The objective of these studies on aconitase is to provide a detailed account of the sequence of conformational changes and proton transfers that occur after the reaction chemistry of the enzyme that are required before another substrate can be processed. This information will contribute to the long term goal of understanding the principles used in evolution to reverse the changes that occur in the active site of all enzymes during the catalytic process and show how an understanding of recycling provides insights into otherwise inaccessible aspects of the reaction itself. Recognition of recycling transitions and the ligands with which they react will specifically be essential for the design of drugs aimed at enzyme targets in vivo. A number of properties of the FeS-aconitase reaction suggest that a study of recycling will be informative by use of noncompetitive inhibitors of the enzyme. Slow recycling may be responsible for the unusually slow turnover of the enzyme, only 23 s(-1). The nature of slow recycling steps will be determined by use of buffers, D(2)O(2), viscogens and potential activators by examining their affects on the Kii and Kis values of specific inhibitors. From the large amount of high-resolution structural information available for aconitase there is nothing to suggest conformational variants with affinity for different ligands. However, it seems probable that the only species that has been studied so far is the non-specific isoform required in all recycling schemes. The kinetic demonstration of forms specific for citrate and for cis-aconitate may lead others to study the nature of the changes in conformation required for the reaction and the recycling process.
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