Crystal structures of two engineered thiol trypsins

Abstract

We have determined the three-dimensional structures of engineered rat trypsins which mimic the active sites of two classes of cysteine proteases. The catalytic serine was replaced with cysteine (S195C) to test the ability of sulfur to function as a nucleophile in a serine protease environment. This variant mimics the cysteine trypsin class of thiol proteases. An additional mutation of the active site aspartate to an asparagine (D102N) created the catalytic triad of the papain-type cysteine proteases. Rat trypsins S195C and D102N,S195C were solved to 2.5 and 2.0 .ANG., respectively. The refined structures were analyzed to determine the structural basis for the 106-fold loss of activity of trypsin S195C and the 108-fold loss of activity of trypsin D102N,S195C, relative to rat trypsin. The active site thiols were found in a reduced state in contrast to the oxidized thiols found in previous thiol protease structures. There are the first reported structures of serine proteases with the catalytic centers of sulfhydryl proteases. Structure analysis revealed only subtle global changes in enzyme conformation. The substrate binding pocket is unaltered, and active site amino acid 102 forms hydrogen bonds of H57 and S214 as well as to the backbone amides of A56 and H57. In trypsin S195C, D102 is a hydrogen-bond acceptor for H57 which allows the other imidazole nitrogen to function as a base during catalysis. In trypsin D102N,S195C, the asparagine at position 102 is a hydrogen-bond donor to H57 which places a proton on the imidazole nitrogen proximal to the nucleophile. This tautomer of H57 is unable to act as a base in catalysis. The 100-fold diminished activity of trypsin D102N,S195C compared to trypsin S195C is most likely due to stabilization of the incorrect tautomer of H57, which is unable to form the critical thiolate-imidazolium ion pair with C195-S.gamma., and to altered electrostatics at the catalytic site due to removal of the negative charge from residue 102. H57 has shifted by 0.2 .ANG. but remains within hydrogen-bonding distance of D/N102 (2.7 .ANG.) and S195 (3.5 .ANG.). The sulfur nucleophile of C195 is larger than the oxygen it replaces but is not sterically hindered. In both structures, C195-S.gamma. may also be to form hydrogen bonds with D193-N (3.7 .ANG.), which is part of the oxyanion hole. The sulfur atom points away from the binding pocket compared to the oxygen atom in trypsin, and the strand of peptide chain which includes residue 193 is closer to the side chain of residue 195 in rat trypsin than it is in cow trypsin. This causes occlusion of the oxyanion hole which could account for the inefficacy of these enzymes. Other possible contributing factors to the reduced activity of the two enzymes are discussed.