Logo of prosciprotein sciencecshl presssubscriptionsetoc alertsthe protein societyjournal home
Protein Sci. 1995 Aug; 4(8): 1498–1506.
PMCID: PMC2143176

Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.


The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (1.6M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anderson RA, Bosron WF, Kennedy FS, Vallee BL. Role of magnesium in Escherichia coli alkaline phosphatase. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2989–2993. [PMC free article] [PubMed]
  • Cathala G, Brunel C. Bovine kidney alkaline phosphatase. Catalytic properties, subunit interactions in the catalytic process, and mechanism of Mg2+ stimulation. J Biol Chem. 1975 Aug 10;250(15):6046–6053. [PubMed]
  • Coleman JE. Structure and mechanism of alkaline phosphatase. Annu Rev Biophys Biomol Struct. 1992;21:441–483. [PubMed]
  • DAYAN J, WILSON IB. THE PHOSPHORYLATION OF TRIS BY ALKALINE PHOSPHATASE. Biochim Biophys Acta. 1964 Mar 9;81:620–623. [PubMed]
  • ENGSTROM L. Incorporation of inorganic phosphate into alkaline phosphatase from Escherichia coli. Biochim Biophys Acta. 1962 Jan 29;56:606–609. [PubMed]
  • Evans SV. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graph. 1993 Jun;11(2):134–128. [PubMed]
  • Kim EE, Wyckoff HW. Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis. J Mol Biol. 1991 Mar 20;218(2):449–464. [PubMed]
  • Ma L, Kantrowitz ER. Mutations at histidine 412 alter zinc binding and eliminate transferase activity in Escherichia coli alkaline phosphatase. J Biol Chem. 1994 Dec 16;269(50):31614–31619. [PubMed]
  • Murphy JE, Xu X, Kantrowitz ER. Conversion of a magnesium binding site into a zinc binding site by a single amino acid substitution in Escherichia coli alkaline phosphatase. J Biol Chem. 1993 Oct 15;268(29):21497–21500. [PubMed]
  • SCHWARTZ JH, LIPMANN F. Phosphate incorporation into alkaline phosphatase of E. coli. Proc Natl Acad Sci U S A. 1961 Dec 15;47:1996–2005. [PMC free article] [PubMed]
  • Simpson RT, Vallee BL. Two differentiable classes of metal atoms in alkaline phosphatase of Escherichia coli. Biochemistry. 1968 Dec;7(12):4343–4350. [PubMed]
  • Sowadski JM, Handschumacher MD, Murthy HM, Foster BA, Wyckoff HW. Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution. J Mol Biol. 1985 Nov 20;186(2):417–433. [PubMed]
  • Sowadski JM, Handschumacher MD, Murthy HM, Kundrot CE, Wyckoff HW. Crystallographic observations of the metal ion triple in the active site region of alkaline phosphatase. J Mol Biol. 1983 Oct 25;170(2):575–581. [PubMed]
  • Tibbitts TT, Xu X, Kantrowitz ER. Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site. Protein Sci. 1994 Nov;3(11):2005–2014. [PMC free article] [PubMed]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...