Kolobe Doris, Sayed Yasien, Dirr Heini W
Protein Structure-Function Research Programme, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
Biochem J. 2004 Sep 1;382(Pt 2):703-9. doi: 10.1042/BJ20040056.
In addition to their catalytic functions, GSTs (glutathione S-transferases) bind a wide variety of structurally diverse non-substrate ligands. This ligandin function is known to result in the inhibition of catalytic function. The interaction between hGSTA1-1 (human class Alpha GST with two type 1 subunits) and a non-substrate anionic ligand, BSP (bromosulphophthalein), was studied by isothermal titration calorimetry and inhibition kinetics. The binding isotherm is biphasic, best described by a set of two independent sites: a high-affinity site and a low-affinity site(s). The binding stoichiometries for these sites are 1 and 3 molecules of BSP respectively. BSP binds to the high-affinity site 80 times more tightly (K(d)=0.12 microM) than it does to the low-affinity site(s) (K(d)=9.1 microM). Binding at these sites is enthalpically and entropically favourable, with no linkage to protonation events. Temperature- and salt-dependent studies indicate the significance of hydrophobic interactions in the binding of BSP, and that the low-affinity site(s) displays low specificity towards the anion. Binding of BSP results in the release of ordered water molecules at these hydrophobic sites, which more than offsets unfavourable entropic changes during binding. BSP inhibition studies show that the binding of BSP to its high-affinity site does not inhibit hGSTA1-1. This site, located near Trp-20, may be related to the buffer-binding site observed in GSTP1-1. The low-affinity-binding site(s) for BSP is most probably located at or near the active site of hGSTA1-1. Binding to this site(s) results in non-competitive inhibition with respect to CDNB (1-chloro-2,4-dinitrobenzene) (K(i)(BSP)=16.8+/-1.9 microM). Given the properties of the H site and the relatively small size of the electrophilic substrate CDNB, it is plausible that the active site of the enzyme can simultaneously accommodate both BSP and CDNB. This would explain the non-competitive behaviour of certain inhibitors that bind the active site (e.g. BSP).
除了具有催化功能外，谷胱甘肽S-转移酶（GSTs）还能结合多种结构各异的非底物配体。已知这种配体结合功能会导致催化功能受到抑制。通过等温滴定量热法和抑制动力学研究了人α类谷胱甘肽S-转移酶1-1（hGSTA1-1，由两个1型亚基组成）与非底物阴离子配体溴磺酞（BSP）之间的相互作用。结合等温线呈双相，最好用一组两个独立的位点来描述：一个高亲和力位点和一个低亲和力位点（或多个低亲和力位点）。这些位点的结合化学计量分别为1个和3个BSP分子。BSP与高亲和力位点的结合比与低亲和力位点（或多个低亲和力位点）的结合紧密80倍（解离常数K(d)=0.12 microM）（低亲和力位点的K(d)=9.1 microM）。在这些位点的结合在焓和熵方面都是有利的，与质子化事件无关。温度和盐依赖性研究表明疏水相互作用在BSP结合中的重要性，并表明低亲和力位点（或多个低亲和力位点）对阴离子的特异性较低。BSP的结合导致这些疏水位点有序水分子的释放，这足以抵消结合过程中不利的熵变。BSP抑制研究表明，BSP与其高亲和力位点的结合并不抑制hGSTA1-1。这个位于色氨酸-20附近的位点可能与在GSTP1-1中观察到的缓冲液结合位点有关。BSP的低亲和力结合位点很可能位于hGSTA1-1的活性位点处或附近。与该位点（或多个位点）的结合导致对1-氯-2,4-二硝基苯（CDNB）的非竞争性抑制（BSP的抑制常数K(i)=16.8±1.9 microM）。鉴于高亲和力位点的特性以及亲电子底物CDNB相对较小的尺寸，酶的活性位点同时容纳BSP和CDNB是合理的。这可以解释某些结合活性位点的抑制剂（如BSP）的非竞争性行为。
