Adachi H, Katayama T, Nakazato H, Tsujimoto M
Suntory Institute for Biomedical Research, Osaka, Japan.
Biochim Biophys Acta. 1993 Apr 21;1163(1):42-8. doi: 10.1016/0167-4838(93)90276-w.
The carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and dansyl-ethylenediamine have shown to inhibit human renal dipeptidase (hrDP) irreversibly in a time-dependent manner. Cilastatin, a competitive inhibitor of the enzyme, partially protected the enzyme from inactivation. To identify the site(s) modified by EEDQ and dansylethylenediamine, the amino-acid sequence of tryptic fragments of modified enzyme were analyzed extensively. A comparison of the determined amino-acid sequences with the predicted primary structure of hrDP revealed that Glu-125 within the Glu115-Arg138 fragment was modified. In consequence, the role of Glu-125 in catalytic activity was investigated by site-directed mutagenesis. Glu-125 was replaced by a glutamine, aspartic acid or cysteine residue. cDNAs for wild-type or mutated enzymes were expressed in CHO cells, and the resulting proteins were purified to apparent homogeneity. The mutated enzyme, Gln-125-hrDP exhibited specific activity of 28.5 U/mg, corresponding to 11.4% of the wild-type. In contrast, Asp-125-hrDP and Cys-125hrDP were found to be inactive (< or = 0.1% of wild-type enzyme). These results suggest that the polarity and/or length of the side chain of Glu-125 residue are important for the enzyme activity.
羧基试剂N-乙氧羰基-2-乙氧基-1,2-二氢喹啉（EEDQ）和丹磺酰乙二胺已被证明能以时间依赖性方式不可逆地抑制人肾二肽酶（hrDP）。西司他丁是该酶的竞争性抑制剂，可部分保护该酶不被失活。为了确定被EEDQ和丹磺酰乙二胺修饰的位点，对修饰酶的胰蛋白酶片段的氨基酸序列进行了广泛分析。将测定的氨基酸序列与hrDP的预测一级结构进行比较，结果显示Glu115-Arg138片段内的Glu-125被修饰。因此，通过定点诱变研究了Glu-125在催化活性中的作用。将Glu-125替换为谷氨酰胺、天冬氨酸或半胱氨酸残基。野生型或突变型酶的cDNA在CHO细胞中表达，所得蛋白质被纯化至表观均一。突变酶Gln-125-hrDP的比活性为28.5 U/mg，相当于野生型的11.4%。相比之下，Asp-125-hrDP和Cys-125hrDP被发现无活性（<或=野生型酶的0.1%）。这些结果表明，Glu-125残基侧链的极性和/或长度对酶活性很重要。
