Substrate-mediated increased reactivity of a critical sulfhydryl group of crystals of cytoplasmic aspartate transaminase.
Substrate-mediated increased reactivity of a critical sulfhydryl group of crystals of cytoplasmic aspartate transaminase.
复制标题
底物介导的细胞质天冬氨酸转氨酶晶体的关键巯基反应性增加。
DOI：
10.1016/0003-9861(80)90470-1
复制
发表时间：
1980
影响因子：
3.9
通讯作者：
Sneden,D
中科院分区：
文献类型：
--
作者：
Martinez-Carrion,M;Sneden,D
天冬氨酸氨基转移酶的线粒体外同工酶（l-天冬氨酸：2-酮戊二酸氨基转移酶EC 2.6.1.1）含有半胱氨酸残基（半胱氨酸-390），在底物存在下，其对巯基试剂显示出增强的反应性。为了深入了解酶在溶液中与其结晶状态相比的结构相似性以及底物诱导的结构变化类型，研究了结晶酶中Cys-390的反应性。从聚乙二醇中结晶的黄色平板形成光谱可检测的酶-底物复合物（C。M. Metzler，D. E. Metzler，D. S.马丁河，巴西-地纽曼，A. Arnone和P. Rodgers，1978，J.Biol.Chem.253，5251 -5254）。在存在和不存在底物对谷氨酸和α-酮戊二酸的情况下，用N-乙基马来酰亚胺或N-乙基[1- 14 C]马来酰亚胺处理晶体，并通过与5，5 ′-二硫代双-2-硝基苯甲酸的比色巯基反应、氨基酸分析、掺入的放射性和酶活性的测量来监测反应的程度。半胱氨酸残基仅在底物存在下被修饰;晶体保持未受损。由于，在酶中的任何大的构象变化将被阻止的晶格或会破坏其完整性，它的结论是，在底物的存在下，半胱氨酸-390的反应性增强必须是由于只有一个小的局部构象变化的底物结合区。
The extramitochondrial isozyme of aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1) contains a cysteinyl residue (cysteine-390) which, in the presence of substrate, displays enhanced reactivity toward sulfhydryl reagents. To gain insight into the structural similarity of the enzyme in solution compared to its crystalline state and into the type of structural change induced by substrates, the reactivity of Cys-390 in the crystalline enzyme has been studied. The flat yellow plates, crystallized from polyethylene glycol, form spectroscopically detectable enzyme-substrate complexes (C. M. Metzler, D. E. Metzler, D. S. Martin, R. Newman, A. Arnone, and P. Rodgers, 1978,J. Biol. Chem.253,5251–5254). The crystals, both in the presence and absence of the substrate pair, glutamate and α-ketoglutarate, were treated withN-ethylmaleimide orN-ethyl[1-14C]maleimide and the extent of the reaction was monitored by the colorimetric sulfhydryl reaction with 5,5′-dithiobis-2-nitrobenzoic acid, by amino acid analysis, by radioactivity incorporated, and by the measurement of enzyme activity. A cysteine residue was modified only in the presence of substrate; the crystals remained undamaged. Since, any large conformational change in the enzyme would either be prevented by the crystalline lattice or would disrupt its integrity, it is concluded that the enhanced reactivity of cysteine-390 in the presence of substrates must be due to only a small local conformational change in the substrate binding region.