Koshland 9405433 The new proposal builds on the previous experience of the laboratory on the structures and functions of protein. The major part of the work will attack the mechanism of isocitric dehydrogenase. The structure of the enzyme has been determined and it has been shown to be covalently regulated by phosphorylation at the active site. Mutations will be made in both the protein and the substrate. Measurements will be made of the binding constants and catalytic turnover numbers of the enzyme containing these mutations. The x-ray structures of the mutant proteins and the wild type with isocitrate and "mutated" substrates will be determined using x-ray crystallography. The binding constants calculated from docking programs and the position of the catalytic groups will then be correlated with the observed properties of catalysis and binding. Through these studies it is hoped that an algorithm can be developed to predict the effect of binding and catalysis on enzymes and receptors in general and this will be applied to a general procedure for altering the specificity of enzymes. The immediate application will be first to convert isocitric dehydrogenase to an isopropylmalate dehydrogenase by modifications of a few residues at the active site and possibly also to convert the isopropyl malate dehydrogenase to an isocitrate dehydrogenase by similar mutations. Since both the structure of the isocitrate and isopropylmalate dehydrogenase are known this should be a moderately straightforward task. If the calculations turn out to be predictive and accurate, it is then proposed to expand the range and apply the techniques to other enzymes. %%% Enzymes, natures catalysts, have specificity and catalytic power capabilities far beyond any man-made catalysts that have been created so far. To understand the mechanisms used by these natural enzymes could therefore be of enormous value in designing new catalysts which could, for example, be applied to creating new materials, cleaning up oil spills, and avoiding toxic side products in chemical synthesis. Two tools, genetic engineering and computer design, have recently been added to the repertoire of tools which could be applied to this problem. In this proposal these new tools are applied to a powerful and vital enzyme in metabolism with the hopes that it can be genetically engineered into a different enzyme. In addition it is hoped that the strategy developed for this example can be generalized for use in a wide variety of other catalysts. ***
