Abstract
Acyl-CoA dehydrogenases are flavoproteins that catalyze the conversion of a fatty acyl thioester substrate to the corresponding α,β-enoyl-CoA product. It has been well established that a glutamate residue in the active site [e.g., E367 in short-chain acyl-CoA dehydrogenase (SCAD) of Megasphaera elsdenii] is responsible for the initial α-proton abstraction. Early studies have also shown that this class of enzymes is capable of catalyzing γ-H abstraction to afford the allylic isomerization between α,β- and β,γ-enone thioesters and/or inactivation by 2- or 3-acetylenic acyl-CoA derivatives. Although a dual role has been proposed for the glutamate residue in both α- and γ-deprotonation, the existence of a second active-site basic group to mediate the observed reactions occurring at γ-C remains a feasible mechanism. In an attempt to discern between these two possibilities, we have prepared a few oxirane-containing acyl-CoA derivatives aimed at trapping active-site bases in the vicinity of the α- and/or γ-C. It was found that 2,3-epoxybutyryl-CoA is a new class-selective irreversible inactivator against SCAD; however, the inability of other oxirane-containing probes to react with these enzymes prompted us to tackle this mechanistic problem by directly studying the role of Glu-367 in SCAD-catalyzed 1,3-isomerization. The effect of E367Q mutation on the proficiency of SCAD to mediate the γ-H exchange of crotonoyl-CoA was examined. The capabilities of the wild-type SCAD and its E367Q mutant to catalyze the γ-H abstraction during the inactivation by 2-butynoyl-CoA was also compared. The fact that the mutant protein fails to promote γ-H exchange/abstraction provides strong evidence supporting a one-base mechanism of this enzyme-catalyzed allylic isomerization. Since the catalysis of acyl-CoA dehydrogenases is closely related, such a one-base mechanism is expected to be conserved within this family of enzymes.
| Original language | English |
|---|---|
| Pages (from-to) | 10971-10979 |
| Number of pages | 9 |
| Journal | Journal of the American Chemical Society |
| Volume | 118 |
| Issue number | 45 |
| DOIs | |
| Publication status | Published - 1996 Nov 13 |
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All Science Journal Classification (ASJC) codes
- Catalysis
- Chemistry(all)
- Biochemistry
- Colloid and Surface Chemistry
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Studies of acyl-CoA dehydrogenase catalyzed allylic isomerization : A one-base or two-base mechanism? / Dakoji, Srikanth; Shin, Injae; Becker, Donald F.; Stankovich, Marian T.; Liu, Hung Wen.
In: Journal of the American Chemical Society, Vol. 118, No. 45, 13.11.1996, p. 10971-10979.Research output: Contribution to journal › Article
TY - JOUR
T1 - Studies of acyl-CoA dehydrogenase catalyzed allylic isomerization
T2 - A one-base or two-base mechanism?
AU - Dakoji, Srikanth
AU - Shin, Injae
AU - Becker, Donald F.
AU - Stankovich, Marian T.
AU - Liu, Hung Wen
PY - 1996/11/13
Y1 - 1996/11/13
N2 - Acyl-CoA dehydrogenases are flavoproteins that catalyze the conversion of a fatty acyl thioester substrate to the corresponding α,β-enoyl-CoA product. It has been well established that a glutamate residue in the active site [e.g., E367 in short-chain acyl-CoA dehydrogenase (SCAD) of Megasphaera elsdenii] is responsible for the initial α-proton abstraction. Early studies have also shown that this class of enzymes is capable of catalyzing γ-H abstraction to afford the allylic isomerization between α,β- and β,γ-enone thioesters and/or inactivation by 2- or 3-acetylenic acyl-CoA derivatives. Although a dual role has been proposed for the glutamate residue in both α- and γ-deprotonation, the existence of a second active-site basic group to mediate the observed reactions occurring at γ-C remains a feasible mechanism. In an attempt to discern between these two possibilities, we have prepared a few oxirane-containing acyl-CoA derivatives aimed at trapping active-site bases in the vicinity of the α- and/or γ-C. It was found that 2,3-epoxybutyryl-CoA is a new class-selective irreversible inactivator against SCAD; however, the inability of other oxirane-containing probes to react with these enzymes prompted us to tackle this mechanistic problem by directly studying the role of Glu-367 in SCAD-catalyzed 1,3-isomerization. The effect of E367Q mutation on the proficiency of SCAD to mediate the γ-H exchange of crotonoyl-CoA was examined. The capabilities of the wild-type SCAD and its E367Q mutant to catalyze the γ-H abstraction during the inactivation by 2-butynoyl-CoA was also compared. The fact that the mutant protein fails to promote γ-H exchange/abstraction provides strong evidence supporting a one-base mechanism of this enzyme-catalyzed allylic isomerization. Since the catalysis of acyl-CoA dehydrogenases is closely related, such a one-base mechanism is expected to be conserved within this family of enzymes.
AB - Acyl-CoA dehydrogenases are flavoproteins that catalyze the conversion of a fatty acyl thioester substrate to the corresponding α,β-enoyl-CoA product. It has been well established that a glutamate residue in the active site [e.g., E367 in short-chain acyl-CoA dehydrogenase (SCAD) of Megasphaera elsdenii] is responsible for the initial α-proton abstraction. Early studies have also shown that this class of enzymes is capable of catalyzing γ-H abstraction to afford the allylic isomerization between α,β- and β,γ-enone thioesters and/or inactivation by 2- or 3-acetylenic acyl-CoA derivatives. Although a dual role has been proposed for the glutamate residue in both α- and γ-deprotonation, the existence of a second active-site basic group to mediate the observed reactions occurring at γ-C remains a feasible mechanism. In an attempt to discern between these two possibilities, we have prepared a few oxirane-containing acyl-CoA derivatives aimed at trapping active-site bases in the vicinity of the α- and/or γ-C. It was found that 2,3-epoxybutyryl-CoA is a new class-selective irreversible inactivator against SCAD; however, the inability of other oxirane-containing probes to react with these enzymes prompted us to tackle this mechanistic problem by directly studying the role of Glu-367 in SCAD-catalyzed 1,3-isomerization. The effect of E367Q mutation on the proficiency of SCAD to mediate the γ-H exchange of crotonoyl-CoA was examined. The capabilities of the wild-type SCAD and its E367Q mutant to catalyze the γ-H abstraction during the inactivation by 2-butynoyl-CoA was also compared. The fact that the mutant protein fails to promote γ-H exchange/abstraction provides strong evidence supporting a one-base mechanism of this enzyme-catalyzed allylic isomerization. Since the catalysis of acyl-CoA dehydrogenases is closely related, such a one-base mechanism is expected to be conserved within this family of enzymes.
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U2 - 10.1021/ja962532e
DO - 10.1021/ja962532e
M3 - Article
VL - 118
SP - 10971
EP - 10979
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 45
ER -