Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF

• Reactivity: Ec
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF Summary

• Details of Functionality: Measured by its ability to phosphorylate N-acetyl-D-glucosamine. The specific activity is >10,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived e. coli N-Acetyl-D-Glucosamine Kinase/NAGK protein
  Met1-Asp303 with C-terminal 6-His tag
• Accession #: P75959
• N-terminal Sequence: Met1
• Protein/Peptide Type: Recombinant Enzymes
• Gene: nagK
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <0.01 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 34 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 37-38 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij-35 and DTT.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MgCl₂, 10 mM CaCl₂, pH 7.0
  • Recombinant E.coli N‑Acetyl‑D‑Glucosamine Kinase/NAGK (rE.coli NAGK) (Catalog # 8020-GK)
  • Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
  • N-acetyl-alpha -D-glucosamine (GlcNAc) (Calbiochem, Catalog # 1079), 1 M stock in deionized water
  • Universal Kinase Activity Kit (Catalog # EA004)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  2. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Prepare a reaction mixture composed of 0.5 mM ATP and 12.5 mM GlcNAc in Assay Buffer.
  4. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.
  5. Dilute rE.coli NAGK to 1 µg/mL in Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 20 µL of the 1 µg/mL rE.coli NAGK into the plate. Include a control containing 20 µL of Assay Buffer.
  8. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to the wells, excluding the standard curve.
  9. Add 20 µL of reaction mixture to the wells, excluding the standard curve.
  10. Cover the plate with a plate sealer and incubate at room temperature for 10 minutes.
  11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  12. Add 100 µL of deionized water to all wells. Mix briefly.
  13. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  14. Read plate at 620 nm (absorbance) in endpoint mode.
  15. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg) x Coupling Rate**)

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for control.
  ** Under these conditions, the coupling rate is 0.475.

  Per Reaction:
  • rE.coli NAGK: 0.020 µg
  • Coupling Phosphatase 4: 0.1 ug
  • ATP: 0.2 mM
  • GlcNAc: 5 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF

• EC 2.7.1.59
• GlcNAc Kinase
• GNK
• GNKGlcNAc kinase
• N-acetyl-D-glucosamine kinase
• N-Acetylglucosamine Kinase
• N-acetylglucosamine kinaseHSA242910
• NAGK

Background

N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) are repeating sugar units of peptidoglycan, the major component of bacterial cell wall structure and a drug target of various antibiotics including penicillin (1). Recently, interest has been generated regarding cell wall peptidoglycan catabolism, because as much as 50% of the peptidoglycan is turned over in one generation of bacterial growth (2). N-acetylglycosamine kinase (nagK) is a key enzyme for the recycling of GlcNAc in E. coli (3). Due to its high activity, it can be used for efficient conversion of GlcNAc to GlcNAc-6-phosphate. The enzyme is assayed using a phosphatase-coupled kinase assay (4).

1. Plumbridge, J. (2009). J. Bacteriol. 191:5641.
2. Reith, J. et al (2011). J. Bacteriol. 193:5386.
3. Uehara, T. and Park, J.T.(2011) J. Bacteriol. 186:7273.
4. Wu, Z.L. (2011) PLoS One 6:e23172.

Bioinformatics

• Gene Symbol: nagK
• Uniprot:
  • P75959()