The structure and the mechanism of substrate recognition of the Lysyl-tRNA synthetase from a mesothermophilic bacteria

(1)测定了嗜热脂肪芽孢杆菌赖氨酰-tRNA合成酶基因的碱基序列。根据该基因的碱基序列推导出该酶(B.s.LysRS)的一级结构。对B.s.Lys RS的基因和蛋白质的一级结构进行了比较研究。并对该酶的热稳定性策略进行了探讨。(2)对一种高纯度的B.s.LysRS制剂进行了结晶分析。得到了一种适合于X射线分析的正交晶。该晶体的X射线衍射值为2.63A。(3)B.s.LysRS的功能分析：在构成底物结合部位的氨基酸残基中，能够与底物L-赖氨酸相互作用的氨基酸残基是通过类比E.coliLysRS的结构推导出来的。通过定点突变的方法将这些残基替换为其他氨基酸。以蛋白质荧光变化为探针，测定了人工突变体LysRS与PPI交换反应的动力学参数和L赖氨酸与突变酶结合的平衡参数，并与野生型酶进行了比较，讨论了取代氨基酸残基的作用。

(1) The base sequence of the cloned gene of the lysyl-tRNA synthetase from a mesothermophile, Bacillus stearothermophilus was determined. The primary structure of the enzyme (B.s.LysRS) was deduced from the base sequence of the gene. Comparative studies were made on the primary structures of the gene and the protein of B.s.Lys RS. and the strategy for thermostability of the enzyme was discussed.(2) A highly purified preparation of B.s.LysRS was subjected to crystallization for the structural analysis. An orthorhombic crystal suitable for the X-ray analysis was obtained. The X-ray diffraction on this crystal gave 2.63A resolution. The model building of B.s.LysRS molecule is underway with these diffraction data by the molecular replacement method with the structure of E. coli LysRS(U) as reference.(3) Functional analysis of B.s.LysRS : Among the amino acid residues that constitute the substrate binding site, those which locate in the position being able to interact with the substrate L-lysine were deduced from the analogy to the structure of E.coli LysRS. Those residues were replaced by other amino acid by the method of site-directed mutagenesis. The kinetic parameters of those artificial mutant LysRS in the ATP-PPi exchange reaction and the equilibrium parameters in the binding of L-lysine and the mutant enzyme, as measured with the protein fluorescence change as probe, were determined and compared with those of the wild type enzyme, The functions of the replaced amino acid residues were discussed.