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| Purification and Characterization of Pyruvate Kinase II from Escherichia coli and Its Application as a Coupling Enzyme |
| LI Hong-yan1, ZHOU Si-tian1, MA Jian-hui1, WANG Yan-xing1,2, SUN Mei-hao1 |
1. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China;
2. Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China |
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Abstract Pyruvate kinase (PK), catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to ADP during glycolysis, which step is essentially irreversible under intracellular conditions and is allosterically controlled. In Escherichia coli, there are two kinds of PKs, namely PKI (PKF) and PKII (PKA) which were activated by fructose 1,6-bisphosphate and adenosine 5'-monophosphate, respectively. PKI had been extensively studied, while PKII had little been investigated largely due to its instability reported by Waywood et al. in 1975. To further characterize PKII, it was prokaryotically overexpressed and purified. Following the determination of kinetic constants for NDP, their catalytic efficiencies (CEs) were calculated and compared. PKII and PK from rabbit muscle (PKr) had similar CEs respect to ADP, GDP and UDP, and PKII had 6.5 times higher CE with CDP than that for PKr. Distinct from published results, little activity decrease was found during 18 months －80℃ storage or 24 h room temperature treatment. High catalytic efficiency and stability enabled PKII as a potentially good coupling enzyme. Together with lactate dehydrogenase, PKII was successfully used as coupling enzyme to determine the kinetic constants of sulfate activating complex (SAC) from Rhodobacter sphaeroides and its mutants, and K409 and S410 were found to be essential for its activity of APSK domain.
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Received: 20 November 2012
Published: 25 May 2013
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