GLK E.coli
Description
Molecular Structure and Recombinant Production
GLK E. coli exists as a homodimer, with each monomer comprising 321 amino acids (native form) or 344 amino acids (recombinant form fused with a 23-residue N-terminal His-tag) . The enzyme folds into two distinct domains:
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Small α/β domain: Residues 2–110 and 301–321, forming a mixed β-sheet flanked by α-helices.
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Larger α+β domain: Residues 111–300, containing the active site .
The active site is situated in a cleft between the domains, where glucose binds via hydrogen interactions with residues Asn99, Asp100, Glu157, His160, and Glu187 . Domain closure occurs upon glucose binding, with a maximum Cα shift of ~10 Å, a mechanism conserved in hexokinases .
| Parameter | Value | Source |
|---|---|---|
| Native monomer length | 321 amino acids | |
| Recombinant length | 344 amino acids (His-tag included) | |
| Molecular weight | 35–37.1 kDa | |
| Quaternary structure | Homodimer |
Biochemical Properties and Activity
GLK E. coli exhibits strict substrate specificity for glucose, with negligible activity toward mannose or galactose . Key kinetic parameters include:
| Parameter | Value | Conditions | Source |
|---|---|---|---|
| (glucose) | 0.78 mM | pH 9.0, 37°C, NADP+ | |
| (ATP) | 3.76 mM | Same as above | |
| 158 U/mg protein | Same as above |
Activity is measured by monitoring NADPH reduction at 340 nm, as glucose is oxidized to G6P . The enzyme operates optimally at pH 9.0 and 37°C .
Functional Role in E. coli Metabolism
GLK’s role is context-dependent:
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PTS-Deficient Strains: GLK compensates for PTS inactivity, enabling glucose utilization via non-PTS transporters like GalP or MglABC .
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Low Glucose Conditions: In chemostat cultures with micromolar glucose, GLK facilitates glucose uptake via ABC transporters, bypassing PTS .
Key Pathways Involving GLK:
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Glycolysis: G6P enters glycolysis, producing energy for growth.
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Regulatory Signaling: Free intracellular glucose modulates the maltose system (Mal) by influencing endogenous inducer formation .
Regulation and Gene Expression
The glk gene is regulated by:
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FruR (Fructose Repressor): A FruR consensus binding motif upstream of glk modulates expression. FruR mutations increase glk transcription, while overexpression of FruR+ represses it .
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Glucose Availability: Growth on glucose reduces glk expression by ~50%, as PTS-mediated glucose uptake dominates .
| Condition | Effect on glk Expression | Mechanism | Source |
|---|---|---|---|
| High glucose | Reduced expression (~50%) | PTS-mediated repression | |
| FruR mutation | Increased expression | Relief of FruR-mediated repression |
Overexpression of glk represses the maltose system, particularly in strains with constitutive mal gene expression (e.g., malK-lacZ fusions). This repression is attributed to elevated intracellular glucose levels, which inhibit MalK (ATPase subunit of the maltose transporter) .
Functional Studies
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Maltose System Interference: Overexpression of glk in malK-lacZ strains reduces mal gene expression by 80-fold, demonstrating glucose’s role in endogenous inducer formation .
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Growth in PTS-Deficient Strains: glk overexpression restores growth on glucose in PTS mutants, highlighting its compensatory role .
Product Specs
Q&A
What is the molecular structure and function of glucokinase in E. coli?
Glucokinase (GLK) in E. coli is a cytoplasmic protein with a molecular weight of approximately 35,000 Da . The enzyme is encoded by the glk gene and catalyzes the phosphorylation of glucose to glucose-6-phosphate using ATP as a phosphoryl donor . This reaction is critical for glucose metabolism, especially when glucose enters the cell through non-phosphorylating transport systems such as the galactose permeases .
