Structural studies of arginase isozymes

Arginase is a binuclear manganese metalloenzyme that catalysis the hydrolysis of L-arginine to form L-ornithine and urea through a metal-activated hydroxide mechanism. In mammals, two isozymes are identified: arginase I is found predominantly in hepatocytes, and arginase II is extrahepatic. The X-ray crystal structure of a fully-active, truncated form of human arginase II complexed with a boronic acid transition state analogue has been determined at 2.7 Å resolution. The structure is consistent with the hydrolysis of L-arginine through a metal-activated hydroxide ion. Human arginase II appears to play a role in regulating L-arginine bioavailability to NO synthase in human penile corpus cavernosum smooth muscle and as such, its inhibition is a potential new strategy for the treatment of erectile dysfunction. Hemodynamic studies conducted with a boronic acid arginase inhibitor in vivo suggest that the extrahepatic arginase II plays a role in both male and female sexual arousal. Therefore, arginase II is a potential target for the treatment of male and female sexual arousal disorders. Chiral L-amino acids bearing sulfonamide side chains have been synthesized and the X-ray crystal structure of arginase I complexed with one of these inhibitors, S-(2-sulfonamidoethyl)-L -cysteine, has been determined at 2.8 Å resolution. The tetrahedral sulfonamide group displaces the metal-bridging hydroxide ion pf the native enzyme and bridges the binuclear manganese cluster with an ionized NH- group. The binding mode of the sulfonamide inhibitor may mimic the binding of the tetrahedral intermediate and its flanking transition states in catalysis. The three-dimensional structures of D128E, D128N, D232A, D232C, D234E, H101N and H101E arginases I have been determined by X-ray crystallographic methods to elucidate the roles of the first-shell metal ligands in the stability and catalytic activity of the enzyme. Substitution of the metal ligands compromises the catalytic activity of the enzyme, either by loss or disruption of the metal cluster or the nucleophilic metal-bridging hydroxide ion. However, the substrate-enzyme affinity as reflected by KM remains relatively invariant across this series of arginase variants, implicating a non-metal binding site for substrate L-arginine in the precatalytic Michaelis complex