Identification of the catalytic residues in the double-zinc aminopeptidase from Streptomyces griseus
Identification of the catalytic residues in the double-zinc aminopeptidase from Streptomyces griseus
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DOI：
10.1016/j.febslet.2004.07.001
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发表时间：
2004-07-30
期刊：
影响因子：
3.5
通讯作者：
Shoham, Y
中科院分区：
文献类型：
--
作者：
Fundoiano-Hershcovitz, Y;Rabinovitch, L;Shoham, Y
从灰色链霉菌（SGAP）的氨肽酶已克隆和表达在大肠杆菌中。通过在18 ℃下在1 M山梨醇存在下培养细胞，获得了可溶性和活性形式的蛋白质。重组SGAP的氨基酸序列含有四个氨基酸，不同于先前公布的序列。天然蛋白质的重新测序表明，天冬酰胺70和184实际上是重组蛋白中的天冬氨酸。基于SGAP的晶体结构，Glu 131和Tyr 246被认为是催化残基。Glu 131的替换导致4-5个数量级的活性损失，与Glu 131作为一般碱基残基一致。Tyr 246的突变导致活性降低约100倍，表明该残基参与过渡态中间体的稳定。(C)2004年欧洲生物化学学会联合会。Elsevier B. V.出版，保留所有权利。
The aminopeptidase from Streptomyces griseus (SGAP) has been cloned and expressed in Escherichia coli. By growing the cells in the presence of 1 M sorbitol at 18 degreesC, the protein was obtained in a soluble and active form. The amino acid sequence of the recombinant SGAP contained four amino acids differing from the previously published sequence. Re-sequencing of the native protein indicated that asparagines 70 and 184 are in fact aspartic acids as in the recombinant protein. Based on the crystal structure of SGAP, Glu131 and Tyr246 were proposed to be the catalytic residues. Replacements of Glu131 resulted in loss of activity of 4-5 orders of magnitude, consistent with Glu131 acting as the general base residue. Mutations in Tyr246 resulted in about 100-fold reduction of activity, suggesting that this residue is involved in the stabilization of the transition state intermediate. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.