Hu Y, Yang X, Yin D H, Mahadevan J, Kuczera K, Schowen R L, Borchardt R T
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045-2106, USA.
Biochemistry. 2001 Dec 18;40(50):15143-52. doi: 10.1021/bi015690d.
S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy), playing an essential role in modulating the cellular Hcy levels and regulating activities of a host of methyltransferases in eukaryotic cells. This enzyme exists in an open conformation (active site unoccupied) and a closed conformation (active site occupied with substrate or inhibitor) [Turner, M. A., Yang, X., Yin, D., Kuczera, K., Borchardt, R. T., and Howell, P. L. (2000) Cell Biochem. Biophys. 33, 101-125]. To investigate the binding of natural substrates during catalysis, the computational docking program AutoDock (with confirming calculations using CHARMM) was used to predict the binding modes of various substrates or inhibitors with the closed and open forms of AdoHcy hydrolase. The results have revealed that the interaction between a substrate and the open form of the enzyme is nonspecific, whereas the binding of the substrate in the closed form is highly specific with the adenine moiety of a substrate as the main recognition factor. Residues Thr57, Glu59, Glu156, Gln181, Lys186, Asp190, Met351, and His35 are involved in substrate binding, which is consistent with the crystal structure. His55 in the docked model appears to participate in the elimination of water from Ado through the interaction with the 5'-OH group of Ado. In the same reaction, Asp131 removes a proton from the 4' position of the substrate after the oxidation-reduction reaction in the enzyme. To identify the residues that bind the Hcy moiety, AdoHcy was docked to the closed form of AdoHcy hydrolase. The Hcy tail is predicted to interact with His55, Cys79, Asn80, Asp131, Asp134, and Leu344 in a strained conformation, which may lower the reaction barrier and enhance the catalysis rate.
S-腺苷同型半胱氨酸（AdoHcy）水解酶催化AdoHcy可逆水解生成腺苷（Ado）和同型半胱氨酸（Hcy），在调节细胞内Hcy水平以及调控真核细胞中众多甲基转移酶的活性方面发挥着至关重要的作用。这种酶存在开放构象（活性位点未被占据）和封闭构象（活性位点被底物或抑制剂占据）[特纳，M. A.，杨，X.，尹，D.，库泽拉，K.，博查德特，R. T.，以及豪厄尔，P. L.（2000年）《细胞生物化学与生物物理学》33卷，第101 - 125页]。为了研究催化过程中天然底物的结合情况，使用计算对接程序AutoDock（并使用CHARMM进行确认计算）来预测各种底物或抑制剂与AdoHcy水解酶的封闭形式和开放形式的结合模式。结果表明，底物与酶的开放形式之间的相互作用是非特异性的，而底物与封闭形式的结合则高度特异性，底物的腺嘌呤部分是主要识别因子。残基苏氨酸57、谷氨酸59、谷氨酸156、谷氨酰胺181、赖氨酸186、天冬氨酸190、甲硫氨酸351和组氨酸35参与底物结合，这与晶体结构一致。对接模型中的组氨酸55似乎通过与Ado的5'-OH基团相互作用参与从Ado中消除水。在同一反应中，天冬氨酸131在酶中的氧化还原反应后从底物的4'位置去除一个质子。为了确定结合Hcy部分的残基，将AdoHcy对接至AdoHcy水解酶的封闭形式。预测Hcy尾部以一种应变构象与组氨酸55、半胱氨酸-文档继续-79、天冬酰胺80、天冬氨酸131、天冬氨酸134和亮氨酸344相互作用，这可能会降低反应屏障并提高催化速率。