Unlike eukaryotic hexokinases, E. coli glucokinase is highly specific for glucose and doesn't significantly phosphorylate other hexoses . The protein lacks a signal sequence, confirming its cytoplasmic localization rather than periplasmic orientation . This distinguishes it from a 47,000 Da glucose-phosphorylating activity that has been reported to be released from the periplasm under certain stress conditions .
What are the kinetic parameters of E. coli glucokinase?
The purified glucokinase from E. coli exhibits the following kinetic parameters:
| Parameter | Value | Units |
|---|---|---|
| Km for glucose | 0.78 | mM |
| Km for ATP | 3.76 | mM |
| Vmax | 158 | U/mg protein |
These parameters indicate that the enzyme has a relatively high affinity for glucose but a lower affinity for ATP . The Km value for glucose (0.78 mM) is consistent with earlier studies using crude extracts, which reported a Km of 0.7 mM .
How is the glk gene regulated in E. coli?
Expression of the glk gene is subject to several regulatory mechanisms:
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Glucose repression: Growth on glucose reduces the expression of glk by approximately 50% .
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FruR regulation: The fruR gene product (also known as Cra, catabolite repressor/activator) appears to play a role in glk regulation. A fruR mutation slightly increases the expression of glk-lacZ, while overexpression of plasmid-encoded fruR+ weakly decreases expression .
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FruR binding site: A FruR consensus binding motif has been identified 123 bp upstream of the potential transcriptional start site of glk, suggesting direct regulation by this transcription factor .
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Other environmental factors: The expression of glk may be influenced by growth conditions such as glucose limitation in chemostat cultures, where glucokinase plays an important role when glucose is present at micromolar levels .
How does overexpression of glk affect other metabolic pathways in E. coli?
Overexpression of glk has been shown to interfere with the expression of the maltose system in E. coli . This interference varies depending on the genetic background:
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Wild-type strains: In wild-type strains growing on maltose, the effect of glk overexpression is minimal .
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Constitutive mal gene expression strains: The repression is strongest in strains exhibiting constitutive mal gene expression due to endogenous induction and in the absence of a functional MalK protein (the ATP-hydrolyzing subunit of the maltose transport system) . In a glk::Tn10(Cam) malK-lacZ fusion strain with constitutive mal gene expression, malK-lacZ expression was reduced 8.2-fold when glk was overexpressed .
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Multiple transporter mutations: The effect was even more dramatic (80-fold reduction) when glk was overexpressed in a strain carrying both a glk mutation and ptsG ptsM double mutations, which lack additional genes encoding glucose-phosphorylating transport systems .
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malT(Con) or malQ mutants: Interestingly, the constitutive expression of malK-lacZ was not reduced at all by overexpressing glk in these mutants .
These findings demonstrate that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system, and glucokinase activity can modulate this process by affecting the levels of free glucose .
What is the relationship between glucokinase and the PTS system in E. coli glucose metabolism?
The PTS (phosphotransferase system) is the primary glucose uptake and phosphorylation pathway in E. coli, while glucokinase provides an alternative route for glucose phosphorylation . Their relationship includes:
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Functional redundancy: E. coli strains with a glk mutation alone show no significant growth defect on glucose, as the PTS pathway can compensate . Severe reduction in growth is observed only when a strain lacks both glucokinase and the ability to phosphorylate glucose via the PTS pathway .
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Internal glucose phosphorylation: There is evidence that enzyme IIGlc of the PTS is also responsible for the utilization of internal glucose, providing another mechanism for glucose phosphorylation independent of glucokinase .
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Low glucose conditions: Under glucose-limited conditions (micromolar levels), glucose uptake occurs preferentially via the galactose-binding protein-dependent ABC transporter as free glucose, highlighting an important role for glucokinase under these specific conditions .
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Disaccharide metabolism: When E. coli utilizes glucose-containing disaccharides such as lactose, maltose, or trehalose, the metabolism involves the formation of glucose inside the cell. Despite the apparent need for glucose phosphorylation, a glk mutation alone does not significantly impair the utilization of these disaccharides .
