CRYSTAL-STRUCTURE OF A DELETION MUTANT OF A TYROSYL-TRANSFER RNA-SYNTHETASE COMPLEXED WITH TYROSINE
CRYSTAL-STRUCTURE OF A DELETION MUTANT OF A TYROSYL-TRANSFER RNA-SYNTHETASE COMPLEXED WITH TYROSINE
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DOI：
10.1016/0022-2836(87)90376-7
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发表时间：
1987-03-20
影响因子：
5.6
通讯作者：
BLOW, DM
中科院分区：
文献类型：
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作者：
BRICK, P;BLOW, DM
嗜热脂肪芽孢杆菌酪氨酰-tRNA合成酶缺失突变体的晶体结构已在2.5埃下测定。使用分子置换技术的分辨率。基因工程分子催化酪氨酸的活化，其动力学特性与野生型酶相似，但不再结合tRNATyr。它含有319个残基，对应于野生型酶晶体中存在可解释电子密度的多肽链区域。野生型酶的部分精制模型用作确定截短突变体结构的起点。新晶体的空间群为P21，在不对称单元内含有分子二聚体。对于8和2.5埃之间的所有反射，改进的模型具有18.7%的晶体学R因子。每个亚基含有两个结构域; α/β结构域。β的含有六链β-的结构域（残基1至220）片和α-螺旋结构域（残基248至319），含有5个螺旋。α/。β的结构域通过非晶体学二分体相关，而α-螺旋结构域在两个亚基中的取向略有不同。酪氨酸底物结合在α/β中间的深活性位点裂缝底部的狭槽中。β的域它被极性侧链和水分子包围，这些分子参与了复杂的氢键网络。α-底物的氨基和羟基与蛋白质形成良好的氢键。氨基与Tyr 169-OH、Asp 78-OD 1和Gln 173-OE 1形成氢键。酚羟基与Asp 76-OD 1和Tyr 34-OH形成氢键。相反，底物羧基不与酶直接相互作用。底物抑制研究和定点诱变实验的结果已被检查在光的精制结构。
The crystal structure of a deletion mutant of tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been determined at 2.5 .ANG. resolution using molecular replacement techniques. The genetically engineered molecule catalyses the activation of tyrosine with kinetic properties similar to those of the wild-type enzyme but no longer binds tRNATyr. It contains 319 residues corresponding to the region of the polypeptide chain for which interpretable electron density is present in crystals of the wild-type enzyme. The partly refined model of the wild-type enzyme was used as a starting point in determining the structure of the truncated mutant. The new crystals are of space group P21 and contain the molecular dimer within the asymmetric unit. The refined model has a crystallographic R-factor of 18.7% for all reflections between 8 and 2.5 .ANG.. Each subunit contains two structural domains; the .alpha./.beta. domain (residues 1 to 220) containing a six-stranded .beta.-sheet and the .alpha.-helical domain (residues 248 to 319) containing five helices. The .alpha./.beta. domains are related by a non-crystallographic dyad while the .alpha.-helical domains are in slightly different orientations in the two subunits. The tyrosine substrate binds in a slot at the bottom of a deep active site cleft in the middle of the .alpha./.beta. domain. It is surrounded by polar side-chains and water molecules that are involved in an intricate hydrogen bonding network. Both the .alpha.-amino and hydroxyl groups of the substrate make good hydrogen bonds with the protein. The amino group forms hydrogen bonds with Tyr169-OH, Asp78-OD1 and Gln173-OE1. The phenolic hydroxyl group forms hydrogen bonds with Asp76-OD1 and Tyr34-OH. In contrast, the substrate carboxyl group makes no direct interactions with the enzyme. The results of both substrate inhibition studies and site-directed mutagenesis experiments have been examined in the light of the refined structure.