Flexibility in the specificity site of serine proteases.

The statistical availability of tryptophan and tyrosine residues with one ring face fully exposed to solvent was examined for two serine proteases and their derivatives by investigating the formation of charge transfer (CT) complexes between the aromatic donor residues of the protein and the acceptor 1-methylnicotinamide chloride. The availability of the ring face of one of the two exposed tryptophan residues in trypsin has been previously shown to be pH dependent and to parallel the acid side of the pH-activity profile of the enzyme. The present results indicate that, in diisopropylphosphoryl-trypsin (DIP-trypsin), this residue [which was identified as Trp-215 in native trypsin (chymotrypsin numbering)] is locked in a relatively rigid, pH-independent conformation with one ring face rotated out toward the solvent. In the zymogen and DIP-zymogen, the ring face is essentially unavailable. Chymotrypsin, like trypsin, has a pH-depent tryptophan residue available for complexation with the CT acceptor, but unlike trypsin, the pH dependence is apparently associated with dimerization of the enzyme. These and other data suggest this residue is the same as in the homologous trypsin structure, i.e., Trp 215, and that the ring face is mostly buried in the zymogen. Comparison of the crystal structure models of chymotrypsin and chymotrypsinogen shows that, as the specificity pocket opens up from its collapsed structure upon zymogen activation, the ring face of Trp-215 moves out and rotates relative to the surface of the enzyme in such a fashion as to become more accessible to solvent. These observations are in accord with the present CT results and provide additional support for the assignment of changes in Trp-215 availability to parallel changes in the conformation of the specificity pocket of these serine proteases. The present investigation also shows that, although a tryptophan ring face is partly exposed in DIP-chymotrypsin, its statistical availability more closely resembles that of the zymogen than the native enzyme. The reverse appears to be true for DIP-trypsin, which suggests the possibility that the specificity pocket in DIP-chymotrypsin may be partially collapsed while the catalytic residues are frozen in the conformation of the acyl-enzyme.