REDESIGNING ENZYME STRUCTURE BY SITE-DIRECTED MUTAGENESIS - TYROSYL TRANSFER-RNA SYNTHETASE AND ATP BINDING
REDESIGNING ENZYME STRUCTURE BY SITE-DIRECTED MUTAGENESIS - TYROSYL TRANSFER-RNA SYNTHETASE AND ATP BINDING
复制标题
DOI：
10.1038/299756a0
复制
发表时间：
1982-01-01
期刊：
影响因子：
64.8
通讯作者：
SMITH, M
中科院分区：
文献类型：
--
作者：
WINTER, G;FERSHT, AR;SMITH, M
我们在这里描述了系统地替换酶中氨基酸的通用方法。这使得可以分析它们在底物结合或催化中的分子作用，并最终可能导致新酶活性的工程设计。编码酶的基因首先被克隆到表达酶的载体中，然后在体外突变以改变特定的核苷酸，从而改变酶的氨基酸序列。我们将嗜热脂肪芽孢杆菌的酪氨酰 tRNA 合成酶基因克隆到源自单链噬菌体 M13 的载体中，以便于使用不匹配的合成寡脱氧核苷酸引物进行诱变。从重组 M13 克隆中，我们获得了在大肠杆菌宿主中表达的高水平酶（约 50% 的可溶性蛋白），并将酶活性位点的半胱氨酸 (Cys35) 转化为丝氨酸。这导致酶活性降低，很大程度上归因于 ATP 的 Km 降低。
We describe here a general method for systematically replacing amino acids in an enzyme. This allows analysis of their molecular roles in substrate binding or catalysis and could eventually lead to the engineering of new enzymatic activities. The gene encoding the enzyme is first cloned into a vector from which the enzyme is expressed and is then mutatedin vitroto change a particular nucleotide and hence the amino acid sequence of the enzyme. We have cloned the gene for the tyrosyl tRNA synthetase ofBacillus stearothermophilusinto a vector derived from the single-stranded bacteriophage M13 to facilitate mutagenesis with mismatched synthetic oligodeoxynucleotide primers. From the recombinant M13 clone we have obtained high levels of the enzyme (∼50% of soluble protein) expressed in theEscherichia colihost and have converted cysteine (Cys35) at the enzyme's active site to serine. This leads to a reduction in enzymatic activity that is largely attributable to a lowerKmfor ATP.