Each individual excretes roughly 10 kg of urea per year, as a result of the hydrolysis of arginine in the final cytosolic step of the urea cycle. This reaction allows the disposal of nitrogenous waste from protein catabolism, and is catalysed by the liver arginase enzyme. In other tissues that lack a complete urea cycle, arginase regulates cellular arginine and ornithine concentrations for biosynthetic reactions, including nitric oxide synthesis: in the macrophage, arginase activity is reciprocally coordinated with that of NO synthase to modulate NO-dependent cytotoxicity. The bioinorganic chemistry of arginase is particularly rich because this enzyme is one of very few that specifically requires a spin-coupled Mn2+-Mn2+ cluster for catalytic activity in vitro and in vivo. The 2.1 angstrom-resolution crystal structure of trimeric rat liver arginase reveals that this unique metal cluster resides at the bottom of an active-site cleft that is 15 angstroms deep. Analysis of the structure indicates that arginine hydrolysis is achieved by a metal-activated solvent molecule which symmetrically bridges the two Mn2+ ions.

The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

The structures reported here clarify aspects of the active site and indicate key features of the catalytic mechanism, including substrate coordination to one of the manganese ions and an orientational role for a neighboring histidine residue. Stereospecificity for L-amino acids is found to depend on their precise recognition at the active-site rim. Identification of a second arginine-binding site, remote from the active site, and associated conformational changes lead us to propose a regulatory role for this site in substrate hydrolysis.

Given the prominence of arginine catabolism in the regulation of diverse metabolic pathways such as ureagenesis 1 and nitric oxide biosynthesis, 2 the synthesis and evaluation of nonreactive arginine analogues as possible enzyme inhibitors or receptor antagonists is a rapidly-growing focus of medicinal chemistry. 2,3 To date, only one enzyme of mammalian arginine catabolism, rat liver arginase, has yielded an X-ray crystal structure to guide structure-based inhibitor design efforts. 4 This trimeric metalloenzyme contains a binuclear manganese cluster in the active site of each subunit required for maximal catalytic activity. 5 Arginine hydrolysis is achieved by a metal-activated solvent molecule that symmetrically bridges the Mn 2+ -Mn 2+ ion pair in the native enzyme. The reaction coordinate of hydrolysis is postulated to proceed through a tetrahedral intermediate resulting from nucleophilic attack of metal-bridging hydroxide ion at the guanidinium carbon of arginine (Figure 1a). 4 The tetrahedral borate anion is a modest, noncompetitive inhibitor of arginase, with K is ) 1.0 mM and K ii ) 0.26 mM; inhibition is even more pronounced in the presence of product ornithine, which is a competitive inhibitor with K i ) 1.0 mM. 6,7 In order to understand the mode of inhibition, we now report the X-ray crystal structure of the ternary arginase-ornithineborate complex. The tetrahedral borate anion mimics binding interactions postulated for the tetrahedral transition state(s) in the catalytic reaction (Figure 1a). 4 This result provides an important foundation for the design, synthesis, and evaluation of the first boronic acid analogue of arginine, 2(S)-amino-6boronohexanoic acid (6). The high affinity of this inhibitor for arginase is proposed to result from the structural similarity between its hydrated form and the proposed tetrahedral intermediate (and flanking transition states) for arginase-catalyzed arginine hydrolysis (Figure 1b).Crystal Structure of the Arginase-Ornithine-Borate Complex. Crystals of rat liver arginase were prepared as described 8 and gradually transferred to a buffer solution containing 10 mM ornithine and 10 mM sodium borate. X-ray diffraction data to 3.0 Å resolution were collected and processed as previously described 4 (28 047 total reflections (20-3 Å), 13 114 unique reflections (9-3 Å) used in refinement, 74% complete with R merge ) 0.062). The atomic coordinates of native rat liver arginase 4 served as the starting model for refinement with X-PLOR. 9 Refinement of the arginase-ornithine-borate complex converged smoothly to a final crystallographic R factor of 0.190 for 9-3 Å data (R free ) 0.301), with root-mean-square deviations from ideal bond lengths and angles of 0.013 Å and 1.6°, respectively.The crystal structure of the arginase-ornithine-borate complex reveals the net displacement of the manganese-bridging solvent molecule of the native enzyme by an oxygen of the tetrahedral borate anion. No other structural changes are observed in the manganese coordination polyhedra, and the...