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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred


IMPACT FACTOR 2018: 3.014
5-year IMPACT FACTOR: 3.162

CiteScore 2018: 3.09

SCImago Journal Rank (SJR) 2018: 1.482
Source Normalized Impact per Paper (SNIP) 2018: 0.820

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1437-4315
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Volume 394, Issue 5

Issues

Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase

Christian Boehme
  • LAROVA GmbH, Loebstedter Str. 80, D-07749 Jena, Germany
  • Faculty of Biology and Pharmacy, Friedrich Schiller University Jena, Institute of Nutrition, Dornburger Str. 25, D-07743 Jena, Germany
  • Other articles by this author:
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/ Frank Bieber / Julia Linnemann / Reinhard Breitling / Stefan Lorkowski
  • Faculty of Biology and Pharmacy, Friedrich Schiller University Jena, Institute of Nutrition, Dornburger Str. 25, D-07743 Jena, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Siegmund Reissmann
  • Corresponding author
  • Faculty of Biology and Pharmacy, Friedrich Schiller University Jena, Institute of Biochemistry and Biophysics, Dornburger Str. 25, D-07743 Jena, Germany
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Published Online: 2013-01-16 | DOI: https://doi.org/10.1515/hsz-2012-0334

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.

This article offers supplementary material which is provided at the end of the article.

Keywords: comparison to native enzyme; enzymatic synthesis of thymidine triphosphate; influence of metal ions; kinetic parameters; purification; recombinant expression

About the article

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: 2012-07-12

Accepted: 2012-12-18

Published Online: 2013-01-16

Published in Print: 2013-05-01


Citation Information: Biological Chemistry, Volume 394, Issue 5, Pages 695–701, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2012-0334.

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