2,4-Dienoyl coenzyme A reductases from bovine liver and Escherichia coli. Comparison of properties.

The chemical mechanism of the 2,4-dienoyl-CoA reductase (EC 1.3.1.34) from rat liver mitochondria has been investigated. This enzyme catalyzes the NADPH-dependent reduction of 2,4-dienoyl-coenzyme A (CoA) thiolesters to the resulting trans-3-enoyl-CoA. Steady-state kinetic parameters for trans-2,trans-4-hexadienoyl-CoA and 5-phenyl-trans-2,trans-4-pentadienoyl-CoA were determined and demonstrated that the dienoyl-CoA and NADPH bind to the 2,4-dienoyl-CoA reductase via a sequential kinetic mechanism. Kinetic isotope effect studies and the transient kinetics of substrate binding support a random order of nucleotide and dienoyl-CoA addition. The large normal solvent isotope effects on V/K ((D)(2)(O)V/K) and V ((D)(2)(O)V) for trans-2,trans-4-hexadienoyl-CoA reduction indicate that a proton transfer step is rate limiting for this substrate. The stability gained by conjugating the phenyl ring to the diene in PPD-CoA results in the reversal of the rate-determining step, as evidenced by the normal isotope effects on V/K(CoA) ((D)V/K(CoA)) and V/K(NADPH) ((D)V/K(NADPH)). The reversal of the rate-determining step was supported by transient kinetics where a burst was observed for the reduction of trans-2,trans-4-hexadienoyl-CoA but not for 5-phenyl-trans-2,trans-4-pentadienoyl-CoA reduction. The chemical mechanism is stepwise where hydride transfer from NADPH occurs followed by protonation of the observable dienolate intermediate, which has an absorbance maximum at 286 nm. The exchange of the C alpha protons of trans-3-decenoyl-CoA, catalyzed by the 2,4-dienoyl-CoA reductase, in the presence of NADP(+) suggests that formation of the dienolate is catalyzed by the enzyme active site.

Section: Determination Of Steady-state Kineticcontrasting

confidence: 79%

The Mechanism of Dienoyl-CoA Reduction by 2,4-Dienoyl-CoA Reductase Is Stepwise: Observation of a Dienolate Intermediate

Fillgrove

Anderson

2001

“…The tight binding of zrans-3-octenoyl-CoA may have physiological significance in that Zrans-3-enoyl-CoA thio esters are intermediate in the metabolism of unsaturated fatty acids with cis double bonds at even-numbered carbon atoms (Dommes & Kunau, 1984a;Cuebas & Schulz, 1982;Mizugaki et al, 1984). Furthermore, the hypoglycemic agent 4-pentenoic acid (Billington et al, 1978) is metabolized to the corresponding 2,4-dienoyl-CoA species (Holland et al, 1973) and then largely reduced to Zrans-3-pentenoyl-CoA (Schulz, 1983) by 2,4-dienoyl-CoA reductase (Dommes & Kunau, 1984a). The accumulation of this metabolite within the mitochondrial matrix might reversibly inhibit the acyl-CoA dehydrogenase (particularly the short-chain enzyme) in ad- figure 4: Spectral changes on the addition of 3-ketooctanoyl-CoA to the medium-chain acyl-CoA dehydrogenase.…”

Section: Resultsmentioning

confidence: 99%

“…In mammals, the enzymes representing the central activities in mitochondrial /3-oxidation (namely, acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-OH-acyl-CoA dehydrogenase, and thiolase) exist in at least two forms to complement the array of acyl chain lengths encountered. In addition to these activities, ancillary enzymes such as a m-3-/Zra«s-2-isomerase (Stoffel et al, 1964;Miesowicz & Bloch, 1979) and a NADPH-linked 2,4-dienoyl-CoA reductase (Dommes & Kunau, 1984a;Cuebas & Schulz, 1982; Mizugaki et al, 1984) are required for the oxidation of unsaturated chains in mitochondria. This multiplicity of enzymes and acyl-CoA intermediates in the mitochondrial matrix poses problems of organization and coordination, and these aspects of fatty acid oxidation are poorly understood.…”

mentioning

confidence: 99%

Interaction of acyl coenzyme A substrates and analogs with pig kidney medium-chain acyl-CoA dehydrogenase

Powell

Lau²,

Killian³

et al. 1987

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)

“…Why the 3-oxidation process should terminate after removal of only four carbons from the carboxyl end of 15-HETE is not apparent. The presence of a c-4 unsaturation does not impede the /3-oxidation of n-6 polyunsaturated fatty acids (Yang et al, 1986;Dommes & Kunau, 1984) for it is encountered in acyl-CoA intermediates during the oxidation of linoleate (c-4-10:1) and arachidonate (c-4,c-7,c-10-16:3). This suggests that either the presence of the hydroxyl group or the conjugated cis-trans unsaturation of the 16:3(11-OH) acyl-CoA intermediate slows /3-oxidation sufficiently to allow the accumulation of this metabolite.…”

Section: Discussionmentioning

confidence: 99%

Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells

Shen¹,

Figard²,

Kaduce³

et al. 1988

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.