Home Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase
Article
Licensed
Unlicensed Requires Authentication

Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase

  • Christian Boehme , Frank Bieber , Julia Linnemann , Reinhard Breitling , Stefan Lorkowski and Siegmund Reissmann EMAIL logo
Published/Copyright: January 16, 2013
Biological Chemistry
From the journal Biological Chemistry Volume 394 Issue 5

Abstract

The stepwise synthesis of thymidine triphosphate (TTP) requires a kinase for phosphorylation in the last step. Because pyruvate kinase (PK) using phosphoenolpyruvate (PEP) as substrate can regenerate adenosine triphosphate and phosphorylate thymidine diphosphate as well, we chose this enzyme for the synthesis of TTP via an enzymatic cascade reaction. The metalloenzyme PK shows pronounced promiscuity and therefore fits well to the conditions of this reaction. PK commonly used today is isolated from rabbit muscle. We cloned and expressed the respective open reading frame in Escherichia coli, purified, and characterized the His-tagged recombinant enzyme. The enzyme has an activity optimum at 37°C and in the pH range from 7.4 to 7.8. Km constants conformed well with the isolated native enzyme for adenosine diphosphate (ADP) to 0.37±0.02 mm and for PEP to 0.07±0.01 mm. The recombinant enzyme shows the following range in its substrate specificity: ADP>dADP>dGDP>dCDP>thymidine diphosphate (TDP). It allows the phosphorylation of TDP to TTP in high yield (up to 95%). The metal ions Mg2+ and K+ are necessary for full enzymatic activity. The addition of transition metal ions such as Mn2+, Cu2+, Co2+, and Ni2+ reduces activity. Storage of the enzyme at -20°C retains full activity.

Keywords: comparison to native enzyme; enzymatic synthesis of thymidine triphosphate; influence of metal ions; kinetic parameters; purification; recombinant expression
Corresponding author: Siegmund Reissmann, Faculty of Biology and Pharmacy, Friedrich Schiller University Jena, Institute of Biochemistry and Biophysics, Dornburger Str. 25, D-07743 Jena, Germany
Received: 2012-7-12
Accepted: 2012-12-18
Published Online: 2013-01-16
Published in Print: 2013-05-01

©2013 by Walter de Gruyter Berlin Boston

Articles in the same Issue

  1. Masthead
  2. Masthead
  3. Guest Editorial
  4. Highlight: European BioEnergetics Conference 2012 in Freiburg
  5. Highlight: European Bioenergetics Conference 2012 in Freiburg
  6. Structure of caa3 cytochrome c oxidase – a nature-made enzyme-substrate complex
  7. Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins
  8. pKa values and redox potentials of proteins. What do they mean?
  9. Cyclophilin D as a potential target for antioxidants in neurodegeneration: the X-ALD case
  10. A critical appraisal of the role of respiratory supercomplexes in mitochondria
  11. Mechanistic aspects of sodium-binding sites in LeuT-like fold symporters
  12. Hydroxynonenal-stimulated activity of the uncoupling protein in Acanthamoeba castellanii mitochondria under phosphorylating conditions
  13. The antiporter-like subunit constituent of the universal adaptor of complex I, group 4 membrane-bound [NiFe]-hydrogenases and related complexes
  14. Alternate pathways for NADH oxidation in Thermus thermophilus using type 2 NADH dehydrogenases
  15. A new trend in the complex I research field
  16. Research Articles/Short Communications
  17. Protein Structure and Function
  18. Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase
  19. Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase
https://doi.org/10.1515/hsz-2012-0334
Keywords for this article
comparison to native enzyme; enzymatic synthesis of thymidine triphosphate; influence of metal ions; kinetic parameters; purification; recombinant expression

Articles in the same Issue

  1. Masthead
  2. Masthead
  3. Guest Editorial
  4. Highlight: European BioEnergetics Conference 2012 in Freiburg
  5. Highlight: European Bioenergetics Conference 2012 in Freiburg
  6. Structure of caa3 cytochrome c oxidase – a nature-made enzyme-substrate complex
  7. Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins
  8. pKa values and redox potentials of proteins. What do they mean?
  9. Cyclophilin D as a potential target for antioxidants in neurodegeneration: the X-ALD case
  10. A critical appraisal of the role of respiratory supercomplexes in mitochondria
  11. Mechanistic aspects of sodium-binding sites in LeuT-like fold symporters
  12. Hydroxynonenal-stimulated activity of the uncoupling protein in Acanthamoeba castellanii mitochondria under phosphorylating conditions
  13. The antiporter-like subunit constituent of the universal adaptor of complex I, group 4 membrane-bound [NiFe]-hydrogenases and related complexes
  14. Alternate pathways for NADH oxidation in Thermus thermophilus using type 2 NADH dehydrogenases
  15. A new trend in the complex I research field
  16. Research Articles/Short Communications
  17. Protein Structure and Function
  18. Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase
  19. Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2025 De Gruyter Brill
Downloaded on 3.10.2025 from https://www.degruyterbrill.com/document/doi/10.1515/hsz-2012-0334/html
Scroll to top button