This proposal focuses on mechanistic studies on arginase which catalyzes the Mn-dependent hydrolysis of L-arginine to produce L-ornithine and urea. In liver, this reaction is catalyzed by type I arginase and constitutes the final step of the urea cycle. Type II arginase is found in nonhepatic tissues such as kidney, mammary gland and macrophages where it provides a source of L-ornithine for the biosynthesis of proline and the polyamines, spermine and spermidine. The type II enzyme may serve as a marker and perhaps a therapeutic target in breast and gastric cancers. Many important features of the metallobiochemistry of manganese as it relates to the catalytic mechanism of arginase are poorly understood. A major goal of the proposed studies is to dissect structure-function relationships for the binuclear Mn center of the enzyme. A high resolution structure of the rat liver enzyme has recently been solved by the Christianson and Ash groups. This has defined the coordination chemistry of Mn(II) and suggested a mechanism for metal ion-promoted attack of water on the guanidinium carbon of substrate arginine. An expression system for the production of rat liver (type I) arginase and human type II arginase in E. coli has been developed for site directed mutagenesis of critical amino acid residues identified in the crystal structure. Arginase variants will be generated to probe the roles of specific amino acids in substrate specificity, chemistry of catalysis and the structure and function of the unusual Mn2 (II,II) center. In addition to catalyzing hydrolytic chemistry, arginase catalyzes the disproportionation of hydrogen peroxide in a reaction that requires an intact binuclear Mn (II) center. Using site-directed and random mutagenesis, redesign of the manganese coordination polyhedra is proposed to enhance the redox activity of arginase. The studies, it is predicted, will provide the basis for understanding divalent cation specificities and the factors that govern the nature of the chemistry, hydrolytic versus redox, that occurs on multinuclear metal centers.
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