Application of capillary electrophoresis-mass spectrometry to synthetic in vitro glycolysis studies

Aya Itoh, Yoshiaki Ohashi, Tomoyoshi Soga, Hirotada Mori, Takaaki Nishioka, Masaru Tomita

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

We propose an approach designed to reconstitute a metabolic pathway composed of multistep biochemical reactions, rather than to dissect the individual reactions that make up the pathway. A synthetic in vitro glycolysis was reconstructed from ten purified Escherichia coli (E. coli) enzymes to obtain a better understanding of the regulation of sequential enzymatic reactions. The key to the success of this approach is the ability to perform direct and simultaneous determination of the diverse metabolic intermediates in the pathway by capillary electrophoresis-mass spectrometry. We observed that the pathway is regulated by a delicate balance between the changing metabolite concentrations and behaves like a natural biological oscillating network that has hitherto not been reported for E. coli glycolysis. The end-product, pyruvate, was periodically synthesized from glucose at an overall efficiency of 30%, corresponding to an average of 90% conversion efficiency for each of the ten steps involved. This approach is likely useful for the synthesis of natural products requiring complex sequential biocatalytic reactions.

Original languageEnglish
Pages (from-to)1996-2002
Number of pages7
JournalELECTROPHORESIS
Volume25
Issue number13
DOIs
Publication statusPublished - 2004 Jul 1

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Keywords

  • Capillary electrophoresis-electrospray ionization-mass spectrometry
  • Glycolysis

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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