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+).
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