SPIROPENTYLACETYL-COA, A MECHANISM-BASED INACTIVATOR OF ACYL-COA DEHYDROGENASES

Acyl-CoA dehydrogenases (ACDs) are FAD-dependent enzymes that catalyze the conversion of an appropriate fatty acyl-CoA thioester substrate t o the corresponding trans-alpha,beta-enoyl-CoA product. Early studies have shown that the dehydrogenation is stereospecific and is initiated by the abstraction of the pro-R alpha-H, followed by the transfer of the pro-R beta-H, as a hydride equivalent, to the bound FAD. However, recent studies of the inactivation of ACDs by a metabolite of hypoglyc in A, (methylenecyclopropyl)acetyl-CoA (MCPA-CoA), led to an alternati ve mechanism in which the reducing equivalent is delivered from the in itially formed alpha-anion to the bound FAD via a single electron tran sfer process. To further explore the observed mechanistic discrepancy, we have reexamined the inhibitory properties of a closely related MCP A-CoA analogue, spiropentylacetyl-CoA (SPA-CoA), which was previously reported as a tight-binding inhibitor for ACDs. In contrast to early r esults, our data showed that SPA-CoA is a mechanism-based inhibitor fo r pig kidney medium-chain acyl-CoA dehydrogenase (MCAD) and Megasphaer a elsdenii short-chain acyl-CoA dehydrogenase (SCAD) and that the inac tivation is time-dependent, active-site-directed, and irreversible. Mo re importantly, both (R)- and (S)-SPA-CoA could effectively inactivate MCAD, and the resulting inhibitor-FAD adducts appear to have one of t he three-membered rings of The spiropentyl moiety cleaved. Since the i nactivation is nonstereospecific with respect to C-beta-C bond scissio n, the ring opening of SPA-CoA leading to enzyme inactivation is likel y initiated by a spiropentylcarbinyl radical. Such a radical-induced r ing fragmentation is expected to be extremely facile and may bypass th e chiral discrimination normally imposed by the enzyme. Thus, these re sults are consistent with our early notion that MCAD is capable of med iating one-electron redox chemistry. Interestingly, it was also found that (R)-SPA-CoA is an irreversible inhibitor for SCAD, while the S-ep imer is only a competitive inhibitor for the same enzyme. The selectiv e inhibition exhibited by these compounds against two closely related dehydrogenases is likely a consequence of the distinct steric and elec tronic demands imposed by the active sites of MCAD and SCAD. Such info rmation is important for the design of novel class-selective inhibitor s to control and/or regulate fatty acid metabolism.