Thia- and oxaoctanoyl-CoA derivatives (substituted at the C-3 and C-4 positions) have been synthesized to prove the reductive half-reaction in the medium-chain acyl-CoA dehydrogenase from pig kidney. 3-Thiaoctanoyl-CoA binds to this flavoenzyme, forming an intense, stable, long-wavelength band (at 804 nm; extinction coefficient = 8.7 mM-1 cm-1 at pH 7.6). The intensity of this band increases about 20% from pH 6.0 to pH 8.8. This long-wavelength species probably represents a charge-transfer complex between bound acyl enolate as the donor and oxidized flavin adenine dinucleotide as the acceptor. Thus, the enzyme catalyzes alpha-proton exchange, and no long-wavelength bands are seen with 3-thiaoctyl-CoA (where the carbonyl moiety is replaced by a methylene group). 3-Oxaoctanoyl-CoA binds comparatively weakly to the dehydrogenase, with a long-wavelength band at 780 nm which is both less intense and less stable than the corresponding thia analogue. These data suggest that the enzyme can accomplish alpha-proton abstraction from certain weakly acidic acyl-CoA derivatives, without concerted transfer of a hydride equivalent to the flavin. 4-Thiaoctanoyl-CoA is dehydrogenated in the standard assay 1.5-fold faster than octanoyl-CoA. Titrations of the medium-chain dehydrogenase with the 4-thia derivative resemble those obtained with octanoyl-CoA, except for the contribution of the strongly absorbing 4-thia-trans-2-octenoyl-CoA product. The corresponding 4-oxa analogue is a much poorer substrate (10% of the rate shown by octanoyl-CoA) but again effects substantially complete reduction of the flavin chromophore in the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
Several alkylthio coenzyme A (CoA) derivatives (from ethyl- to hexadecyl-SCoA) have been synthesized to probe the substrate binding site in the flavoprotein medium-chain acyl-CoA dehydrogenase from pig kidney. All bind to apparently equivalent sites with a stoichiometry of four per tetramer. A plot of log Kd vs: hydrocarbon chain length is linear from 2 to 16 carbons with a free energy of binding of 390 cal/methylene group. These data suggest an acyl-binding site of moderate hydrophobicity and imply that the observed substrate specificity of the medium-chain dehydrogenase is not achieved simply by the length of the hydrocarbon binding pocket. Extrapolation of the graph to zero chain length predicts a Kd of 1 mM for the CoA moiety. The difference between this value and the experimentally determined value of 206 microM may be attributed to a contribution from the ionization of the sulfhydryl group in CoASH. The interaction of several eight-carbon intermediates of beta-oxidation (trans-2- and trans-3-octenoyl-CoA and L-3-hydroxy- and 3-ketooctanoyl-CoA) with the dehydrogenase has also been studied. All but the L-3-OH derivative bind tightly to the enzyme (with Kd values in the 50-90 nM range) and are very effective inhibitors of the dehydrogenation of octanoyl-CoA. The trans-3-enoyl analogue produces an immediate, intense, long-wavelength band (lambda max = 820 nm), which probably represents a charge-transfer interaction between the delocalized alpha-carbanion donor and oxidized flavin as the acceptor. The L-3-OH analogue is a reductant of the flavin, yielding 3-ketooctanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)
4-Thiaacyl-CoA analogues, in which the 4-methylene group is replaced by a thioether sulfur atom, represent new chromophoric substrates of acyl-CoA dehydrogenases and oxidase. The corresponding 4-thia-trans-2-enoyl-CoA products exhibit a strong new absorption band (extinction coefficient 22 mM-1 cm-1) that is red shifted from 312 to 338 nm upon binding to the medium-chain acyl-CoA dehydrogenase. 4-Thiaoctanoyl-CoA reduces the dehydrogenase several-fold slower than octanoyl-CoA, although in turnover it is dehydrogenated 1.5-fold faster. The redox potential of 4-thia analogues is some 30 mV more negative than that of their unsubstituted counterparts. 4-Thia-trans-2-enoyl-CoA derivatives are slowly hydrated by enoyl-CoA hydratase (EC 4.2.1.17) to the corresponding thiohemiacetal which fragments nonenzymatically to 1 equiv each of malonylsemialdehyde-CoA and alkanethiol. This fragmentation reaction might explain the release of methanethiol during the transamination pathway of methionine degradation. 4-Oxaoctanoyl-CoA is a much poorer substrate and kinetic reductant of acyl-CoA dehydrogenase and oxidase than the 4-thia analogue. The corresponding enoyl-CoA product is also fragmented by the hydratase, yielding butanol and malonylsemialdehyde-CoA. Thus, 4-heterosubstituted acyl-CoA derivatives provide new tools for the study of beta-oxidation enzymes.
ABSTRACT:The flavoprotein medium-chain acyl coenzyme A (acyl-CoA) dehydrogenase from pig kidney exhibits an intrinsic hydratase activity toward crotonyl-CoA yielding L-3-hydroxybutyryl-CoA. The maximal turnover number of about 0.5 min-' is 500-1000-fold slower than the dehydrogenation of butyryl-CoA using electron-transferring flavoprotein as terminal acceptor. trans-2-Octenoyl-and trans-2-hexadecenoyl-CoA are not hydrated significantly. Hydration is not due to contamination with the short-chain enoyl-CoA hydratase crotonase. Several lines of evidence suggest that hydration and dehydrogenation reactions probably utilize the same active site. These two activities are coordinately inhibited by 2-octynoyl-CoA and (methylenecyclopropy1)acetyLCoA [whose targets are the protein and flavin adenine dinucleotide (FAD) moieties of the dehydrogenase, respectively]. The hydration of crotonyl-CoA is severely inhibited by octanoyl-CoA, a good substrate of the dehydrogenase. The apoenzyme is inactive as a hydratase but recovers activity on the addition of FAD. Compared with the hydratase activity of the native enzyme, the 8-fluoro-FAD enzyme exhibits a roughly 2-fold increased activity, whereas the 5-deaza-FAD dehydrogenase is only 20% as active.
A cytochrome P450 with low affinity (2.9 x 10(3) M-1) for CO appears to be the major microsomal P450 in some plant tissues. The presence of low CO affinity cytochrome P450 correlates with its lack of NADPH reducibility and with the presence of high levels of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate peroxidase activity. This activity and low CO affinity are retained by purified tulip cytochrome P450, which appears to be catalytically identical to a flaxseed-derived fatty acid allene oxide synthase P450 described previously [Song, W.-C., & Brash, A.R. (1991) Science 253, 781-784]. Other heme-binding ligands, such as CN- and imidazoles, bind weakly to the allene oxide synthase P450s, suggesting that axial coordination in the heme distal pocket may be hindered. We conclude that low CO affinity is characteristic of the allene oxide synthase P450s and that these P450s constitute a major portion of the microsomal P450 in a variety of plant tissues, particularly from monocot species.
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