Work

The Second-Shell Metal Ligands of Human Arginase Affect Coordination of the Nucleophile and Substrate

Year: 2010

Type: article

Abstract: 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 of l-arginine and the subsequent production of urea and l-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.

Source: Biochemistry

DOI: 10.1021/bi101542t

Authors: Everett M. Stone, Lynne Chantranupong, George Georgiou

Institution: The University of Texas at Austin

Language: English

Topic: Amino Acid Enzymes and Metabolism

Subfield: Biochemistry

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,500

Funder: National Cancer Institute