PMID:15237399

Citation	Itoh, A, Ohashi, Y, Soga, T, Mori, H, Nishioka, T and Tomita, M (2004) Application of capillary electrophoresis-mass spectrometry to synthetic in vitro glycolysis studies. Electrophoresis 25:1996-2002
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.
Links	PubMed Online version:10.1002/elps.200305905
Keywords	Catalysis; Cloning, Molecular; Electrophoresis, Capillary/methods; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/metabolism; Gene Expression; Genetic Engineering; Glucose/metabolism; Glycolysis; Mass Spectrometry/methods; Pyruvic Acid/metabolism; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Analysis, DNA; Time Factors
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