Crystallographic studies of two proteases: The aminopeptidase from Vibrio proteolyticus (AAP) and gamma-chymotrypsin

Proteases are ubiquitous and important biological molecules. They perform the chemistry of hydrolyzing peptide bonds in a variety of different cellular pathways: among them are blood coagulation, simple digestion, pre-hormone processing, and protein processing in general. With this in mind, it is important to understand in detail the kinetic and chemical mechanisms of proteases, such that drug design might be facilitated.; The aminopeptidase from Vibrio proteolyticus (AAP) is a bimetallic dizinc protease. It is an ideal enzyme for crystallographic studies of a bimetallic protease as it is not large (291 amino acids), easily crystallizes and diffracts in many cases to atomic resolution. The kinetic mechanism of the enzyme is still not fully understood nor is it explicitly known how the two zinc cations aid in carrying out the protease chemistry. The probable general acid/base has been identified as glutamate 151. In this work three mutants of AAP have been studied: E151D-AAP, E151A-AAP and E151H-AAP have been characterized crystallographically. Some insights into the chemical mechanism have been made from these studies and have resulted in observation of the product complex, in good agreement with the previously proposed mechanism. In additional experiments, a Tris-based inhibitor of AAP is here described both kinetically and crystallographically. Tris had been demonstrated to bind to the bimetallic zinc center of AAP and competitively inhibit the enzyme. It is shown that Tris can act as a general scaffold for the design of inhibitors for this bimetallic enzyme and that Tris derivatives can be more potent inhibitors than Tris itself. The inhibitor binds to AAP in a manner analogous to Tris itself.; Also here reported is a novel vanadate-based inhibitor of chymotrypsin. A complex of vanadate and benzohydroxamic acid is shown both kinetically and crystallographically to inhibit chymotrypsin in a competitive fashion. The inhibitor binds covalently to the enzyme in a manner similar to that of transition state analogues. It provides direct evidence that vanadate-based inhibitors are effective against serine proteases.