Arginase catalyzes the Mn(II) dependent hydrolysis of L-arginine to produce L-ornithine and urea in the final reaction of the urea cycle. In liver and non-hepatic tissues, this reaction also supplies L-ornithine for the synthesis of L-Proline and the polyamines, spermine and spermidine. Many important features of the metallobiochemistry of Mn(II) as it relates to the catalytic mechanism of arginase are poorly understood. The role of the intrinsic Mn(II) in arginase structure and function is not known. A major goal of these studies will be to define the function of inorganic cofactors in this reaction. Electron paramagnetic resonance experiments will probe the structure of the binuclear Mn(II) center of arginase recently discovered in this laboratory. Additional studies will assess the effect of the intrinsic Zn(II) on protein conformation and stability. Rat liver arginase has been crystallized in a form eminently suitable for X-ray crystallographic studies. Analysis of crystals diffracting to 2.4. Angstroms resolution has established that rat liver arginase is a trimer. The three dimensional structure of arginase will be determined to high resolution. An expression system for the production of rat liver arginase in E. coli has been developed as source of large quantities of enzyme and for site- directed mutagenesis of critical amino acid residues identified bh chemical modifications and X-ray crystallography. The specific roles of histidine residues in the chemistry of catalysis and metal binding will be established in a series of biochemical, biophysical, and molecular biological studies. We have recently shown that arginase has a catalase activity that requires an intact binuclear Mn(II) center. The catalytic mechanism of this novel activity will be determined through kinetic, spectroscopic and mutagenic approaches. These studies will provide a three dimensional structure of the enzyme, from which the molecular basis for divalent cation specificity and function can be determined. Arginase will serve as a paradigm in structure-function analyses of the enigmatic class of Mn-metalloenzymes, including the Mn-catalase.
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