Catalytic Mechanism and Role of Hydroxyl Residues in the Active Site of Theta Class Glutathione S-Transferases

Abstract

Spectroscopic and kinetic studies have been per-formed on the Australian sheep blowfly Lucilia cuprina glutathione S-transferase (Lucilia GST; EC 2.5.1.18) to clarify its catalytic mechanism. Steady state kinetics of Lucilia GST are non-Michaelian, but the quite hyper-bolic isothermic binding of GSH suggests that a steady state random sequential Bi Bi mechanism is consistent with the anomalous kinetics observed. The rate-limiting step of the reaction is a viscosity-dependent physical event, and stopped-flow experiments indicate that prod-uct release is rate-limiting. Spectroscopic and kinetic data demonstrate that Lucilia GST is able to lower the pK a of the bound GSH from 9.0 to about 6.5. Based on crystallographic suggestions, the role of two hydroxyl residues, Ser-9 and Tyr-113, has been investigated. Re-moval of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pK a of bound GSH to about 7.6, and a very low turnover number (about 0.5% of that of wild type) is observed. This inactivation may be ex-plained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an in-volvement in the stabilization of the ionized GSH. This serine residue is highly conserved in the Theta class GSTs, so the present findings may be applicable to all of the family members. Tyr-113 appears not to be essential for the GSH acti-vation. Stopped-flow data indicate that removal of the hydroxyl group of Tyr-113 does not change the rate-limiting step of reaction but causes an increase of the rate constants of both the formation and release of the GSH conjugate. Tyr-113 resides on ␣-helix 4, and its hy-droxyl group hydrogen bonds directly to the hydroxyl of Tyr-105. This would reduce the flexibility of a protein region that contributes to the electrophilic substrate binding site; segmental motion of ␣-helix 4 possibly mod-ulates different aspects of the catalytic mechanism of the Lucilia GST. Glutathione S-transferases (GSTs; 1 EC 2.5.1.18) are a wide group of isoenzymes able to catalyze the conjugation of GSH with a variety of electrophilic molecules (1– 6). The cytosolic GSTs are dimeric enzymes subdivided into at least five main classes, Alpha, Mu, Pi (7), Theta (8), and Sigma (9, 10), and are characterized by a low sequence homology (less than 30%). Despite this heterogeneity, the overall polypeptide fold is very similar among the crystal structures so far obtained (6), and all GSTs are highly selective for the GSH molecule. An important conserved residue between classes is a tyrosine near the N-terminal region (Tyr-6 in rat GST M1–1 (11), Tyr-7 in human GST P1–1 (12), and Tyr-8 in human GST A1–1 (13)) that has been proposed to activate the bound GSH by stabilizing its thiolate form. The blowfly Lucilia cuprina infests Australian sheep flocks, and the GST isoenzyme purified from this species (14) is possibly involved in the mechanism of insecticide resist-ance. The Lucilia GST has been classified as a Theta class GST on the basis of its primary structure, but the crystal structure shows the equivalent tyrosine residue within the N-terminal region (Tyr-5) to be 13.9 Å away from the thiol group of GSH (15). Therefore, other residues could replace Tyr-5 and be in-volved in the activation of GSH. From the crystal structure, Ser-9 is found to be within hydrogen bond distance of the sulfur atom of GSH (3.9 Å), and the hydroxyl group of Tyr-113 may also form a hydrogen bond with the GSH sulfur atom through a water molecule. This tyrosine residue is not conserved in all of the Theta class isoenzymes, and it is absent in the human Alpha class GST. Data so far obtained on L. cuprina GST show that Tyr-113 is not significantly involved in catalysis (16). Nevertheless, an equivalent tyrosine residue plays an impor-tant catalytic role in the Mu class GST. X-ray diffraction of GST M1–1 in complex with a transition state analogue shows ␴-complex stabilization by hydrogen-bonding interactions with the hydroxyl groups of Tyr-6 and Tyr-115 (17); moreover, the hydroxyl group of Tyr-115 is involved both in chemical and physical steps of catalysis, and its removal has different effects, depending on which of these steps is rate-limiting (18). In the GST P1–1, the equivalent Tyr-108 shows a multifunctional role in catalysis (19); the hydroxyl function of Tyr-108 stabilizes the transition state for the Michael addition of GSH to ethacrynic acid, whereas it has a negative influence when 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole is used as cosubstrate.