logo

Volume 138, Issue 4

Purification and characterization of pyruvate decarboxylase from Free

Abstract

Summary: Pyruvate decarboxylase from the obligate anaerobe was purified eightfold. The subunit was 57000 ± 3000 as estimated from SDS-PAGE, and the native estimated by gel filtration on a Superose 6 column was 240000, indicating that the enzyme is a tetramer. The values are comparable to those for pyruvate decarboxylase from and , which are also tetrameric enzymes. The enzyme was oxygen stable, and had a pH optimum within the range 6·3-6·7. It displayed sigmoidal kinetics for pyruvate, with a of 13 m, kinetic properties also found for pyruvate decarboxylase from yeast and differing from the Michaelis-Menten kinetics of the enzyme from No activators were found. -Chloromercuri-benzoate inhibited activity and the inhibition was reversed by the addition of dithiothreitol, indicating that cysteine is important in the active site. The N-terminal amino acid sequence of pyruvate decarboxylase was more similar to the sequence of than pyruvate decarboxylase.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Funding
This study was supported by the:
  • National Science Foundation and Technology Center (Award DE-FG02- 87ER-13719)
© Society for General Microbiology 1992
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-4-803
1992-04-01
2024-04-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/4/mic-138-4-803.html?itemId=/content/journal/micro/10.1099/00221287-138-4-803&mimeType=html&fmt=ahah

References

  1. Boiteux A., Hess B. 1970; Allosteric properties of yeast pyruvate decarboxylase. FEBS Letters 9:293–296
     [Google Scholar]
  2. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
     [Google Scholar]
  3. De Ley J., Schell J. 1959; Studies on the metabolism of Acetobacter peroxydans. II. The enzymic mechanism of lactate metabolism. Biochimica et Biophysica Acta 35:154–165
     [Google Scholar]
  4. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12:387–395
     [Google Scholar]
  5. Flatau S., Fischer G., Kleinpeter E., Schellenberger A. 1988; 31P NMR investigations on free and enzyme bound thiamine pyrophosphate. FEBS Letters 233:379–382
     [Google Scholar]
  6. Goodwin S., Zeikus J. G. 1987; Physiological adaptations of anaerobic bacteria to low pH: metabolic control of proton motive force in Sarcina ventriculi . Journal of Bacteriology 169:2150–2157
     [Google Scholar]
  7. Gounaris A. D., Turkenkopf I., Buckwald S., Young A. 1971; Pyruvate decarboxylase. I. Protein dissociation into subunits under conditions in which thiamine pyrophosphate is released. Journal of Biological Chemistry 246:1302–1309
     [Google Scholar]
  8. Gounaris A. D., Turkenkopf I., Civerchia L. L., Greenlie J. 1975; Pyruvate decarboxylase. III. Specificity restrictions for thiamine pyrophosphate in the protein association step, sub-unit structure. Biochimica et Biophysica Acta 405:492–499
     [Google Scholar]
  9. Haq A., Dawes E. A. 1971; Pyruvic acid metabolism and ethanol formation in Erwinia amylovora . Journal of General Microbiology 68:295–306
     [Google Scholar]
  10. Hoppner T. C., Doelle H. W. 1983; Purification and kinetic characteristics of pyruvate decarboxylase and ethanol dehydrogenase from Zymomonas mobilis in relation to ethanol production. European Journal of Applied Microbiology and Biotechnology 17:152–157
     [Google Scholar]
  11. Jordan F., Kuo D. J., Monse E. U. 1978; A pH-rate determination of the activity-pH profile of enzymes. Application to yeast pyruvate decarboxylase demonstrating the existence of multiple ionizable groups. Analytical Biochemistry 86:298–302
     [Google Scholar]
  12. Kellerman E., Seeboth P. G., Hollenberg C. P. 1986; Analysis of the primary structure and promoter function of a pyruvate decarboxylase gene (PDC1) from Saccharomyces cerevisiae . Nucleic Acids Research 14:8963–8977
     [Google Scholar]
  13. King T., Cheldelin V. H. 1954; Pyruvic carboxylase of Acetobacter suboxydans . Journal of Biological Chemistry 208:821–831
     [Google Scholar]
  14. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
     [Google Scholar]
  15. Lowe S. E., Zeikus J. G. 1991; Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi . Archives of Microbiology 155:325–329
     [Google Scholar]
  16. Moench T. T., Zeikus J. G. 1983; An improved preparation method for a titanium(III) media reductant. Journal of Microbiological Methods 1:199–202
     [Google Scholar]
  17. Neale A. D., Scopes R. K., Wettenhall R. E. H., Hoogenraad N. J. 1987a; Pyruvate decarboxylase of Zymomonas mobilis: isolation, properties, and genetic expression in Escherichia coli . Journal of Bacteriology 169:1024–1028
     [Google Scholar]
  18. Neale A., D” Scopes R. K., Wettenhall R. E. H., Hoogenraad N. J. 1987b; Nucleotide sequence of the pyruvate decarboxylase gene from Zymomonas mobilis . Nucleic Acids Research 15:1753–1761
     [Google Scholar]
  19. Stephenson M. P., Dawes E. A. 1971; Pyruvic acid and formic acid metabolism in Sarcina ventriculi and the role of ferredoxin. Journal of General Microbiology 69:331–343
     [Google Scholar]
  20. Thauer R. K., Rupprecht E., Jungermann K. 1970; Glyoxylate inhibition of clostridial pyruvate synthase. FEBS Letters 9:271–273
     [Google Scholar]
  21. Uhlemann H., Schellenberger A. 1976; Glyoxylic acid as an active site marker of yeast pyruvate decarboxylase. FEBS Letters 63:37–39
     [Google Scholar]
  22. Ullrich J., Donner I. 1970; Kinetic evidence for two active sites in cytoplasmic yeast pyruvate decarboxylase. Hoppe-Seyler's Zeit- schrift fur Physiologische Chemie 350:1026–1029
     [Google Scholar]
  23. Ullrich J., Wittorf J. H., Gubler C. J. 1966; Molecular weight and coenzyme content of pyruvate decarboxylase from brewer's yeast. Biochimica et Biophysica Acta 113:595–604
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-138-4-803
Loading
Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi
Microbiology 138, 803 (1992); https://doi.org/10.1099/00221287-138-4-803
/content/journal/micro/10.1099/00221287-138-4-803
/content/journal/micro/10.1099/00221287-138-4-803
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error