This project, Structural and Function of Biologically Active Molecules, is focussed on activity of three allosteric enzymes: fructose-1,6 bisphosphatase (FBPase) in the gluconeogenic pathway, yeast chorismate mutase (CMase) in the pathway to tyrosine and phenylalanine and aspartate transcarbamylase ATCase in the pyrimidine biosynthetic pathway. FBPase levels are elevated in Type 2 diabetics. Starting from the three-dimensional structures of the T (less active) form and R (more active) forms, attempts are in progress to develop analogues of AMP, the allosteric inhibitor, in order to lower levels of glucose in diabetics. The stages use cycles of synthesis, structure of the enzyme-inhibitor complex by X-ray diffraction, model building and redesign for new synthesis. Inhibitors highly specific for FBPase have been found in the 20nM range for Kd, substantially more strongly bound than AMP (Kd = 1 mu M). Other aspects of structure-function studies of FBPase include establishing the catalytic and regulatory mechanism, especially the roles of the divalent metal cations. Also, as a result of structure determinations active site inhibitors for allosteric (CMase can be designed as herbicides, bacteriocides or fungicides which are harmless to humans who do not make this enzyme. Likewise inhibitors of ATCase, such as PALA (N-phosphonyl-L-aspartate) can be improved upon for control of the pyrimidine pathway in certain cancers. These two regulatory enzymes merit further study, proposed here, in order to understand the structural basis for aspects of allosteric regulation not explainable using the present day models, such as the concerted or sequential models.
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