INHIBITOR REARRANGEMENT FOLLOWING COMPLEX FORMATION BETWEEN ACARBOSE AND HUMAN PANCREATIC [[alpha]]-AMYLASE. Yaoguang Luo, Stephen G. Withers, Chris M. Overall and Gary D. Brayer, Departments of Biochemistry and Molecular Biology, Chemistry, and Clinical Dental Science, University of British Columbia, Vancouver, B. C. Canada V6T 1Z3.

The complex sugar starch forms the principal source of glucose in the human diet. Initial starch digestion is provided by a salivary [[alpha]]-amylase, and then upon reaching the gut these degradation products are more extensively hydrolyzed by an [[alpha]]-amylase secreted by the pancreas. The salivary and pancreatic [[alpha]]-amylases are closely related isozymes which are expressed in a tissue-specific manner. Each of these enzymes are composed of a single polypeptide chain (MW=55,000) consisting of 496 amino acids. To gain a comprehensive understanding of the catalytic mechanism of human pancreatic [[alpha]]-amylase, we have completed the 1.8Å structure of this enzyme using x-ray diffraction techniques. These studies show this enzyme is composed of three structural domains. The core of the most prominent of these consists of an 8-stranded parallel [[beta]]-barrel surrounded by extensive [[alpha]]-helical segments. To one end of this domain is located the active site region and a chloride binding site. A second domain is constructed around a calcium binding site which is essential for catalytic activity. The third structural domain is only loosely attached to the core of the enzyme and its functional role remains unclear. These structural studies have allowed for a detailed mapping of catalytic elements directly in the active site region and associated residues within the nearby elongated surface substrate binding groove. Further work has focused on determining the structure of the complex formed by the clinically utilized inhibitor acarbose and human pancreatic [[alpha]]-amylase. This work has shown this enzyme catalyzes a rearrangement of the individual components of acarbose to produce a stable enzyme-product complex. An important part of this process involves a concerted movement of a surface polypeptide chain loop to closely interact with the bound inhibitor. On the basis of these results attempts are in progress to develop novel high affinity inhibitors of human [[alpha]]-amylase. The goal of this work is not only to provide additional insight into the catalytic mechanism of this enzyme, but also to identify potential enhanced therapeutic agents.