Walter P, Despons L, Laforet M, Ebel J P, Fasiolo F
Institut de Biologie, Moléculaire et Cellulaire du CNRS, Laboratoire de Biochimie, Strasbourg, France.
Biochimie. 1990 Aug;72(8):537-44. doi: 10.1016/0300-9084(90)90118-z.
Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step. The Km for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration of the catalytic residues. The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.
酵母甲硫氨酰 - tRNA合成酶具有一个长的N端延伸区，该延伸区与同源大肠杆菌酶N端的单核苷酸结合结构域融合。我们通过在MESI中创建几个保留正确阅读框的内部缺失来研究该多肽区域对酶活性的贡献。结果表明，185个氨基酸对于活性和稳定性是可有可无的。去除接下来的5个残基会影响酶的活性。这种影响在tRNA氨基酰化步骤上比在腺苷酸形成步骤上更为明显。ATP和甲硫氨酸的Km未改变，表明酶的整体结构得以维持。tRNA的Km略有增加，增加了3倍，这表明tRNA在分子表面的定位未受影响。然而，对酶的Vmax有很大影响。对同源大肠杆菌甲硫氨酰 - tRNA合成酶的三维结构研究表明，单核苷酸结合结构域之前的氨基酸区域不直接参与催化裂隙。然而，它可能通过传播催化残基的突变改变在一定距离外起作用。大肠杆菌酶的tRNA(Met)反密码子结合区域最近已被表征。通过对酵母酶中拓扑等效区域进行诱变，我们可以鉴定出特异性改变tRNA氨基酰化的残基。Leu 658提供了一个范德华接触，这对于酵母tRNA的识别至关重要。
