Our objectives are to establish the basis for activity and allosteric regulation of enzymes, using three-dimensional structures as determined by X-ray diffraction methods. Structures are established of complexes of the enzymes with substrate analogues, allosteric effectors, inhibitors, and other molecules and ions, including mutants and other modified forms of the enzymes. The allosteric enzymes under study are fructose-1, 6- bisphosphatase (FBPase), aspartate transcarbamylase (ATCase), and chorismate mutase (CMase) from Saccharomyces cerevisiae. We will also continue studies of the non-allosteric enzyme leucine aminopeptidase (LAP and pepA). The LAP structure at 1.6 angstroms guides us in the two-metal mechanism of FBPase. In the allosteric enzymes, structural studies are continued of binding of various ligands in order to elucidate the mechanisms and pathways for the transmission of conformational information from the allosteric sites to the active sites. Although many such studies have been completed for FBPase and ATCase, the detailed pathways for informational transfer are not yet established. At this early stage both T and R structures are known for allosteric CMase. The structural basis for design of an analogue of the allosteric inhibitor (AMP) of FBPase is being established in order to develop a drug for treatment of Type II diabetes. A highly specific analogue (K(d)=0.1 microM) of AMP (K(d)=1 microM) has been obtained as an inhibitor for FBPase, an enzyme which is elevated in Type II diabetes; the method consisted of repeated consecutive cycles of structures of FBPase bound to the inhibitor followed by model building of a newly designed inhibitor from the X-ray results, followed by chemical synthesis. Active site inhibitors can be designed from structures for ATCase (control pyrimidine synthesis), CMase (to make herbicides, bacteriocides or fungicides), and leucine aminopeptidase (to control protein degradation and hormone processing).
Showing the most recent 10 out of 63 publications
