Acyl-CoA complexes of general acyl-CoA dehydrogenase and electron transfer flavoprotein.

We have investigated the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction via rapid-scanning stopped-flow (RSSF) UV/vis spectroscopy, combined with the single-wavelength stopped-flow technique, utilizing 3-indolepropionyl-CoA (IPCoA) and trans-3-indoleacryloyl-CoA (IACoA) as chromophoric pseudosubstrates. The RSSF spectral data reveal that formation of an intermediary species with an absorbance maximum at 400 nm and a broad charge-transfer band around 600 nm accompanies the reduction of MCAD-FAD by IPCoA. In the presence of high concentrations of enzyme ([MCAD] >> [IPCoA]) the intermediary spectral band at 400 nm remains unperturbed, whereas in the presence of low concentrations of enzyme ([MCAD] << [IPCoA]) it slowly shifts to an absorption band with an absorbance maximum at 370 nm. Appearance and disappearance of this intermediary species coincides with the appearance and disappearance of the charge-transfer band. Single-wavelength stopped-flow studies, performed under similar high and low enzyme conditions, were consistent with one (1/tau 1) and two (1/tau 1 > 1/tau 2) relaxation rate constants, respectively. These findings, combined with relaxation studies performed in the reverse directions as well as substrate and product binding studies with the oxidized and reduced forms of the enzyme, have allowed us to conclude the following: (1) the intermediary species possesses the properties of reduced flavin and highly conjugated reaction product IACoA (absorbance maximum = 400 nm); (2) this intermediary species collapses into an MCAD-FADH2-IACoA complex (absorbance maximum = 370 nm) in the presence of excessive concentrations of IPCoA; the collapse is being driven by the competitive binding of IPCoA with the reduced form of the enzyme; (3) the 400-nm absorption band and the charge-transfer band are given by the same intermediary species formed during the enzyme-catalyzed reaction pathway. The role of protein conformational changes in modulating the substrate/product structures during the MCAD-catalyzed reaction is discussed.

Section: Resultssupporting

confidence: 78%

Detection and identification of a chromophoric intermediate during the medium-chain fatty acyl-CoA dehydrogenase-catalyzed reaction via rapid-scanning UV/visible spectroscopy

Johnson¹,

Srivastava²

1993

“…These spectral changes show biphasic kinetics with 85% of the total amplitude occurring with a rate constant of 0.43 min™1 and the remainder showing a rate of 0.04 min™1. The fast phase in Figure 1A is some 105-fold slower than the corresponding rate observed with a preferred substrate of the enzyme, octanoyl-CoA (Hall et al, 1979;Gorelick et al, 1985;Lau et al, 1989). Clearly a methyl substituent at the C-3 position of octanoyl-CoA is very poorly tolerated in the reductive halfreaction of the medium chain enzyme.…”

Section: Resultsmentioning

confidence: 85%

3-Methylene-octanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA: Two new mechanism-based inhibitors of medium chain acyl-CoA dehydrogenase from pig kidney

Cummings¹,

Thorpe²

1994

The medium chain acyl-CoA dehydrogenase catalyzes the FAD-dependent oxidation of a variety of acyl-CoA substrates to the corresponding trans-2-enoyl-CoA thioesters. This work identifies 3-methyleneoctanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA as representatives of a new class of mechanism-based inhibitor of the dehydrogenase. One equivalent of either compound generates an inactive reduced flavin species with low absorption at 450 nm and a shoulder at 320 nm suggestive of an N-5 adduct. Reduction is rapid with the 3-methylene analogue (10/s at 1 degree C), but comparatively slow for 3-methyl-trans-2-octenoyl-CoA (1.1 x 10(-4)/s, under the same conditions). The reduced species is very stable, but the adduct can be slowly displaced with a large excess of octanoyl-CoA. The reduced adduct resists oxidation by the facile one-electron oxidant of the dehydrogenase, ferricenium hexafluorophosphate. Evidence that both isomeric inhibitors generate the same reduced flavin species includes an essentially identical visible spectrum, the same kinetics of displacement using octanoyl-CoA, and the same mixture of products on HPLC after denaturation of the treated enzyme with trichloroacetic acid, methanol, or by boiling. Experiments with the corresponding shorter analogues of these inhibitors, 3-methylenebutanoyl-CoA and 3-methyl-2-butenoyl-CoA confirm and extend these findings. These reduced adducts are less stable, allowing the dehydrogenase to catalyze the interconversion of the unconjugated 3-methylenebutanoyl-CoA to the more stable conjugated 3-methyl-2-butenoyl-CoA thioester (Keq ca. 150). These data suggest that alpha-proton abstraction from the 3-methylene derivatives or gamma-proton removal from the 3-methyl-2-enoyl analogues generates a common carbanionic intermediate which attacks oxidized flavin. As would be expected, the unconjugated 3-methylene derivatives are more effective inhibitors of the dehydrogenase than the thermodynamically more stable 3-methylenoyl analogues.

