Posisjon  1

Structural basis for the primary specificity of serine proteinases of the chymotrypsin family The factors which determine protein interaction and substrate specificity are poorly understood because opposing energies are involved: The interactions are entropically unfavourable due to e.g. reduced flexibility, but they can be associated with gain in enthalpy due to burial of e.g. hydrophobic surfaces, or formation of favourable electrostatic interactions. Knowledge of protein structure and ligand association is important in several fields, such as: - Diagnostics of diseases - Approaches to drug design - Development of biological catalysts for industrial purposes - Protein design Structure studies of protein complexes, in with combination experimental biochemical data, mutational studies and molecular dynamics simulations, may provide valuable information about the factors influencing the function of the protein. Such knowledge is of importance when trying to design proteins with highly specific functions. The biophysical properties one wants to change can be: - Increased efficiency - Increased or decreased stability vs. temperature, pH, salt etc. - Increased or decreased specificity Trypsin and chymotrypsin catalyse the hydrolysation of peptide bonds. In order to compare the specificity of the two enzymes, the primary binding residue of Bovine Pancreatic Trypsin Inhibitor (BPTI), lysine15, was mutated to the other naturally occurring amino acids (except proline). BPTI mimics a natural substrate, but in contrast to a normal catalysis, the product is not released from the enzyme after hydrolysis. Orthorhombic crystals of Trypsin and BPTI Hexagonal crystals of Chymotrypsin and BPTI The association energies between the BPTI and bovine trypsin and chymotrypsin have been measured, and it was found that the primary binding residue is responsible for ca 70% of the binding energy in trypsin, and ca 50% in chymotrypsin. The 3-dimensional crystal structures of 10 variants of BPTI in complex with trypsin and chymotrypsin have been determined. The crystal structures show that the secondary binding interactions are almost completely conserved in all complexes, and the P1 residues of the inhibitor occupy almost identical conformations in the complexes with the two enzymes. Three amino acids in the primary binding pocket of the two enzymes differ, and this results in slightly different fold and polarity of the pockets. The perturbation of the solvent network in the pocket and unequal conformational strain imposed on the primary binding residues by the pocket walls are the main cause of the differences in binding energies and the different discrimination between primary residues of various size and polarity. Active site of trypsin with 4 mutants of BPTI: Red: glycine Green: valine Blue: leucine Yellow: Phenylalanine Active site of chymotrypsin with 4 mutants of BPTI: Red: glycine Green: valine Blue: leucine Yellow: Phenylalanine References: Krowarsch et al. 1999. J Mol Biol 289(1), 175-186. Helland et al. 1999. J. Mol. Biol. 287, 923 - 942. Helland et al. 2003. J. Mol. Biol. 333, 845 - 861. Czapinska et al. 2004 J. Mol. Biol. 344, 1005-1020.