eng URI http://hdl.handle.net/10795/2664 φαρμακευτικό προϊόν ένζυμο βιοχημεία βιοτεχνολογία Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/ CBL-GSH complex was solved at 2.1 A° resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the Cterminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated. 11 pp. LOMv1.0 creator LOMv1.0 publisher LOMv1.0 Project Final Beneficiary LOMv1.0 Project Executing Organisation 2013-02-27 Digital Library of the Operational Programme "Education and Lifelong Learning" abstract types Text Glutathione transferases Chemotherapeutic drugs Enzyme kinetics Cellular detoxification Crystal structure Kinetic analysis Molecular dynamics Chlorambucil MDR GTs CBL http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0056337 751048 application/pdf http://repository.edulll.gr/edulll/bitstream/10795/2664/2/2664_Paper%204.pdf URI http://hdl.handle.net/10795/2664 LOMv1.0creator 2016-04-15T07:25:11Z LOMv1.0validator 2016-04-15T07:25:11Z LOMv1.0 gre no no Copyright EYD-EPEDBM (Operational Programme "Education and Lifelong Learning")