The Catalytic Tyr-9 of Glutathione S-Transferase A1-1 Controls the Dynamics of the C terminus

Abstract

The glutathione S-transferase enzymes (GSTs) have a tyrosine or serine residue at their active site that hydrogen bonds to and stabilizes the thiolate anion of glutathione, GS ؊ . The importance of this hydrogen bond is obvious, in light of the enhanced nucleophilicity of GS ؊ versus the protonated thiol. Several A-class GSTs contain a C-terminal segment that undergoes a ligand-dependent local folding reaction. Here, we demonstrate the effects of the Y9F substitution on binding affinity for glutathione conjugates and on rates of the order-disorder transition of the C terminus in rat GST A1-1. The equilibrium binding affinity of the glutathione conjugate, GS-NBD (NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole), was decreased from 4.09 M to 0.641 M upon substitution of Tyr-9 with Phe. This result was supported by isothermal titration calorimetry, with K d values of 1.51 M and 0.391 M for wild type and Y9F, respectively. The increase in binding affinity for the mutant is associated with dramatic decreases in rates for the C-terminal order-disorder transition, based on a stopped-flow kinetic analysis. The same effects were observed, qualitatively, for a second GSH conjugate, GSethacrynic acid. Apparently, the phenolic hydroxyl group of Tyr-9 is critical for orchestrating C-terminal dynamics and efficient product release, in addition to its role in lowering the pK a of GSH.The importance of protein dynamics and flexibility to the energetics of ligand binding is well appreciated and has been illustrated in a number of systems (1-5). In many cases, a protein-ligand interaction appears to drive a flexible segment to fold into a rigid, ligand-bound conformation (6, 7). An analogy between global protein folding and ligand binding has been only recently described (8 -11). The concept of folding funnels, where folding progresses via multiple routes rather than a single pathway, can be likened to binding where a ligand drives an ensemble of local states to a single conformation. However, the detailed mechanism of any ligand-dependent, localized folding process remains elusive and is probably less well understood than global folding (12).The glutathione S-transferase (GST) 1 isoform A1-1 provides an outstanding opportunity to dissect the mechanism of a liganddependent local folding reaction. The GSTs are a family of xenobiotic metabolizing enzymes that play a critical role in the detoxication of various endogenous and exogenous compounds. The mammalian cytosolic GSTs consist of seven classes based on sequence similarity and substrate selectivity: ␣ (A), (P), (M), (T), (K), (S), and (Z) (13-17). The catalytic activity of GSTs is based on deprotonation of GSH to form the thiolate (GS Ϫ ), which is a superior nucleophile compared with the protonated thiol. X-ray crystallographic structures and site-directed mutagenesis studies illustrate that each GST contains a conserved tyrosine or serine residue that contributes to thiolate formation through a hydrogen bond (OH⅐⅐⅐⅐ Ϫ SG) and reduces the pK a of bound GSH. Although mutation a...

Tertiary Interactions Stabilise the C-terminal Region of Human Glutathione Transferase A1-1: a Crystallographic and Calorimetric Study

“…Although access to the active-site is hindered by the C-terminal region of the apo protein, 25 deletion of the region, while increasing the rate of ligand binding, 10,11,25 brings about an almost complete loss (O99%) of catalytic function. 14,24 Prior to its ligand-induced localisation at the active-site, a mobile and positionally displaced C-terminal region provides for rapid access to the site.…”

“…14,22,23 Given that the catalytic and ligandin functions of GST A1-1 are tightly coupled to the C-terminal region, insight into the dynamics of the region is important to understanding the mechanisms by which the protein functions. 11,14,16,17,[24][25][26][27][28] In the present study, we investigated further the importance of tertiary interactions in determining the conformational dynamics of the C-terminal region in apo and ligand-complexed hGST A1-1.…”

Regulation of Cell Physiology and Pathology by Protein S-Glutathionylation: Lessons Learned from the Cardiovascular System

“…Although GSTs are known to catalyze these detoxification reactions, their role in S-glutathionylation of protein cysteines represents a previously uncharacterized function in redox signaling. The three major GST groups, alpha, mu and pi, utilize the ionization of GSH by hydrogen bonding to the active site tyrosine residue of GST (119). These enzymes among others may allow for S-glutathionylation of higher pKa protein cysteines within the cell.…”

The Anomalous pK of Tyr-9 in Glutathione S-Transferase A1-1 Catalyzes Product Release

“…1). Indirect evidence from several laboratories suggests an intimate link between the ionization state of Tyr-9 and the dynamics of the C terminus (14,15,17,18). One possible implication is that ligands will preferentially bind to or disso-ciate from the ionized state of the enzyme.…”