Probing the general base catalysis in the first step of BamHI action by computer simulations

M. Fuxreiter, R. Osman

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

BamHI is a type II restriction endonuclease that catalyzes the scission of the phoshodiester bond in the GAGTCC cognate sequence in the presence of two divalent metal ions. The first step of the reaction is the preparation of water for nucleophilic attack by Glu-113, which has been proposed to abstract the proton from the attacking water molecule. Alternatively, the 3′-phosphate group to the susceptible phosphodiester bond has been suggested to play a role as the general base. The two hypotheses have been tested by computer simulations using the semiempirical protein dipoles Langevin dipoles (PDLD/S) method. Deprotonation of water by Glu-113 has been found to be less favorable by 5.7 kcal/mol than metalcatalyzed deprotonation with a concomitant proton transfer to bulk solvent. The preparation of the nucleophile by the 3′-phosphate group is less favorable by 12.3 kcal/mol. These results suggest that both the general base and the substrate-assisted mechanisms in the first step of BamHI action are less likely than the metal-catalyzed reaction. The metal ions in the active site of BamHI make the largest contributions to the reduction of the free energy of hydroxide ion formation. On the basis of these findings we propose that the first step of endonuclease catalysis does not require a general base; rather, the essential attacking nucleophile in BamHI catalytic action is stabilized by the metal ions.

Original languageEnglish
Pages (from-to)15017-15023
Number of pages7
JournalBiochemistry
Volume40
Issue number49
DOIs
Publication statusPublished - Dec 11 2001

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Catalysis
Computer Simulation
Metal ions
Deprotonation
Nucleophiles
Metals
Water
Computer simulation
Type II Site Specific Deoxyribonucleases
Ions
Phosphates
Protons
Proton transfer
Endonucleases
Free energy
Catalytic Domain
Molecules
Substrates
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Probing the general base catalysis in the first step of BamHI action by computer simulations. / Fuxreiter, M.; Osman, R.

In: Biochemistry, Vol. 40, No. 49, 11.12.2001, p. 15017-15023.

Research output: Contribution to journalArticle

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