AN ESSENTIAL CYSTEINE RESIDUE LOCATED IN THE VICINITY OF THE FAD-BINDING SITE IN SHORT-CHAIN, MEDIUM-CHAIN, AND LONG-CHAIN ACYL-COA DEHYDROGENASES FROM RAT-LIVER MITOCHONDRIA

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  • 1 January 1985
    • journal article
    • research article
    • Vol. 260  (2) , 1338-1345
Abstract
Medium-chain and long-chain acyl-CoA dehydrogenases from rat liver were purified in 2 forms, holoenzymes containing FAD and apoenzymes which do not contain this cofactor. In contrast, short-chain acyl-CoA dehydrogenase can only be isolated as the holoenzyme. Marked differences in the reactivity to organic sulfhydryl reagents were observed between the apo and holo forms of these enzymes. While the 2 apoenzymes were severely inactivated by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB) and iodoacetate (IAA), the 2 corresponding holoenzymes were not susceptible to these reagents. The inactivation of the 2 apoenzymes by NEM followed pseudo-1st order kinetics. Incubation of the apoenzymes with FAD completely prevented the inactivation by the organic sulfhydryl reagents. Methylmercury halides (iodide or chloride) inactivated both the apo and holo forms of medium-chain and long-chain acyl-CoA dehydrogenases. Holo-short-chain acyl-CoA dehydrogenase behaved somewhat differently from the other 2 holoenzymes in that it was inactivated by pCMB (but not NEM or IAA) following a pseudo-1st order process. The titration of the 2 apoenzymes with [14C]NEM and that of the holo-short-chain acyl-CoA dehydrogenase with [14C]pCMB indicated that all 3 acyl-CoA dehydrogenases contain a single essential cysteine residue/subunit. In the inactivation of holo-medium-chain and holo-long-chain acyl-CoA dehydrogenases with methylmercury halide, the same essential cysteine residue was modified without perturbing or releasing the enzyme-bound FAD. The inactivations of the 3 holoenzymes by appropriate organic sulfhydryl reagents were prevented by prior incubation with substrate. The essential cysteine residue is probably located in the vincinity of the FAD- and substrate-binding sites within the active center of the enzymes. It appears, however, that this cysteine residue does not participate directly in FAD binding.