Enteropeptidase (also known as enterokinase) is a protease of the intestinal brush border that specifically cleaves trypsinogen to yield trypsin, which then cleaves and activates other pancreatic zymogens. This regulatory mechanism confines the activity of digestive hydrolases to the gut. Enteropeptidase is synthesized as a single chain zymogen, whereas active enteropeptidase contains a approximately 47 kDa serine protease domain (light chain) and a disulfide-linked approximately 120 kDa heavy chain. Enteropeptidase is conserved among vertebrates, and congenital deficiency causes intestinal malabsorption. Understanding the targeting and structure-function relationships of this ancient, essential protease is critical to understanding the regulation of digestive enzymes in vivo.
Specific Aim 1 is to characterize the signals that specify apical targeting of enteropeptidase. Preliminary studies have identified two motifs that contain apical targeting information. These signals will be dissected by mutagenesis and expression of chimeric proteins in polarized MDCK cells. In addition, the role of sphingolipid and cholesterol-rich """"""""raft"""""""" domains in enteropeptidase sorting will be defined.
Specific Aim 2 is to define the structural basis for the recognition by enteropeptidase of substrates and inhibitors. Enteropeptidase recognizes substrates that resemble the trypsinogen activation peptide, Val-Asp-Asp-Asp-Asp-Lys, but enteropeptidase cleaves trypsinogen with approximately 500-fold greater catalytic efficiency than it cleaves similar model peptides. This specificity for trypsinogen is determined by """"""""exosites"""""""" on both the light chain and the noncatalytic heavy chain that are distinct from the catalytic center. These exosites will be characterized by targeted mutagenesis and by selection of optimal substrates from phage display libraries.
Specific Aim 3 is to determine the three-dimensional structure of the enteropeptidase catalytic domain complexed with substrate analogs and inhibitors. The crystallographic structure is being refined of the recombinant enteropeptidase catalytic domain (L-BEK) complexed with an analog of the trypsinogen activation peptide, Val-Asp-Asp-Asp-Asp-Lys-chloromethane. This structure provides a framework to understand the specificity of enteropeptidase in atomic detail and to predict the effects of mutagenesis. Additional structures will be determined for mutant enzymes with novel specificity and for selected enzyme-inhibitor complexes, including L-BEK (Lys99A1a), L-BEK-STI, and L-BEK-BPTI. The complementary approaches of mutagenesis, kinetic analysis and structure determination will provide new insight into the range of properties that evolution can confer on trypsin-like serine proteases.
