Theoretical calculation of pk(a) reveals an important role of Arg(205) in the activity and stability of Streptomyces sp N174 chitosanase

Based on the crystal structure of chitosanase from Streptomyces sp, N174, w e have calculated theoretical pK(alpha) values of the ionizable groups of t his protein using a combination of the boundary element method and continuu m electrostatics. The pK(alpha) value obtained for Arg(205), which is locat ed in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases pos sessing mutations in this position (R205A, R205H, and R205Y), produced by S treptomyces lividans expression system, were found to have less than 0.3% o f the activity of the wild type enzyme and to possess thermal stabilities 4 -5 kcal/mol lower than that of the wild type protein. In the crystal struct ure, the Arg205 Side chain is in close proximity to the Asp(145) Side chain (theoretical pK(alpha), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(alpha), 17,7), These theoretical pK(alpha) values ar e abnormal, suggesting that both of these residues may participate in the A rg(205) interaction network. Activity and stability experiments using Asp(1 45)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experiment al data supporting the hypothesis derived from the theoretical pK(alpha) da ta and prompt the conclusion that Arg(205) forms a strong interaction netwo rk with Asp(145) and Arg(190) that stabilizes the catalytic cleft.