STUDIES OF ACYL-COA DEHYDROGENASE CATALYZED ALLYLIC ISOMERIZATION - AONE-BASE OR 2-BASE MECHANISM

Acyl-CoA dehydrogenases are flavoproteins that catalyze the conversion of a fatty acyl thioester substrate to the corresponding alpha,beta-e noyl-CoA product. It has been well established that a glutamate residu e in the active site [e.g., E367 in short-chain acyl-CoA dehydrogenase (SCAD) of Megasphaera elsdenii] is responsible for the initial alpha- proton abstraction. Early studies have also shown that this class of e nzymes is capable of catalyzing gamma-H abstraction to afford the ally lic isomerization between alpha,beta- and beta,gamma-enone thioesters and/or inactivation by 2- or 3-acetylenic acyl-CoA derivatives. Althou gh a dual role has been proposed for the glutamate residue in both alp ha- and gamma-deprotonation, the existence of a second active-site bas ic group to mediate the observed reactions occurring at gamma-C remain s a feasible mechanism. In an attempt to discern between these two pos sibilities, we have prepared a few oxirane-containing acyl-CoA derivat ives aimed at trapping active-site bases in the vicinity of the alpha- and/or gamma-C. It was found that 2,3-epoxybutyryl-CoA is a new class -selective irreversible inactivator against SCAD; however, the inabili ty of other oxirane-containing probes to react with these enzymes prom pted us to tackle this mechanistic problem by directly studying the ro le of Glu-367 in SCAD-catalyzed 1,3-isomerization. The effect of E367Q mutation on the proficiency of SCAD to mediate the gamma-H exchange o f crotonoyl-CoA was examined. The capabilities of the wild-type SCAD a nd its E367Q mutant to catalyze the gamma-H abstraction during the ina ctivation by 2-butynoyl-CoA was also compared. The fact that the mutan t protein fails to promote gamma-H exchange/abstraction provides stron g evidence supporting a one-base mechanism of this enzyme-catalyzed al lylic isomerization. Since the catalysis of acyl-CoA dehydrogenases is closely related, such a one-base mechanism is expected to be conserve d within this family of enzymes.