Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase.

Abstract Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK a values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK a value obtained for Arg205, which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced byStreptomyces lividans expression system, were found to have less than 0.3% of 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 structure, the Arg205 side chain is in close proximity to the Asp145 side chain (theoretical pK a, -1.6), which is in turn close to the Arg190 side chain (theoretical pK a, 17.7). These theoretical pK a values are abnormal, suggesting that both of these residues may participate in the Arg205 interaction network. Activity and stability experiments using Asp145- and Arg190-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK a data and prompt the conclusion that Arg205 forms a strong interaction network with Asp145 and Arg190 that stabilizes the catalytic cleft.