“…A question arose immediately concerning whether the above feature is an intrinsic property of the IPC0A/IAC0A as the pseudo substrate/product pair of the enzyme or is a property of all of the other acyl-CoA/enoyl-Co A pairs in general. While pondering this possibility, we realized that despite the recognition that octanoyl-CoA is one of the most efficient (and physiological) substrates for the enzyme, very little attempt has been made toward elucidation of its detailed oxidoreductivepathway (Hall et al, 1979). Besides, octanoyl-…”

Section: E-fadmentioning

confidence: 99%

“…1990); the natural electron acceptor for this process, under physiological conditions, is the electron-transferring flavoprotein (ETF; Beinert, 1963b) (eq 1). The enzyme is known to E-FAD + acyl-CoA ^E-FADH2-enoyl-CoA (1 A) E-FADH2-enoyl-CoA + ETF-FAD Ê-FAD-enoyl-CoA + ETF-FADH2 (IB) exhibit a fairly broad range of substrate specificity, catalyzing the oxidation of aliphatic-CoA substrates ranging from chain length C4 (butyryl-CoA) to Cíe (palmitoyl-CoA) (Beinert & Page, 1957;Hall et al, 1979). Of these, C$ (octanoyl-CoA) through C10 (decanoyl-CoA) are oxidized by the enzyme with maximum efficiencies, and thus they are considered to be the physiological substrates for the MCAD-catalyzed reaction (Crane et al, 1956;Hall, et al, 1979).…”

mentioning

confidence: 99%

“…The enzyme is known to E-FAD + acyl-CoA ^E-FADH2-enoyl-CoA (1 A) E-FADH2-enoyl-CoA + ETF-FAD Ê-FAD-enoyl-CoA + ETF-FADH2 (IB) exhibit a fairly broad range of substrate specificity, catalyzing the oxidation of aliphatic-CoA substrates ranging from chain length C4 (butyryl-CoA) to Cíe (palmitoyl-CoA) (Beinert & Page, 1957;Hall et al, 1979). Of these, C$ (octanoyl-CoA) through C10 (decanoyl-CoA) are oxidized by the enzyme with maximum efficiencies, and thus they are considered to be the physiological substrates for the MCAD-catalyzed reaction (Crane et al, 1956;Hall, et al, 1979). The ratio of fccat to Km (octanoyl-CoA) has been measured to be 1.64 X 107 M_1 s_1 when utilizing ETF as the electron acceptor (Beinert, 1963a).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Reductive Half-Reaction of Medium-Chain Fatty Acyl-CoA Dehydrogenase Utilizing Octanoyl-CoA/Octenoyl-CoA as a Physiological Substrate/Product Pair: Similarity in the Microscopic Pathways of Octanoyl-CoA Oxidation and Octenoyl-CoA Binding

Nr¹,

Dk²

1994

Of the different chain length fatty acyl-CoA substrates, octanoyl-CoA has been known as one of the most efficient (and physiological) substrates for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction of MCAD-FAD with octanoyl-CoA ([MCAD-FAD] << [octanoyl-CoA]), measured via the stopped-flow technique, at 5 degrees C was characterized by a biphasic decrease and increase in absorptions at 450 and 545 nm, respectively. The average values of the fast (1/tau 1) and slow (1/tau 2) relaxation rate constants, derived from the data at these wavelengths, were found to be 319.7 +/- 33.5 and 28.8 +/- 12.5 s-1, respectively, and both of these relaxation rate constants remained invariant between 8 and 200 microM concentrations of octanoyl-CoA. Under identical experimental conditions, we measured time courses for the interaction of MCAD-FAD with octenoyl-CoA ([MCAD-FAD] << [octenoyl-CoA]) by monitoring the absorption changes at 299, 394, and 440 nm. The binding profile was consistent with a biphasic decrease (at 440 nm) and increase (at 299 and 394 nm) in absorbance, with similar magnitudes of fast [1/tau 1 (average) = 382.3 +/- 39.8 s-1] and slow [1/tau 2 (average) = 14.3 +/- 7.4 s-1] relaxation rate constants. The observed relaxation rate constants were, once again, found to be invariant with changes in the octenoyl-CoA concentration from 40 to 150 microM.(ABSTRACT TRUNCATED AT 250 WORDS)