Molecular design of trypsin mutants towards higher substrate specificity for arginine or ly-sine type substrates was studied. The difference in side chain pKa of arginine and lysine was utilized in redesigning trypsin. If the enzyme could react effectively at a pH higher than lysine’s pKa but lower than that of arginine, it would react more selectively with arginine-type substrates, since in that pH range, the side chain of arginie remain protonated, while that of lysine is deprotonated. For trypsin. the change of histidine (57)’s pKa reflects the shift in reaction optimum pH. Electrostatic calculations showed that when surface positive residues were mutated into neutral or negative ones, the pKa of histidine(57) would be raised and those surface charges within a cone of 70 degree around histidine(57) have strong influence on its pKa. Several sites were suggested in rat trypsin which might serve as potential mutation locations to make trypsin active at a higher pH, thus more selective towards arginine
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