Abstract: Kirkwood theory was applied to investigate the influence of substituting surface charged groups in protein engineering on pK_a of active site residues in enzymes. An expression was obtained to calculate the change of pK_a quartitatively, which was successfully used in calculating the change of pK_a of histidine in the active site in subtilisin and trypsin. The results of subtilisin agrees with experimental data well When surface positive residues were changed to neutral or negative ones, the pK_a would be raised; and when surface negative residues were changed to neutral or positive ones, the pK_a would be lowered. There is an obvious relationship between the position of surface chares and the change of pK_a. For trypsin, when surface charges are within a cone with a base angle of 70°around His (57), the pK_a of His (57) will be changed remarkablly. Four mutants in rat-trypsin were selected as potential sites to raise the pK_a of His (57).