Adang A E, Brussee J, van der Gen A, Mulder G J
Department of Organic Chemistry, University of Leiden, The Netherlands.
Biochem J. 1990 Jul 1;269(1):47-54. doi: 10.1042/bj2690047.
The GSH-binding site of glutathione S-transferase (GST) isoenzymes was studied by investigating their substrate-specificity for three series of GSH analogues; further, a model of the interactions of GSH with the G-site is proposed. Twelve glycyl-modified GSH analogues, four ester derivatives of GSH and three cysteinyl-modified GSH analogues were synthesized and tested with purified forms of rat liver GST (1-1, 2-2, 3-3 and 4-4). The glycyl analogues exhibited spontaneous chemical reaction rates with 1-chloro-2,4-dinitrobenzene comparable with the GSH rate. In contrast, the enzymic rates (Vmax.) differed greatly, from less than 1 up to 140 mumol/min per mg; apparently, a reaction mechanism is followed that is very sensitive to substitutions at the glycyl domain. No correlation exists between the chemical rates and Vmax. values for the analogues. Analogues of GSH in which L-cysteine was replaced by D-cysteine, L-homocysteine or L-penicillamine showed little or no capacity to replace GSH as co-substrate for the GSTs. GSH monomethyl and monoethyl esters showed Vmax. values greater than the Vmax. measured with GSH: the Vmax. for the monoethyl ester of GSH and GST 3-3 was 5-fold that for GSH. The data obtained in this and previous studies [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724; Adang, Meyer, Brussee, van der Gen, Ketterer & Mulder (1989) Biochem. J. 264, 759-764] allow a model of the interactions of GSH in the G-site in GSTs to be postulated. The gamma-glutamyl site is the main binding determinant: the alpha-carboxylate group is obligatory, whereas shifting of the amino group and shortening of the peptide backbone only decreased kcat./Km. Furthermore, the GSTs appear to be very critical with respect to a correct orientation of the thiol group of the GSH analogue. The glycyl site is the least restrictive domain in the G-site of GSTs: amino acid analogues all showed Km values between 0.2 and 0.6 mM (that for GSH is 0.2-0.3 mM), but large differences in Vmax. exist. The glycyl carboxylate group is not essential for substrate recognition, since decarboxy analogues and ester derivatives showed high activities. The possible mechanisms for an increased Vmax. in some analogues are briefly discussed.
通过研究谷胱甘肽S-转移酶（GST）同工酶对三类谷胱甘肽（GSH）类似物的底物特异性，对其GSH结合位点进行了研究；此外，还提出了一个GSH与G位点相互作用的模型。合成了12种甘氨酰修饰的GSH类似物、4种GSH的酯衍生物和3种半胱氨酰修饰的GSH类似物，并用纯化的大鼠肝脏GST（1-1、2-2、3-3和4-4）进行了测试。甘氨酰类似物与1-氯-2,4-二硝基苯的自发化学反应速率与GSH的速率相当。相比之下，酶促反应速率（Vmax）差异很大，从每毫克每分钟不到1微摩尔到140微摩尔不等；显然，所遵循的反应机制对甘氨酰结构域的取代非常敏感。类似物的化学反应速率与Vmax值之间不存在相关性。用D-半胱氨酸、L-高半胱氨酸或L-青霉胺取代L-半胱氨酸的GSH类似物，作为GSTs的共底物取代GSH的能力很小或没有。GSH单甲酯和单乙酯的Vmax值大于用GSH测得的Vmax值：GSH单乙酯和GST 3-3的Vmax值是GSH的5倍。本研究及之前研究[Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721 - 724; Adang, Meyer, Brussee, van der Gen, Ketterer & Mulder (1989) Biochem. J. 264, 759 - 764]中获得的数据，使得可以推测出GSH在GSTs的G位点中的相互作用模型。γ-谷氨酰位点是主要的结合决定因素：α-羧基是必需的，而氨基的移动和肽主链的缩短只会降低kcat./Km。此外，GSTs对于GSH类似物硫醇基团的正确取向似乎非常关键。甘氨酰位点是GSTs的G位点中限制最少的结构域：氨基酸类似物的Km值都在0.2至0.6 mM之间（GSH的Km值为0.2 - 0.3 mM），但Vmax存在很大差异。甘氨酰羧基对于底物识别不是必需的，因为脱羧类似物和酯衍生物表现出高活性。简要讨论了一些类似物中Vmax增加的可能机制。
