The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate.
The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate.
复制标题
人精氨酸酶的第二壳金属配体影响亲核试剂和底物的配位。
DOI：
10.1021/bi101542t
复制
发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Georgiou,George
中科院分区：
文献类型：
--
作者：
Stone,EverettM;Chantranupong,Lynne;Georgiou,George
The active sites of eukaryotic arginase enzymes are strictly conserved, especially the first- and second-shell ligands that coordinate the two divalent metal cations that generate a hydroxide molecule for nucleophilic attack on the guanidinium carbon ofl-arginine and the subsequent production of urea andl-ornithine. Here by using comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I, we demonstrate that several metal binding ligands are actually quite tolerant to amino acid substitutions. Of >2800 double mutants of first- and second-shell residues analyzed, we found more than 80 unique amino acid substitutions, of which four were in first-shell residues. Remarkably, certain second-shell mutations could modulate the binding of both the nucleophilic water/hydroxide molecule and substrate or product ligands, resulting in activity greater than that of the wild-type enzyme. The data presented here constitute the first comprehensive saturation mutagenesis analysis of a metallohydrolase active site and reveal that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution.
