Ca2+ binding in the active site of HincII: Implications for the catalytic mechanism

Christopher Etzkorn, Nancy C Horton

Research output: Contribution to journalArticle

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Abstract

The 2.8 Å crystal structure of the type II restriction endonuclease HincII bound to Ca2+ and cognate DNA containing GTCGAC is presented. The DNA is uncleaved, and one calcium ion is bound per active site, in a position previously described as site I in the related blunt cutting type II restriction endonuclease EcoRV [Horton, N. C., Newberry, K. J., and Perona, J. J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13489-13494], as well as that found in other related enzymes. Unlike the site I metal in EcoRV, but similar to that of PvuII, NgoMIV, BamHI, BglII, and BglI, the observed calcium cation is directly ligated to the pro-Sp oxygen of the scissile phosphate. A calcium ion-ligated water molecule is well positioned to act as the nucleophile in the phosphodiester bond cleavage reaction, and is within hydrogen bonding distance of the conserved active site lysine (Lys 129), as well as the pro-Rp oxygen of the phosphate group 3′ of the scissile phosphate, suggesting possible roles for these groups in the catalytic mechanism. Kinetic data consistent with an important role for the 3′-phosphate group in DNA cleavage by HincII are presented. The previously observed sodium ion [Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47] persists in the active sites of the Ca 2+-bound structure; however, kinetic data show little effect on the single-turnover rate of DNA cleavage in the absence of Na+ ions.

Original languageEnglish (US)
Pages (from-to)13256-13270
Number of pages15
JournalBiochemistry
Volume43
Issue number42
StatePublished - Oct 26 2004

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Catalytic Domain
Type II Site Specific Deoxyribonucleases
Phosphates
Ions
DNA Cleavage
DNA
Calcium
Oxygen
Nucleophiles
Kinetics
Chemical bonds
Hydrogen Bonding
Lysine
Cations
Hydrogen bonds
Crystal structure
Metals
Sodium
Molecules
Water

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ca2+ binding in the active site of HincII : Implications for the catalytic mechanism. / Etzkorn, Christopher; Horton, Nancy C.

In: Biochemistry, Vol. 43, No. 42, 26.10.2004, p. 13256-13270.

Research output: Contribution to journalArticle

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