This US-Hungary biochemical research project on "Structurally Related Serine Proteases" is between Dr. William J. Rutter of The Hormone Research Institute, University of California-San Francisco, and Dr. Gabor Pal of Eotvos University, Budapest. Serene proteases, such as trypsin and chymotrypsin, share a 3-dimensional structure and possess an identical catalytic apparatus but display strikingly different substrate specificities. As such, they are ideal models to study how the substrate recognition process (ground state stabilization) relates to catalysis (transition state stabilization). Via site-directed mutagenesis, a series of alterations of the substrate binding pocket of trypsin (residues 189195, 214-220, 225-228) were made to produce mutants with chymotrypsin-like specificity. One of the trypsin mutants (D189S, YG217-219SGG, Q192M) is of particular interest. Its affinity toward various peptide substrates of chymotrypsin (P1=Leu, Phe, Tyr, Trp) is comparable to that of chymotrypsin, while its catalytic efficiency (Kcat/Km) is 3-4 orders of magnitude less than that of chymotrypsin or trypsin. Thus the redesigned and apparently tight enzyme-substrate interactions in the ground state do not seem to properly contribute to transition state stabilization. One interpretation of these results is that the interaction of trypsin and chyumotrypsin with their own substrates might induce differential conformational changes crucial for optimal transition state stabilization. In this proposal, the researchers suggest that the structural unit that mediates such differential structural changes upon substrate binding may be identical with the so-called "activation domain" of trypsin and chymotryupsin. The researchers' view is that the different conformational flexibility of these domains in trypsin and chymotrypsin may represent the structural basis for different substrate specificities of these two proteases. Thus, to interchange the substrate specificities of trypsin and chymotrypsin (without a dramatic loss of activity), the activation domains rather than just the substrate binding pockets have to be interchanged. The researchers will test this hypothesis. This project in biochemistry fulfills the program objective of advancing scientific knowledge by enlisting leading experts in the United States and Eastern Europe to continue complementary talents and pool resources in areas of strong mutual interest and competence.
