The cyclopropyl group is an important structural entity found in a wide variety of natural products. Because of its highly strained structural features and associated unique physical properties, study of its chemistry has continuously been an intriguing and challenging subject. Taking advantage of its unique reactivity, the cyclopropyl group in recent years has been shown to be an effective irreversible inhibitor and, thus, an important mechanistic probe as well to explore the catalytic mechanisms of the target enzymes. The inactivation is presumedly related to the chemical reactivity of loci at or adjacent to the cyclopropane equivalent. However, as in most other enzymatic inhibition cases, the exact modes of inactivation is still mood. Outlined in this proposal are the experiments designed to study the inactivation of a number of important enzymes by cyclopropane derivatives, with emphasis placed on the mechanistic and stereochemical aspects. Initial efforts will be directed to study the inactivation of acy1- CoA dehydrogenases by (methylenecyclopropane) acetyl-CoA (MCPA- CoA). This inactivation process is directly responsible for the hypoglycin toxicity found in Jamaican vomiting sickness. Specifically, we will 1) purify the enzymes, general acyl-CoA dehydrogenase from pig kidney and butyryl-CoA dehydrogenase from Megasphaera elsdenii, in large quantities; 2) synthesize isotopically labeled substrates and potential alternate substrates/inhibitors containing mechanistically informative functionalities at key sites of the molecules; 3) incubate these compounds with the enzymes and determine their competence as substrates and/or inhibitors. The results of these experiments will be used to address the following issues: a) the stereochemical course and the mechanism of the inactivation, b) the chemical nature of the covalently modified coenzyme adducts; c) the possible derivatization of the active-site amino acids; d) the general catalytic mechanism of the target enzyme (hydride transfer vs. stepwise radical transfer). With everything well under way we will proceed with the study of the inactivation of other enzymes systems, such as the inactivation of quinoproteins by cyclopropanol, etc. An understanding of the molecular bases of these inactivation phenomena in chemical terms can aid not only in the delineation of how chemical transformations are effected by these enzymes, but also the assessment of the potential of the cyclopropyl group as a mechanistic probe in general, and the rational design of drugs with maximal in vivo specificity.
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