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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Characterization of a bifunctional HPr kinase/phosphorylase from Leuconostoc mesenteroides SY1
Jae-Yong Park1, Kang Wook Lee, Ae Ran Lee
1Institute of Agriculture & Life Science, Gyeongsang National University,Jinju 660-701, Korea.
Abstract:
The hprK gene encoding bifunctional HPrK/P (kinase/ phosphorylase) was cloned from L. mesenteroides SY1, a strain isolated from kimchi. hprK was transcribed as a monocistronic gene. His-tagged HPrH16A and HPrK/P were produced in E. coli BL21(DE3) using pET26b(+) and purified. HPrK/P phosphorylation assay with purified proteins showed that the kinase activity of HPrK/P increased at slightly acidic pHs. Divalent cations such as Mg2+ and Mn2+ and glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and phosphoenolpyruvate (PEP) increased the kinase activity of HPrK/P, but inorganic phosphate strongly inhibited it. Kinetic studies for the kinase activity of HPrK/P showed that the apparent Km values were 0.18 and 14.57 microM for ATP and HPr, respectively. The Km value for the phosphorylase activity of HPrK/P was 14.16 microM for P-Ser-HPr (HPr phosphorylated at the serine residue).
Insights
The bifunctional HPrK/P enzyme from L. mesenteroides SY1, involved in carbohydrate metabolism, shows increased kinase activity at acidic pH and is regulated by various molecules. Its kinetic properties for both kinase and phosphorylase activities were characterized.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- The hprK gene encodes the bifunctional HPrK/P enzyme, crucial for carbohydrate metabolism.
- This study focuses on L. mesenteroides SY1, a strain isolated from kimchi.
Purpose of the Study:
- To clone and characterize the hprK gene and its encoded HPrK/P enzyme.
- To investigate the enzymatic activities and regulatory mechanisms of HPrK/P.
Main Methods:
- Gene cloning and protein expression in E. coli.
- Purification of His-tagged HPrH16A and HPrK/P.
- Enzymatic assays to determine kinase and phosphorylase activities.
- Kinetic studies to determine Km values for substrates.
Main Results:
- The hprK gene was cloned and HPrK/P was successfully produced and purified.
- Kinase activity was optimal at slightly acidic pH and enhanced by Mg2+, Mn2+, FBP, and PEP, but inhibited by inorganic phosphate.
- Kinetic parameters for ATP, HPr, and P-Ser-HPr were determined for kinase and phosphorylase activities, respectively.
Conclusions:
- HPrK/P from L. mesenteroides SY1 is a bifunctional enzyme with characterized kinase and phosphorylase activities.
- Its activity is modulated by pH, divalent cations, glycolytic intermediates, and inorganic phosphate, suggesting a role in metabolic regulation.
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