Recognition and catalysis by proteases figure prominently in a host of biological processes of medical importance. Little is known about the details of protease-substrate recognition at atomic level. We will determine structural mechanisms for recognition in protease through our experiments with trypsin, collagenase, ecotin and enteropeptidase. The pancreatic proteases, elastase, chymotrypsin and trypsin are thought to derive their substrate discrimination from a specificity pocket adjacent to the catalytic machinery of these enzymes. A distant homolog, serine collagenase, has binding sites distinct from these enzymes that provide the remarkable catalytic power to degrade the collagen triple helix, a protein that is resistant to all the pancreatic proteases. The activator of the pancreatic proenzymes, enteropeptidase, is strikingly specific for its target sequence, Asp Asp Asp Asp Lys. It rivals restriction endonucleases in its selectivity. These pancreatic and collagenolytic proteases are inhibited by ecotin, an unusual protein of 282 amino acids from E. coli. This protein uses unknown interactions to inhibit all of these proteases. We will determine the atomic level mechanisms of these interactions using the tools of protein engineering and X-ray crystallography. This knowledge can also be used to understand general mechanisms for enzyme specificity and rate enhancement and can also be valuable in designing novel enzymes that can serve as therapeutic or diagnostic agents.
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