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001-es BibID:	BIBFORM031388
Első szerző:	Böcskei Z.
Cím:	Crystallization and preliminary X-ray analysis of porcine muscle prolyl oligopeptidase / Z. Böcskei, M. Fuxreiter, G. Náray-Szabó, E. Szabó, L. Polgar
Dátum:	1998
Megjegyzések:	Prolyl oligopeptidase from pig muscle has been crystallized in complex with an inhibitor, using PEG 8000 and calcium acetate as precipitants. The crystals are orthorombic and the space group is P212121 with cell dimensions a = 111.8, b = 101.8, c = 72.4 Å. The asymmetric unit contains a single chain of prolyl oligopeptidase, corresponding to a specific volume of 2.55 Å3 Da-1 and a solvent content of 52%. The observed diffraction pattern extends to 2.3 Å resolution and the native crystals are well suited for structural analysis by X-ray diffraction methods.
Tárgyszavak:	Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban egyetemen (Magyarországon) készült közlemény
Megjelenés:	Acta crystallographica. Section D, Biological crystallography. - 54 : S2 (1998), p. 1414-1415. -
További szerzők:	Náray-Szabó Gábor Szabó E. Polgár László Fuxreiter Mónika (1969-) (kutató vegyész)
Internet cím:	DOI
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001-es BibID:	BIBFORM031413
035-os BibID:	PMID:9188736 WOS:A1997XD03700007
Első szerző:	Fuxreiter Mónika (kutató vegyész)
Cím:	Role of electrostatics at the catalytic metal binding site in xylose isomerase action : Ca(2+)-inhibition and metal competence in the double mutant D254E/D256E / Fuxreiter M., Bocskei Z., Szeibert A., Szabo E., Dallmann G., Naray-Szabo G., Asboth B.
Dátum:	1997
ISSN:	0887-3585
Megjegyzések:	The catalytic metal binding site of xylose isomerase from Arthrobacter B3728 was modified by protein engineering to diminish the inhibitory effect of Ca2+ and to study the competence of metals on catalysis. To exclude Ca2+ from Site 2 a double mutant D254E/D256E was designed with reduced space available for binding. In order to elucidate structural consequences of the mutation the binary complex of the mutant with Mg2+ as well as ternary complexes with bivalent metal ions and the open-chain inhibitor xylitol were crystallized for x-ray studies. We determined the crystal structures of the ternary complexes containing Mg2+, Mn2+, and Ca2+ at 2.2 to 2.5 Angstrom resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all metals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca2+ to that of the cofactors as well as results with Be2+ weaken the assumption that geometry differences should account for the catalytic noncompetence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca2+ inhibition was accompanied by a similar reduction in the enzymatic activity. Qualitative argumentation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significantly reduces the electrostatic stabilization of the transition state. Furthermore, due to the smaller size of the catalytic metal site, no water molecule, coordinating the metal, could be observed in ternary complexes of the double mutant. Consequently, the proton shuttle step in the overall mechanism should differ from that in the wild type. These effects can account for the observed decrease in catalytic efficiency of the D254E/D256E mutant enzyme.
Tárgyszavak:	Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban egyetemen (Magyarországon) készült közlemény
Megjelenés:	Proteins. - 28 : 2 (1997), p. 183-193. -
További szerzők:	Szeibert A. Szabó E. Dallmann, G. Náray-Szabó Gábor Asboth, B. Böcskei Z.
Internet cím:	DOI
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001-es BibID:	BIBFORM049596
Első szerző:	Lábas Anikó
Cím:	Optimization of reorganization energy drives evolution of the designed Kemp eliminase KE07 / A. Labas, E. Szabo, L. Mones, M. Fuxreiter
Dátum:	2013
ISSN:	1570-9639
Megjegyzések:	Understanding enzymatic evolution is essential to engineer enzymes with improved activities or to generate enzymes with tailor-made activities. The computationally designed Kemp eliminase KE07 carries out an unnatural reaction by converting of 5-nitrobenzisoxazole to cyanophenol, but its catalytic efficiency is significantly lower than those of natural enzymes. Three series of designed Kemp eliminases (KE07, KE70, KE59) were shown to be evolvable with considerable improvement in catalytic efficiency. Here we use the KE07 enzyme as a model system to reveal those forces, which govern enzymatic evolution and elucidate the key factors for improving activity. We applied the Empirical Valence Bond (EVB) method to construct the free energy pathway of the reaction in the original KE07 design and the evolved R7 1/3H variant. We analyzed catalytic effect of residues and demonstrated that not all mutations in evolution are favorable for activity. In contrast to the small decrease in the activation barrier, in vitro evolution significantly reduced the reorganization energy. We developed an algorithm to evaluate group contributions to the reorganization energy and used this approach to screen for KE07 variants with potential for improvement. We aimed to identify those mutations that facilitate enzymatic evolution, but might not directly increase catalytic efficiency. Computational results in accord with experimental data show that all mutations, which appear during in vitro evolution were either neutral or favorable for the reorganization energy. These results underscore that distant mutations can also play role in optimizing efficiency via their contribution to the reorganization energy. Exploiting this principle could be a promising strategy for computer-aided enzyme design. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:	Biochimica et Biophysica Acta (BBA). Proteins and Proteomics. - 1834 : 5 (2013), p. 908-917. -
További szerzők:	Szabó E. Mones, Letif Fuxreiter Mónika (1969-) (kutató vegyész)
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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