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Estimation of phosphoenolpyruvate carboxylation mediated by phosphoenolpyruvate carboxykinase (PCK) in engineered Escherichia coli having high ATP

Paper ID Volume ID Publish Year Pages File Format Full-Text
17174 42649 2013 5 PDF Available
Title
Estimation of phosphoenolpyruvate carboxylation mediated by phosphoenolpyruvate carboxykinase (PCK) in engineered Escherichia coli having high ATP
Abstract

•Kinetic parameters of recombinant PCK and PPC for PEP carboxyation reaction in an engineered condition were provided.•PEP carboxylation activities by native PCK and PPC were compared.•Extra ATP formation by PCK activity and biotechnological benefits thereby is discussed.

We have previously reported that phosphoenolpyruvate carboxykinase (PCK) overexpression under glycolytic conditions enables Escherichia coli to harbor a high intracellular ATP pool resulting in enhanced recombinant protein synthesis. To estimate how much PCK-mediated phosphoenolpyruvate (PEP) carboxylation is contributed to the ATP increase under engineered conditions, the kinetics of PEP carboxylation by PCK and substrate competing phosphoenolpyruvate carboxylase (PPC) were measured using recombinant enzymes. The PEP carboxylation catalytic efficiency (kcat/Km) of the recombinant PCK was 660 mM−1 min−1, whereas that of the recombinant PPC was 1500 mM−1 min−1. Under the presence of known allosteric effectors (fructose 1,6-bisphosphate, acetyl-CoA, ATP, malate, and aspartate) close to in vivo conditions, the catalytic efficiency of PCK-mediated PEP carboxylation (84 mM−1 min−1) was 28-folds lower than that of PPC (2370 mM−1 min−1). To verify the above results, an E. coli strain expressing native PCK and PPC under control of identical promoter was constructed by replacing PCK promoter region with that of PPC in chromosome. The native PCK activity (33 nmol/mg-protein min) was 5-folds lower than PPC activity (160 nmol/mg-protein min) in the cell extract from the promoter-exchanged strain. Intracellular modifications of ATP concentration by PCK activity and the consequences for biotechnology are further discussed.

Keywords
Phosphoenolpyruvate carboxylkinase (PCK); Phosphoenolpyruvate carboxylase (PPC); High intracellular ATP concentration; Enzyme kinetics
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Estimation of phosphoenolpyruvate carboxylation mediated by phosphoenolpyruvate carboxykinase (PCK) in engineered Escherichia coli having high ATP
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 53, Issue 1, 10 June 2013, Pages 13–17
Authors
, , , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us