Caccuri A M, Antonini G, Nicotra M, Battistoni A, Lo Bello M, Board P G, Parker M W, Ricci G
Department of Biology, University of Rome "Tor Vergata," 00133 Rome, Italy and Children's Hospital IRCCS "Bambin Gesú," 00165 Rome, Italy.
J Biol Chem. 1997 Nov 21;272(47):29681-6. doi: 10.1074/jbc.272.47.29681.
Spectroscopic and kinetic studies have been performed 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 hyperbolic 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 product release is rate-limiting. Spectroscopic and kinetic data demonstrate that Lucilia GST is able to lower the pKa 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. Removal of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pKa 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 explained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an involvement 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 activation. 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 alpha-helix 4, and its hydroxyl 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 alpha-helix 4 possibly modulates different aspects of the catalytic mechanism of the Lucilia GST.
已对澳大利亚绵羊绿蝇（Lucilia cuprina）谷胱甘肽S-转移酶（Lucilia GST；EC 2.5.1.18）进行了光谱和动力学研究，以阐明其催化机制。Lucilia GST的稳态动力学不符合米氏方程，但谷胱甘肽（GSH）相当双曲线的等温结合表明，稳态随机顺序双底物双产物机制与观察到的异常动力学一致。反应的限速步骤是一个依赖于粘度的物理事件，停流实验表明产物释放是限速步骤。光谱和动力学数据表明，Lucilia GST能够将结合的GSH的pKa从9.0降低到约6.5。基于晶体学的提示，研究了两个羟基残基Ser-9和Tyr-113的作用。通过定点诱变去除Ser-9的羟基会使结合的GSH的pKa升高到约7.6，并且观察到极低的周转数（约为野生型的0.5%）。这种失活可能是由于Ser-9羟基对GSH的有效结合有很大贡献，并参与了离子化GSH的稳定。这个丝氨酸残基在Theta类GST中高度保守，因此目前的发现可能适用于所有家族成员。Tyr-113似乎对GSH的活化不是必需的。停流数据表明，去除Tyr-113的羟基不会改变反应的限速步骤，但会导致GSH共轭物形成和释放的速率常数增加。Tyr-113位于α-螺旋4上，其羟基直接与Tyr-105的羟基形成氢键。这将降低有助于亲电底物结合位点的蛋白质区域的灵活性；α-螺旋4的片段运动可能调节Lucilia GST催化机制的不同方面。
