The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate.
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.
Department of Chemical Engineering, University of Texas, Austin, Texas 78712, United States.
Everett M
EM
Stone
Lynne
L
Chantranupong
George
G
Georgiou
D000595:Amino Acid Sequence; D001119:Arginase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity / Q000235:genetics
D008024:Ligands; D003035:Cobalt; D008345:Manganese; D001119:Arginase