Evaluation of functionally important amino acids in L-aspartate ammonia-lyase from Escherichia coli.
Evaluation of functionally important amino acids in L-aspartate ammonia-lyase from Escherichia coli.
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DOI：
10.1021/bi970452x
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发表时间：
1997-07
期刊：
影响因子：
2.9
通讯作者：
M. M. Jayasekera-M.;W. Shi;G. Farber;R. Viola
中科院分区：
文献类型：
--
作者：
M. M. Jayasekera-M.;W. Shi;G. Farber;R. Viola
最近已经确定了来自大肠杆菌的l-天冬氨酸氨裂解酶的高分辨率结构[Shi，W.，Dunbar，J.，Jayasekera，M. M. K.，维奥拉河E、& Farber，G. K.（1997）Biochemistry 36，9136-9144]。已经进行了推定的活性位点的检查，活性位点位于含有功能上重要的赖氨酸327的裂缝中。根据其与该活性位点赖氨酸的接近程度、与天冬氨酸酶-富马酸酶家族其他成员的序列同源性比较以及拟议作用的化学合理功能的必要性，生成了潜在活性位点残基的列表。五个最有可能的候选人在推定的活性位点裂缝已被检查的定点诱变，以测试其可行性，无论是底物结合或酸碱催化作用。精氨酸和赖氨酸残基已被确定，似乎在酶活性位点的天冬氨酸的方向和结合中起作用。一些尝试性的任务也已作出的酸和碱催化基团，建议参与脱氨基反应。
The high-resolution structure of l-aspartate ammonia-lyase from Escherichia coli has recently been determined [Shi, W., Dunbar, J., Jayasekera, M. M. K., Viola, R. E., & Farber, G. K. (1997) Biochemistry 36, 9136-9144]. An examination of the putative active site has been carried out, with the active site located in a cleft that contains the functionally significant lysine 327. A list of potential active site residues has been generated based on their proximity to this active site lysine, sequence homology comparisons with other members of the aspartase-fumarase enzyme family, and the necessity for chemically reasonable functionalities for the proposed roles. The five most likely candidates in the putative active site cleft have been examined by site-directed mutagenesis to test their feasibility for either substrate binding or acid-base catalytic roles. Arginine and lysine residues have been identified that appear to function in the orientation and binding of aspartic acid at the enzyme active site. Some tentative assignments have also been made of the acid and base catalytic groups that are proposed to be involved in the deamination reaction.