Brick P, Blow D M
J Mol Biol. 1987 Mar 20;194(2):287-97. doi: 10.1016/0022-2836(87)90376-7.
The crystal structure of a deletion mutant of tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been determined at 2.5 A 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 P2(1) 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 A. 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.
利用分子置换技术，已在2.5埃分辨率下测定了嗜热栖热放线菌酪氨酰 - tRNA合成酶缺失突变体的晶体结构。这种基因工程分子催化酪氨酸的活化，其动力学性质与野生型酶相似，但不再结合tRNATyr。它包含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。野生型酶的部分精修模型被用作确定截短突变体结构的起点。新晶体属于空间群P2(1)，在不对称单元中包含分子二聚体。对于8至2.5埃之间的所有反射，精修模型的晶体学R因子为18.7%。每个亚基包含两个结构域：含有六股β折叠的α/β结构域（残基1至220）和含有五个螺旋的α螺旋结构域（残基248至319）。α/β结构域通过一个非晶体学二倍体相关，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结构域中间一个深活性位点裂缝底部的狭槽中。它被参与复杂氢键网络的极性侧链和水分子包围。底物的α氨基和羟基都与蛋白质形成良好的氢键。氨基与Tyr169 - OH、Asp78 - OD1和Gln173 - OE1形成氢键。酚羟基与Asp76 - OD1和Tyr34 - OH形成氢键。相比之下，底物羧基与酶没有直接相互作用。已根据精修结构检查了底物抑制研究和定点诱变实验的结果。
