L-arginine binding to liver arginase requires proton transfer to gateway residue His141 and coordination of the guanidinium group to the dimanganese(II,II) center.
Rat liver arginase contains a dimanganese(II,II) center per subunit that is required for catalytic hydrolysis of l-arginine to form urea and l-ornithine. A recent crystallographic study has shown that the Mn2 center consists of two coordinatively inequivalent manganese(II) ions, MnA and MnB, bridged by a water (hydroxide) molecule and two aspartate residues [Kanyo et al. (1996) Nature 383, 554-557]. A conserved residue, His141, is located near the proposed substrate binding region at 4.2 A from the bridging solvent molecule. The present EPR studies reveal that there is no essential alteration of the Mn2 site upon mutation of His141 to an Asn residue, which lacks a potential acid/base residue, while the catalytic activity of the mutant enzyme is 10 times lower vs wild-type enzyme. The binding affinity of l-lysine, l-arginine (substrate), and Nomega-OH-l-arginine (type 2 binders) increases inversely with the pKa of the side chain. Binding of l-lysine is more than 10 times weaker, and the substrate Michaelis constant (Km) is >6-fold greater (weaker binding) in the His141Asn mutant than in wild-type arginase. L-Lysine and Nomega-OH-L-arginine, type 2 binders, induce extensive loss of the EPR intensity, suggesting direct coordination to the Mn2 center. From these data and the pH dependence of type 2 binders, we conclude that His141 functions as the base for deprotonation of the side-chain amino group of L-lysine and the substrate guanidinium group, -NH-C(NH2)2+ and that the unprotonated side chain of these amino acids is responsible for binding to the active site. A different class of inhibitors (type 1), including L-isoleucine, L-ornithine, and L-citrulline, suppresses enzymatic activity, producing only minor change in the zero-field splitting of the Mn2 EPR signal and no change in the EPR intensity, suggestive of minimal conformational transformation. We propose that type 1 alpha-amino acid inhibitors do not bind directly to either Mn ion, but interact with the recognition site on arginase for the alpha-aminocarboxylate groups of the substrate. A new mechanism for the arginase-catalyzed hydrolysis of L-arginine is proposed which has general relevance to all binuclear hydrolases: (1) Deprotonation of substrate l-arginine(H+) by His141 permits entry of the neutral guanidinium group into the buried Mn2 region. Binding of the substrate imino group (>C=NH), most likely to MnB, is coupled to breaking of the MnB-(mu-H2O) bond, forming a terminal aquo ligand on MnA. (2) Proton transfer from the terminal MnA-aqua ligand to the substrate Ndelta-guanidino atom forms the nucleophilic hydroxide on MnA and the cationic NdeltaH2+-guanidino leaving group. Protonation of the substrate -NdeltaH2+-group is likely assisted by hydrogen bonding to the juxtaposed anionic carboxylate group of Glu277. (3) Attack of the MnA-bound hydroxide at the electrophilic guanidino C-atom forms a tetrahedral intermediate. (4) Formation of products is initiated by cleavage of the Cepsilon-NdeltaH2+ bond, yielding urea and L-ornithine(H+).
Khangulov
S V
SV
Department of Chemistry, Henry H. Hoyt Laboratory, Princeton University, New Jersey 08544, USA.
Sossong
T M
TM
Ash
D E
DE
Dismukes
G C
GC
D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites; D001881:Borates / Q000494:pharmacology; D002956:Citrulline / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D019791:Guanidine / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009952:Ornithine / Q000494:pharmacology; D011522:Protons; D051381:Rats; D013379:Substrate Specificity
D001881:Borates; D011522:Protons; D007532:Isoleucine; D002956:Citrulline; D008345:Manganese; D006639:Histidine; C068309:N(omega)-hydroxyarginine; D001216:Asparagine; D001120:Arginine; D009952:Ornithine; D001119:Arginase; D019791:Guanidine; D008239:Lysine