In order to establish a base of structural knowledge concerning the structure, function, and regulation of the enigmatic family of manganese metalloproteins, we have selected the metallohydrolase rat liver arginase as a target for X-ray crystallographic structure determination, protein engineering, and inhibitor design experiments. This enzyme contains a binuclear, spin-coupled manganese cluster in its active site, and this cluster is required for the catalytic hydrolysis of arginine into ornithine plus urea. Additionally, this cluster participates in the catalytic disproportionation of hydrogen peroxide into water plus molecular oxygen. Hence, arginase is truly extraordinary in that it is a hydrolyase as well as an oxidoreductase, and both activities of this novel bifunctional catalyst require an intact Mn2+2 cluster. To our knowledge, no other metalloenzyme is capable of catalyzing hydrolytic and redox reactions via the same metal site, and the three-dimensional structure of arginase promises to reveal important structure-function relationships that confer these dual activities upon the manganese cluster. We have crystallized native arginase isolated from the rat liver, and we have crystallized recombinant wild-type arginase and a single-point variant expressed in E. coli (a T7 expression system allows for the production of large amounts of wild-type and variant arginases). We have determined the space group and unit cell parameters for these crystals, and we have prepared heavy atom derivatives in order to phase an initial electron density map. Herein, we request support for the complete, three- dimensional structure determinations of native rat liver and recombinant wild-type arginase, as well as certain enzyme variants. Additionally, using arginase as the prototype, we will develop a structure-assisted rationale for the development of potent inhibitors of metalloenzymes which exploit 2 transition metal ions in catalytic hydrolysis reactions. Importantly, arginase is implicated as an inhibitor of liver transplant rejection through its role in maintaining cellular arginine pools. Additionally, arginase activity is implicated in cancer biology. Therefore, the structural biology of arginase not only advances our understanding of manganese in biological systems, but it also complements clinical studies involving novel therapeutic applications.
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