Alkaline phosphatase

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Alkaline Phosphatase is a hydrolytic enzyme found in the periplasmic space of E.coli that removes inorganic phosphates from phosphate monoesters. They are commonly used to remove the 5' phosphate from DNA or RNA. The homodimeric enzyme consists of two identical subunits, 429 a.a in size, each with an active site containing 3 metal binding sites; M1, M2 and M3. M1 and M2 have a Zn²+ attached creating Zn1 and Zn2. M3 has an associated Mg²+ ion. These three binding sites form a catalytic metal triad which catalyses two chemical displacement steps in order to remove the phosphate. Alkaline Phosphatases are so named because of their high activity rate at alkaline pH.

DNA Manipulation

Alkaline Phosphatases are useful in the manipulation of DNA in order to insert a DNA fragment into a plasmid or a bacteriophage vector. They remove the 5' phosphate group from the ends of the plasmid or phage that have been cut with a restriction endonuclease to prevent the ligation of the original parent without the insertion of foreign DNA - so called self-ligation [1][2][3][4][5].

References

  1. Structure and mechanism of alkaline phosphatase Coleman JE; (1992); 21:441-83
  2. Reaction mechanism of alkaline phosphatase based on crystal structures. Two - metal ion catalysis. Kimm EE, Wyckoff HW; (1991); 218(2):449-64
  3. A revised mechanism for the alkaline phosphatase reaction involving three metal ions. Stec B, Holtz KM, Kantrowitz ER; (2000); 299(5):1303-11
  4. Phosphate monoester hydrolysis by trinuclear alkaline phosphatase; DPT study of transition states and reaction mechanism. Chen SL, Liao RZ; (2014, Aug 4); 15(11):2321-30
  5. Alkaline Phosphatase (2000, Feb 19) http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/phosphatase.html
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