Abstract
The mechanisms of the initial interactions of three rat liver acyl-CoA dehydrogenases (short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases) and their fatty acyl-CoA substrate were studied using enzyme-catalyzed deuterium exchange. The reaction products were identified and quantitated using mass spectroscopy and 1H-NMR. When fatty acyl-CoA substrates were incubated with catalytic amounts of acyl-CoA dehydrogenase in D2O in the absence of an electron acceptor, a rapid monodeuteration of the substrate occurred to replace one of the prochiral C-2 hydrogens, while no C-3 hydrogens were exchanged with deuterium. The C-2 monodeuteration proceeded to the extent of 80% of the total amount of substrate added at 90 min and almost to completion at 120 min. The pKa values and optimum pD values for the C-2 proton/deuteron exchange reactions were 6.0 and 7.5, respectively, for each of the three acyl-CoA dehydrogenases. The apparent turnover numbers were 3.0, 3.3, and 0.5 s-1 for short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases, respectively. These results provide the first direct evidence for carbanion formation via abstraction of a C-2 hydrogen by a base in the enzyme, as the first step of the catalytic pathway of acyl-CoA dehydrogenation. When the acyl-CoA dehydrogenases were reacted with moderate excesses of acyl-CoA substrates in D2O in the absence of an electron acceptor, maximum bleaching of the FAD absorbance and the appearance of the long wavelength absorbance, attributed to a charge transfer complex, were observed. However, the dehydrogenation products, 2-enoyl-CoAs, were produced either not at all or in an amount which represented only a minor fraction of the amount of the enzyme added, while the substrates in the enzyme-substrate complexes rapidly turned over as indicated by the extensive monodeuteration which concomitantly occurred. Unlike previous hypothesis, these results indicate that the hydride ion transfer from C-3 of the substrate to the enzyme-FAD is not yet complete in the charge-transfer complex. The transfer of the hydride ion to alloxazine N-5 and the release of products are completed only in the presence of electron-transfer flavoprotein or another suitable electron acceptor.
Highlights
The mechanisms of the initial interactions of three The overall reaction of the ETF’-linkedacyl-CoA dehydror a t liver acyl-CoA dehydrogenases trated as shown in Equation 1
Strates wereincubated with catalytic amountosf acylCoA dehydrogenase in D20 in the absenceof an elec- In this reaction, fattaycyl-CoA thioesters aredehydrogenated tron acceptor, a rapid monodeuterationof the substrate at C-2 and C-3, resulting in the formation of trans-2-enoyloccurred to replace oonfethe prochiral C-2 hydrogensC, oAs [1].Electrons taken up by ETF are further transferred while no C-3 hydrogens were exchanged with deuter- to the main mitochondrial electron-transport chainvia E T F
A mass range from 50 to 300 atomic mass Catalytic amountsof the holoenzyme of short-chain, mediumunits was scanned throughout ata rate of 0.60 s cycle" for capillary runs and 2.40 s cycle" for packed column runs
Summary
Demonstration of Acyl-CoA Dehydrogenase-catalyzed Monport, transferline, and ionsource temperatures were kept at 225,270, odeuteration of Substrate in D20Using Mass Spectroscopyand 130 "C, respectively. A chemical ionization/mass chromatogram mode was used to accurately quantitate the deuterium enrichment in the molecular ions of methyl valerate. In the case of medium-chain acyl-CoA deof authentic unlabeled methyl valerate. Uct, taken at 90 min, shifted tom/z 117 (Fig. 1B).This shift Mass Spectral Identification of 2-Monodeuterated Carboxylic of the molecular ion was accompanied by the similar shiftof Acids Produced by the Enzyme-catalyzed DeuteriumExthe M - 31 ion from m/z 85 (methyl valerate) tom/z 86. The loss of intensity of the C-2-proton signal, as measured by integrationa, ndthe changes of its shape and chemical shift in the labeled compound all indicated that one of the C-2 protons was almost
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