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 ² Sergei V. Khangulov,* ,‡ Thomas M. Sossong, Jr., § David E. Ash, § and G. Charles Dismukes* ,‡ Department of Chemistry, Henry H. Hoyt Laboratory, Princeton UniVersity, Princeton, New Jersey 08544, and Department of Biochemistry, Temple UniVersity, School of Medicine, Philadelphia, PennsylVania 19140 ReceiVed NoVember 25, 1997; ReVised Manuscript ReceiVed March 16, 1998 ABSTRACT: 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 Mn 2 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 Mn 2 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 Mn 2 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(NH 2 ) 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 Mn 2 EPR signal and no change in the EPR intensity, suggestive of minimal conformational transformation. We propose that type 1 R-amino acid inhibitors do not bind directly to either Mn ion, but interact with the recognition site on arginase for the R-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 Mn 2 region. Binding of the substrate imino group (>CdNH), most likely to Mn B , is coupled to breaking of the Mn B -(μ-H 2 O) 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 H 2 + -guanidino leaving group. Protonation of the substrate -N δ H 2 + -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 ǫ -N δ H 2 + bond, yielding urea and L-ornithine(H + ). L-Arginine is utilized in living cells for the biosynthesis of proteins. It is a precursor to the polyamines, used as growth factors, and a precursor to nitric oxide (NO), used for intracellular communication (1-3). L-Arginine is also a primary intermediate of the urea cycle, which allows many organisms to metabolize nitrogen-containing compounds with excretion of urea. The concentration of L-arginine in tissues is regulated by arginase, making this enzyme extremely important for a variety of biochemical reactions and bio- medical applications. Arginases from all sources require a divalent cation activator. The mammalian enzyme can be activated by Mn(II), Co(II), Ni(II), Fe(II), and Cd(II), although the physiological activator is Mn(II) (4, 5). Rat liver arginase is a homotrimeric enzyme (6, 7), containing one binuclear Mn(II) center, Mn 2 (II), in each subunit (8) that is required ² This research was supported by the National Institutes of Health, Institute of Digestive Diseases and Kidney, Grants DK45414 (G.C.D.) and DK44841 (D.E.A.). * Address correspondence to S.V.K. at fax, (609) 258-1980; e-mail, khang@chemvax.princeton.edu. Address correspondence to G.C.D. at phone, (609) 258-3949; fax, (609) 258-1980; e-mail, dismukes@ chemvax.princeton.edu. ‡ Princeton University. § Temple University. 8539 Biochemistry 1998, 37, 8539-8550 S0006-2960(97)02874-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/21/1998