How does glucokinase activity impact carbon catabolite repression in E. coli?
Glucokinase activity can influence carbon catabolite repression mechanisms through its effect on internal glucose levels:
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Free internal glucose signaling: By controlling the phosphorylation of free glucose inside the cell, glucokinase affects the concentration of this important signaling molecule .
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Maltose system regulation: The strong repression of the maltose system observed when glucokinase is overexpressed indicates that the enzyme plays a role in modulating catabolite repression of the mal genes, likely by reducing the levels of free internal glucose that contribute to endogenous inducer formation .
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Integration with other regulatory networks: The regulation of glk by FruR (Cra) connects glucokinase activity to the broader carbon flux sensing network in E. coli .
What approaches can be used to clone and characterize the glk gene from E. coli?
The following methodological approach has been successfully used to clone and characterize the glk gene:
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Selection strategy: Use a strain that is mutated in glk and deleted for ptsI, ptsH, and crr (UE79). This strain cannot grow on glucose even in the presence of D-fucose (a non-metabolizable inducer of galactose permeases) .
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Library construction: Create a gene bank of partially digested Sau3A DNA fragments from a wild-type E. coli strain, ligated into a suitable vector such as pBR322 linearized with BamHI .
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Transformation and selection: Transform the library into the selection strain and plate on minimal medium containing glucose and D-fucose. Colonies that grow represent potential glk-complementing clones .
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Verification: Verify glucokinase activity in the selected clones through enzyme assays, and confirm the identity of the glk gene through restriction mapping and sequencing .
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Expression analysis: Create glk-lacZ fusions to monitor gene expression under various conditions, such as growth on different carbon sources or in different genetic backgrounds .
What are effective methods for measuring glucokinase activity in E. coli?
Glucokinase activity can be measured using the following approaches:
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Coupled enzyme assay: The standard method involves coupling glucose-6-phosphate formation to NADP+ reduction via glucose-6-phosphate dehydrogenase, measuring the increase in absorbance at 340 nm due to NADPH formation .
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Reaction conditions: Typical assay conditions include:
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Buffer: 100 mM Tris-HCl, pH 7.5
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Substrates: 10 mM glucose, 5 mM ATP
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Cofactors: 10 mM MgCl2
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Coupling enzyme: Glucose-6-phosphate dehydrogenase
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Detection: NADPH formation at 340 nm
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Kinetic analysis: For determining Km and Vmax values, assays are performed with varying concentrations of substrates (glucose and ATP) .
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Protein purification: For detailed characterization, glucokinase can be purified from overexpression strains using:
How can researchers design experiments to study the effects of glk mutations or overexpression?
To study the effects of glk mutations or overexpression, researchers can employ the following experimental approaches:
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Construction of glk null mutants:
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Overexpression systems:
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Reporter gene fusions:
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Growth phenotype analysis:
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Analysis of maltose system expression:
Product Science Overview
Recombinant glucokinase is often expressed in Escherichia coli (E. coli) due to the bacterium’s well-characterized genetics, rapid growth, and ability to express high levels of recombinant proteins . The recombinant glucokinase protein expressed in E. coli is typically used for research purposes, including studies on enzyme kinetics, glucose metabolism, and diabetes .
Glucokinase has a high Km for glucose, meaning it is effective only when glucose is abundant . This property makes it a key regulator of glucose levels in the body. In pancreatic beta cells, glucokinase acts as a glucose sensor, modulating insulin secretion in response to blood glucose levels . In the liver, it facilitates the uptake and conversion of glucose, acting as an insulin-sensitive determinant of hepatic glucose usage .
Mutations in the glucokinase gene (GCK) can lead to various metabolic disorders. For instance, certain mutations are associated with non-insulin-dependent diabetes mellitus (NIDDM), maturity-onset diabetes of the young (MODY2), and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) . Understanding the function and regulation of glucokinase is therefore critical for developing therapeutic strategies for these conditions.
