Structural determinants of trypsin affinity and specificity for cationic inhibitors

The binding free energies of four inhibitors to bovine beta-trypsin are cal culated. The inhibitors use either ornithine, lysine, or arginine to bind t o the S-1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson-Boltzmann equ ation, the contribution of nonpolar interactions is calculated using a free energy-surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy val ues are of a reasonable magnitude and the relative affinity of the four inh ibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine- an d lysine-based inhibitors, the salt bridge formed between their charged gro up and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsi n binding site results in highly specific recognition of substrates and inh ibitors. Specifically, partially burying Asp189 in the inhibitor-free enzym e decreases the penalty for desolvation of this group upon complexation. Wa ter molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion p air formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, re latively immobile in the inhibitor-free state, thus reducing the entropic c ost of complexation. The implications of the results for the general proble m of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to bin ding that are more favorable than oppositely charged molecules due to enhan ced interactions with the solvent in the highly charged complex that is for med.