S‐(2,3‐Dichlorotriazinyl)glutathione

Abstract

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 I by SDTG at pH 6.5 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 (K d 107.9 ± 2.1 lM for the fast phase, and 224.5 ± 4.2 lM for the slow phase) before irreversible modification with maximum rate constants of 0.049 ± 0.002 min )1 and 0.0153 ± 0.001 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), % 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 motif. The ability of SDTG to inactivate other glutathione-binding enzymes and GST isoenzymes was also investigated, and it was concluded that this new reagent may have general applicability as an affinity reagent for other enzymes with glutathione-binding sites.

The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

“…This indicated that the reaction obeyed pseudo-first-order saturation kinetics and was consistent with reversible binding of reagent prior to covalent modification according to the following equation:where E represents the free enzyme, E∶CBL is the reversible complex and E-CBL is the covalent product. The steady-state rate equation for the interaction is [30], [33], [34], [46], [47]:where k obs is the observed rate of enzyme inactivation for a given concentration of CBL, k 3 is the maximal rate of inactivation (min −1 ), and K D is the apparent dissociation constant of the E∶CBL complex. From the data shown in Figure 6, K D values of 2.95±0.5 mM and 6.05±0.8 mM, for the fast and slow reactions, respectively, were determined.…”