tailieunhanh - Báo cáo khóa học: S-(2,3-Dichlorotriazinyl)glutathione A new affinity label for probing the structure and function of glutathione transferases

S-(2,3-Dichlorotriazinyl)glutathione (SDTG) was synthes-ized and shown to be an effective alkylating affinity label for recombinant maize glutathioneS-transferase I (GST I). InactivationofGST IbySDTGatpH followedbiphasic pseudo-first-order saturation kinetics. The biphasic kinetics can be described in terms of a fast initial phase of inactiva-tion followed by a slower phase, leading to 42 ± 3% residual activity. | Eur. J. Biochem. 271 3503-3511 2004 FEBS 2004 doi 5- 2 3-Dichlorotriazinyl glutathione A new affinity label for probing the structure and function of glutathione transferases Georgia A. Kotzia and Nikolaos E. Labrou Laboratory of Enzyme Technology Department of Agricultural Biotechnology Agricultural University of Athens Greece S- 2 3-Dichlorotriazinyl glutathione SDTG was synthesized and shown to be an effective alkylating affinity label for recombinant maize glutathione S-transferase I GST I . Inactivation of GST IbySDTGatpH followed biphasic pseudo-first-order saturation kinetics. The biphasic kinetics can be described in terms of a fast initial phase of inactivation followed by a slower phase leading to 42 3 residual activity. The rate of inactivation for both phases exhibits nonlinear dependence on SDTG concentration consistent with the formation of a reversible complex with the enzyme Kj M for the fast phase and M for the slow phase before irreversible modification with maximum rate constants of min-1 and min-1 for the fast and slow phases respectively. Protection from inactivation was afforded by substrate analogues demonstrating the specificity of the reaction. When the enzyme was inactivated 42 residual activity w 1 mol SDTG per mol dimeric enzyme was incorporated. Amino-acid analysis molecular modelling and site-directed mutagenesis studies suggested that the modifying residue is Met121 which is located at the end of a-helix H 3 and forms part of the xenobiotic-binding site. The results reveal an unexpected structural communication between subunits which consists of mutually exclusive modification of Met residues across enzyme subunits. Thus modification of Met121 on one subunit prevents modification of Met121 on the other subunit. This communication is governed by Phe51 which is located at the dimer interface and forms part of the hydrophobic lock-and-key intersubunit .