Substrate binding stabilizes S-adenosylhomocysteine hydrolase in a closed conformation.
Substrate binding stabilizes S-adenosylhomocysteine hydrolase in a closed conformation.
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底物结合使 S-腺苷高半胱氨酸水解酶稳定在闭合构象。
DOI：
10.1021/bi000595a
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Squier,TC
中科院分区：
文献类型：
--
作者：
Yin,D;Yang,X;Hu,Y;Kuczera,K;Schowen,RL;Borchardt,RT;Squier,TC
s -腺苷型同型半胱氨酸（adhcy）水解酶在无底物，NAD+形式下的晶体结构比较[Hu， Y., Komoto， J., Huang， Y., Gomi， T., Ogawa， H., Takata， Y., Fujioka， M.和Takusagawa， F.（1999）生物化学38,8323−8333]和底物结合，NADH形式[Turner， m.a ., Yuan， C.-S.]。，博查特，r.t.，赫什菲尔德，m.s.，史密斯，g.d.，和豪威尔，p.l.（1998）。结构体。biol . 5,369−376]表明催化和NAD+结合域的空间排列存在很大差异。无底物的NAD+形式以“开放”形式存在于催化和NAD+结合结构域，而有底物结合的NADH形式以封闭形式存在于这些结构域。为了解决结构域闭合是由底物结合还是其随后的氧化引起的，我们测量了催化和羧基末端结构域内与cys113和cys421共价结合的光谱探针的旋转动力学。在底物结合之前，一个独立的结构域运动与催化结构域有关，这表明在催化和NAD+结合结构域之间存在一个柔性铰链元件。随着底物（即腺苷或neplanocin A）或非底物（即3 ' -脱氧腺苷）的结合，与催化结构域相关的独立结构域运动基本上被废除。同样，在早期的晶体学研究中观察到，在底物结合和氧化后，蛋白质的平均水动力体积大幅减少，这与同四聚体酶的总体尺寸减少是一致的。因此，催化结构域和NAD+结合结构域在底物氧化之前通过与底物的相互作用稳定形成封闭的活性位点。
Comparison of crystal structures ofS-adenosylhomocysteine (AdoHcy) hydrolase in the substrate-free, NAD+form [Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (1999)Biochemistry38, 8323−8333] and a substrate-bound, NADH form [Turner, M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998)Nat. Struct. Biol.5, 369−376] indicates large differences in the spatial arrangement of the catalytic and NAD+binding domains. The substrate-free, NAD+form exists in an “open” form with respect to catalytic and NAD+binding domains, whereas the substrate-bound, NADH form exists in a closed form with respect to those domains. To address whether domain closure is induced by substrate binding or its subsequent oxidation, we have measured the rotational dynamics of spectroscopic probes covalently bound to Cys113and Cys421within the catalytic and carboxyl-terminal domains. An independent domain motion is associated with the catalytic domain prior to substrate binding, suggesting the presence of a flexible hinge element between the catalytic and NAD+binding domains. Following binding of substrates (i.e., adenosine or neplanocin A) or a nonsubstrate (i.e., 3‘-deoxyadenosine), the independent domain motion associated with the catalytic domain is essentially abolished. Likewise, there is a substantial decrease in the average hydrodynamic volume of the protein that is consistent with a reduction in the overall dimensions of the homotetrameric enzyme following substrate binding and oxidation observed in earlier crystallographic studies. Thus, the catalytic and NAD+binding domains are stabilized to form a closed active site through interactions with the substrate prior to substrate oxidation.