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001-es BibID:	BIBFORM031392
035-os BibID:	PMID:11732923 WOS:000172608100033
Első szerző:	Fuxreiter Mónika (kutató vegyész)
Cím:	Probing the general base catalysis in the first step of BamHI action by computer simulations / Monika Fuxreiter, Roman Osman
Dátum:	2001
ISSN:	0006-2960
Megjegyzések:	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 metal-catalyzed 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.
Tárgyszavak:	Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban külföldön készült közlemény
Megjelenés:	Biochemistry. - 40 : 49 (2001), p. 15017-15023. -
További szerzők:	Osman, Roman
Internet cím:	Intézményi repozitóriumban (DEA) tárolt változat DOI
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001-es BibID:	BIBFORM031390
035-os BibID:	PMID:10423235 WOS:000081768900004
Első szerző:	Fuxreiter Mónika (kutató vegyész)
Cím:	Role of active site residues in the glycosylase step of T4 endonuclease V. Computer simulation studies on ionization states / Monika Fuxreiter, Arieh Warshel, Roman Osman
Dátum:	1999
ISSN:	0006-2960
Megjegyzések:	T4 Endonuclease V (EndoV) is a base excision repair enzyme that removes thymine dimers (TD) from damaged DNA. To elucidate the role of the active site residues in catalysis, their pK(a)'s were evaluated using the semimicroscopic version of the protein dipoles-Langevin dipoles method (PDLD/S). Contributions of different effects to the pK(a) such as charge-charge interactions, conformational rearrangement, protein relaxation, and DNA binding were analyzed in detail. Charging of the active site residues was found to be less favorable in the complex than in the free enzyme. The pK(a) of the N-terminus decreased from 8.01 in the free enzyme to 6.52 in the complex, while the pK(a) of Glu-23 increased from 1. 52 to 7.82, which indicates that the key residues are neutral in the reactant state of the glycosylase step. These pK(a)'s are in agreement with the optimal pH range of the reaction and support the N-terminus acting as a nucleophile. The Glu-23 in its protonated form is hydrogen bonded to O4' of the sugar of 5' TD and can play a role in increasing the positive charge of C1' and, hence, accelerating the nucleophilic substitution. Furthermore, the neutral Glu-23 is a likely candidate to protonate O4' to induce ring opening required to complete the glycosylase step of EndoV. The positively charged Arg-22 and Arg-26 provide an electrostatically favorable environment for the leaving base. To distinguish between S(N)1 and S(N)2 mechanisms of the glycosylase step the energetics of protonating O2 of 5' TD was calculated. The enzyme was found to stabilize the neutral thymine by approximately 3.6 kcal/mol, whereas it destabilizes the protonated thymine by approximately 6.6 kcal/mol with respect to an aqueous environment. Consequently, the formation of a protonated thymine intermediate is unlikely, indicating an S(N)2 reaction mechanism for the glycosylase step.
Tárgyszavak:	Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban egyetemen (Magyarországon) készült közlemény
Megjelenés:	Biochemistry. - 38 : 30 (1999), p. 9577-9589. -
További szerzők:	Warshel, Arieh Osman, Roman
Internet cím:	Intézményi repozitóriumban (DEA) tárolt változat Szerző által megadott URL DOI
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001-es BibID:	BIBFORM031412
035-os BibID:	PMID:18020376 WOS:000251547700018
Első szerző:	Mones, Letif
Cím:	Probing the two-metal ion mechanism in the restriction endonuclease BamHI / Letif Mones, Petr Kulhánek, Jan Florián, István Simon, Monika Fuxreiter
Dátum:	2007
ISSN:	0006-2960
Megjegyzések:	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.
Tárgyszavak:	Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban külföldön készült közlemény
Megjelenés:	Biochemistry. - 46 : 50 (2007), p. 14514-14523. -
További szerzők:	Kulhánek, Petr Florián, Jan Simon István Fuxreiter Mónika (1969-) (kutató vegyész)
Pályázati támogatás:	T049073 OTKA F046164 OTKA
Internet cím:	Intézményi repozitóriumban (DEA) tárolt változat DOI
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