PERTURBING THE POLAR ENVIRONMENT OF ASP102 IN TRYPSIN - CONSEQUENCES OF REPLACING CONSERVED SER214

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser2l4 adjacent to AspI02. Ser2l4 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-angstrom crystal structure (R = 0.1 50) of trypsin S214K shows that Lys2l4 occupies the position held by Ser2l4 and a buried water molecule in the buried polar cave. Lys2l4-N-zeta is solvent inaccessible and is less than 5 angstrom from the catalytic Aspl02. The side chain of Glu2l4 (2.8 angstrom, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter k(cat) demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.