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

Sergei V. Khangulov, Thomas M. Sossong, David E. Ash, G. Charles Dismukes

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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, Mn(A) and Mn(B), 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 Å 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 N(ω)-OH-L-arginine (type 2 binders) increases inversely with the pK(a) of the side chain. Binding of L-lysine is more than 10 times weaker, and the substrate Michaelis constant (K(m)) is >6-fold greater (weaker binding) in the His141Asn mutant than in wild-type arginase. L-Lysine and N(ω)-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 α-amino acid inhibitors do not bind directly to either Mn ion, but interact with the recognition site on arginase for the α-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 Mn(B), is coupled to breaking of the Mn(B)-(μ-H2O) bond, forming a terminal aquo ligand on Mn(A). (2) Proton transfer from the terminal Mn(A)-aqua ligand to the substrate N(δ)- guanidino atom forms the nucleophilic hydroxide on Mn(A) and the cationic N(δ)H2/+-guanidino leaving group. Protonation of the substrate- N(δ)H2/+-group is likely assisted by hydrogen bonding to the juxtaposed anionic carboxylate group of Glu277. (3) Attack of the Mn(A)-bound hydroxide at the electrophilic guanidino C-atom forms a tetrahedral intermediate. (4) Formation of products is initiated by cleavage of the C(ε)-N(δ)H2/± bond, yielding urea and L-ornithine(H+).

Original languageEnglish
Pages (from-to)8539-8550
Number of pages12
Issue number23
Publication statusPublished - Jun 9 1998

ASJC Scopus subject areas

  • Biochemistry

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