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

Biochemistry. 2010 Dec 14;49(49):10582-8. doi: 10.1021/bi101542t. Epub 2010 Nov 12.


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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arginase / antagonists & inhibitors
  • Arginase / genetics
  • Arginase / metabolism*
  • Binding Sites / genetics
  • Cobalt / chemistry*
  • Cobalt / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Humans
  • Ligands
  • Magnetic Resonance Spectroscopy
  • Manganese / chemistry*
  • Manganese / metabolism
  • Molecular Sequence Data
  • Spectrophotometry, Ultraviolet
  • Substrate Specificity / genetics


  • Ligands
  • Cobalt
  • Manganese
  • Arginase