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@article{839383,
author = {Mones, Letif and Kulhánek, Petr and Florian, Jan and Simon, Istvan and Fuxreiter, Monika},
article_number = {46(50)},
keywords = {SUBSTRATE-ASSISTED CATALYSIS; DIVALENT METAL-IONS; DNA-POLYMERASE-I; FREE-ENERGY; ECORV ENDONUCLEASE; CRYSTAL-STRUCTURE; ANGSTROM RESOLUTION; ENZYMATIC-REACTIONS; AQUEOUS-SOLUTION; BINDING-SITES},
language = {eng},
issn = {0006-2960},
journal = {Biochemistry},
title = {Probing the two-metal ion mechanism in the restriction endonuclease BamHI},
volume = {2007},
year = {2007}
}
TY - JOUR
ID - 839383
AU - Mones, Letif - Kulhánek, Petr - Florian, Jan - Simon, Istvan - Fuxreiter, Monika
PY - 2007
TI - Probing the two-metal ion mechanism in the restriction endonuclease BamHI
JF - Biochemistry
VL - 2007
IS - 46(50)
SP - 14514-14523
EP - 14514-14523
SN - 00062960
KW - SUBSTRATE-ASSISTED CATALYSIS
KW - DIVALENT METAL-IONS
KW - DNA-POLYMERASE-I
KW - FREE-ENERGY
KW - ECORV ENDONUCLEASE
KW - CRYSTAL-STRUCTURE
KW - ANGSTROM RESOLUTION
KW - ENZYMATIC-REACTIONS
KW - AQUEOUS-SOLUTION
KW - BINDING-SITES
N2 - The choreography of restriction endonuclease catalysis is a long-standing paradigm in molecular biology. Bivalent metal ions are required almost for all PD..D/ExK type enzymes, but the number of cofactors essential for the DNA backbone scission remained ambiguous. On the basis of crystal structures and biochemical data for various restriction enzymes, three models have been developed that assign critical roles for one, two, or three metal ions during the phosphodiester hydrolysis. To resolve this apparent controversy, we investigated the mechanism of BamHI catalysis using quantum mechanical/molecular mechanical simulation techniques and determined the activation barriers of three possible pathways that involve a Glu-113 or a neighboring water molecule as a general base or an external nucleophile that penetrated from bulk solution. The extrinsic mechanism was found to be the most favorable with an activation free energy of 23.4 kcal/mol, in reasonable agreement with the experimental data. On the basis of the effect of the individual metal ions on the activation barrier, metal ion A was concluded to be pivotal for the reaction, while the enzyme lacking metal ion B still has moderate efficiency. Thus, we propose that the catalytic scheme of BamHI does not involve a general base for nucleophile generation and requires one obligatory metal ion for catalysis that stabilizes the attacking nucleophile and coordinates it throughout the nucleophilic attack. Such a model may also explain the variation in the number of metal ions in the crystal structures and thus could serve as a framework for a unified catalytic scheme of type II restriction endonucleases.
ER -
MONES, Letif, Petr KULHÁNEK, Jan FLORIAN, Istvan SIMON a Monika FUXREITER. Probing the two-metal ion mechanism in the restriction endonuclease BamHI. \textit{Biochemistry}. 2007, roč.~2007, 46(50), s.~14514-14523, 9 s. ISSN~0006-2960.
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