Altering the binuclear manganese cluster of arginase diminishes thermostability and catalytic function

Arginase is a thermostable (T-m=75 degrees C) binuclear manganese metalloenzyme which hydrolyzes L-arginine to form L-ornithine and urea. The three-dimensional structures of native metal-depleted arginase, metal-loaded H101N arginase, and metal-depleted H101N arginase have been determined by X-ray crystallographic methods to probe the roles of the manganese ion in site A (Mn-A(2+)) and its ligand H101 in catalysis and thermostability. We correlate these structures with thermal stability and catalytic activity measurements reported here and elsewhere [Cavalli, R. C., Burke, C. J., Kawamoto, S., Soprano, D. R., and Ash, D. E. (1994) Biochemistry, 33, 10652-10657]. We conclude that the substitution of a wild-type histidine ligand to Mn-A(2+) compromises metal binding, which in turn compromises protein thermostability and catalytic function. Therefore, a fully occupied binuclear manganese metal cluster is required for optimal catalysis and thermostability.