GLK E.Coli, Active
Description
Introduction to GLK E.Coli, Active
GLK E.Coli (glucokinase) is an ATP-dependent enzyme from Escherichia coli that phosphorylates glucose to glucose-6-phosphate. While not essential for glucose uptake via the phosphoenolpyruvate:sugar phosphotransferase (PTS) system, it plays a critical role in metabolizing glucose liberated from disaccharides like maltose or lactose . Recombinant GLK E.Coli is produced in E. coli with an N-terminal His-tag for purification and is widely used in biochemical studies .
Molecular Properties
Key Features:
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His-Tag: Facilitates purification via affinity chromatography .
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Active Site: Residues Asn99, Asp100, Glu157, His160, and Glu187 bind glucose, structurally conserved in hexokinases .
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Dimerization: Forms a homodimer with two domains: a small α/β domain (residues 2–110 and 301–321) and a larger α+β domain (residues 111–300) .
Gene Regulation and Expression
GLK is regulated by FruR, a global carbon metabolism regulator. A FruR consensus binding motif is located 123 bp upstream of the glk promoter .
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Expression Patterns:
Interactions with Metabolic Systems:
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Maltose System: Overexpression of GLK represses mal gene expression, particularly in strains with constitutive maltose transport (e.g., malK mutants) . This repression highlights GLK’s role in modulating catabolite repression.
Biological Role in E. coli Metabolism
Primary Functions:
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Disaccharide Utilization: Phosphorylates glucose released from maltose, trehalose, or lactose .
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Acid Stress Response: GLK, alongside PykF and Pgk, elevates ATP levels under weakly acidic conditions, enhancing survival .
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Regulatory Cross-Talk: Competes with the PTS system for glucose metabolism, influencing carbon source preference .
Functional Redundancy:
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glk mutations alone show minimal growth defects, but combined with ptsG ptsM mutations (blocking PTS-mediated glucose uptake), severe growth impairment occurs .
Research Applications and Mutational Studies
Adaptive Enzyme Engineering:
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NanK Mutations: L84P/R substitutions in nanK enhance glucokinase activity (6.1–6.9× increase in k<sub>cat</sub>/K<sub>m</sub> for glucose), with minor trade-offs in native N-acetylmannosamine phosphorylation .
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Metabolic Engineering: GLK’s role in acid stress adaptation makes it a target for optimizing E. coli survival in industrial fermentation .
Experimental Uses:
Product Specs
Glucokinase, ECK2384, JW2385.
Escherichia Coli.
MGSSHHHHHH SSGLVPRGSH MGSMTKYALV GDVGGTNARL ALCDIASGEI SQAKTYSGLD YPSLEAVIRV YLEEHKVEVK DGCIAIACPI TGDWVAMTNH TWAFSIAEMK KNLGFSHLEI INDFTAVSMA IPMLKKEHLI QFGGAEPVEG KPIAVYGAGT GLGVAHLVHV DKRWVSLPGE GGHVDFAPNS EEEAIILEIL RAEIGHVSAE RVLSGPGLVN LYRAIVKADN RLPENLKPKD ITERALADSC TDCRRALSLF CVIMGRFGGN LALNLGTFGG VFIAGGIVPR FLEFFKASGF RAAFEDKGRF KEYVHDIPVY LIVHDNPGLL GSGAHLRQTL GHIL
Product Science Overview
Recombinant DNA technology involves inserting the gene encoding glucokinase into E. coli, which then expresses the enzyme. This method allows for the production of large quantities of the enzyme with high purity and activity. The recombinant glucokinase produced in E. coli is typically a single, non-glycosylated polypeptide chain containing 344 amino acids, with a molecular mass of approximately 37.1 kDa .
The enzyme is often tagged with a His-tag at the N-terminus to facilitate purification using affinity chromatography techniques . The resulting product is highly pure, with a purity greater than 95% as determined by SDS-PAGE .
The biological activity of recombinant glucokinase is measured by its ability to catalyze the phosphorylation of glucose to glucose-6-phosphate. The specific activity of the enzyme is typically greater than 70 units/mg, where one unit is defined as the amount of enzyme that will oxidize 1.0 µmole of glucose to glucose-6-phosphate per minute in the presence of beta-NADP at pH 9.0 and 37°C .
Recombinant glucokinase from E. coli is used in various biochemical and physiological studies, particularly those related to glucose metabolism and regulation. It is also employed in high-throughput screening assays for drug discovery and development, as well as in the study of enzyme kinetics and mechanisms .
For optimal stability, the recombinant glucokinase should be stored at 4°C if it will be used within 2-4 weeks. For longer-term storage, it is recommended to store the enzyme at -20°C, with the addition of a carrier protein such as 0.1% HSA or BSA to prevent degradation. It is important to avoid multiple freeze-thaw cycles to maintain the enzyme’s activity .
