The cyclopropyl group is an important-structural entity found in a wide variety of natural products. Because of its highly strained structural features and the associated unusual physical properties, study of its chemistry has continuously been an intriguing and challenging subject. Inspired by the unique biological activities possessed by many cyclopropane-containing compounds and the increasingly popular utilization of these highly strained systems in the design of novel enzyme inhibitors as well as mechanistic probes, we have initiated a study to examine their effects on the catalysis of several important enzymes. Our efforts in the past few years have been focused on the mechanistic studies of the inactivation of acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl- CoA (MCPA-CoA) which is the causative agent of Jamaican vomiting sickness. Although our earlier results appear to support a radical mechanism for this inactivation, further investigation has accumulated data that are inconsistent with such a one-electron oxidation pathway. Thus, the actual mechanism is still elusive. As a continuation of our ongoing efforts, this proposal outlines our future plans to fully elucidate the course of this inactivation. Since the particular chemical reaction sequence mediated by the target enzyme is crucial for unraveling the latent functionality, a cyclopropyl group in this case, understanding the chemical basis of the overall inactivation process would certainly aid in delineating the actual catalytic mechanism of the target enzymes as well. The second system to be examined is a PLP linked enzyme, 1-aminocyclopropane-l-carboxylate (ACPC) deaminase, which catalyzes the ring cleavage of a variety of cyclopropyl amino acids, a reaction unique to vitamin B6 dependent catalysts. The inactivation of 3-oxoacyl-CoA thiolase by (methylenecyclopropyl)formyl-CoA (MCPF-CoA) will also be investigated. The mechanistic knowledge gained from these studies will not only provide invaluable information for designing approaches to control and/or mimic the catalytic roles of the target enzymes, but will also enhance our understanding pertaining to the potentials of cyclopropane group as a mechanistic probe. To reach our intended goals, a combinatorial approach is required encompassing the expression and purification of the desired enzymes, the construction of a variety of alternative substrates, inhibitors, and cofactors containing mechanistically informative functionalities at key sites of the molecules, the isolation and characterization of enzymatic incubations with these compounds, and the use of physical and spectroscopic methods for the visualization of reaction intermediates and kinetic determinations. From the proposed studies, our further understanding of the molecular basis of inactivation phenomena, for each particular example, will directly reflect upon the enzymatic mechanism, the potentials of the rational design of drugs with maximal in vivo specificity, and the assessment of the potentials of the cyclopropyl group as a general mechanistic probe. Thus, our anticipated results will have a stimulating impact upon a broad range of applications related to mechanistic enzymology.
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