Enoyl coenzyme A reduction by bovine mammary fatty acid synthetase. Specificity and other characteristics.

The NADPH-dependent enoyl coenzyme A reductase activity of bovine mammary fatty acid synthetase has been characterized with regard to substrate specificity and the product formed. A relatively high specificity for an unsubstituted, four-carbon, 2,3-enoyl chain in trans configuration is obtained. Reduction of trans-crotonyl-CoA results in butyrate, 50% of which is coenzyme A-bound. The reaction is subject to product inhibition, specifically by butyryl-CoA and NADP. Free coenzyme A, on the other hand, is an activator. The pH profile, susceptibility to inhibition by -SH reagents, the results of the relative activities obtained with substrate analogues and homologues, and the ready use of crotonyl-CoA as a primer in fatty acid synthesis are consistent with a mechanism in which the crotonyl group is transferred to an -SH group, is reduced, and then is either transferred back to CoA or hydrolyzed.     

Beta-oxidation of very-long-chain fatty acids and their coenzyme A derivatives by human skin fibroblasts

The beta-oxidation of lignoceric acid (C24:0), hexacosanoic acid (C26:0), and their coenzyme A derivatives was investigated in human skin fibroblast homogenates. The cofactor requirements for oxidation of lignoceric acid and hexacosanoic acid were identical but were different from their coenzyme A derivatives. For example, lignoceric acid and hexacosanoic acid oxidation was strictly ATP dependent whereas the oxidation of the corresponding coenzyme A derivatives was ATP independent. Also the rate of oxidation of coenzyme A derivatives of lignoceric acid or hexacosanoic acid was much higher compared to the free fatty acids. In patients with Zellweger's syndrome, X-linked adrenoleukodystrophy and infantile Refsum's disease, the beta-oxidation of lignoceric and hexacosanoic acids was defective whereas the oxidation of their corresponding coenzyme A derivatives was nearly normal. The results presented in this communication suggest strongly that the beta-oxidation of very-long-chain fatty acids occurs exclusively in peroxisomes. However, the coenzyme A derivatives of very-long-chain fatty acids can be oxidized in mitochondria as well as in peroxisomes. The inability of the mitochondrial system to oxidize free fatty acids may be due to its inability to convert them to their corresponding coenzyme A derivatives. Our results suggest that a specific very-long-chain fatty acyl CoA synthetase may be required for the activation of the free fatty acids and that this synthetase may be deficient in patients with Zellweger's syndrome and possibly X-linked adrenoleukodystrophy, as well. The results presented suggest that substrate specificity and the subcellular localization of the synthetase may regulate the beta-oxidation of very-long-chain fatty acids in the cell.     

Resonance Raman study of flavins and the flavoprotein fatty acyl coenzyme A dehydrogenase.

The resonance Raman (RR) spectra of FMN, FAD, FAD in D2O, and 7,8-dimethyl-1, 10-ethyleneisoalloxazinium perchlorate have been obtained by employing KI as a collisional fluorescence-quenching agent. The spectra are very similar to those obtained recently by using the CARS technique to eliminate fluorescence. Spectra have also been obtained for several species in which flavin is known to fluoresce only weakly. We report RR spectra of protonated FMN, FMN semiquinone cation, the general fatty acyl-CoA dehydrogenase, and two "charge-transfer" complexes of fatty acyl-CoA dehydrogenase. Tentative assignment of several vibrational bands can be made on the basis of our flavin spectra. RR spectra of fatty acyl-CoA and its complexes are consistent with the previous hypothesis that visible spectral shifts observed during formation of acetoacetyl-CoA and crotonyl-CoA complexes of fatty acyl-CoA dehydrogenase result from charge-transfer interactions in which the ground state is essentially nonbonding as opposed to interactions in which complete electron transfer occurs to form FAD semiquinone. The only significant change in the RR spectrum of FAD on binding to enzyme occurs in the 1250-cm-1 region of the spectrum, a region associated with delta N--H of N-3. The position of this band in fatty acyl-CoA dehydrogenase and the other flavoproteins studied to date is discussed in terms of hydrogen bonding between flavin and protein.     

Induction of acyl coenzyme A synthetase and hydroxyacyl coenzyme A dehydrogenase during fatty acid degradation in Neurospora crassa

Neurospora crassa is able to use long-chain fatty acids as the sole carbon and energy source. After growth on oleate there was nearly a 10-fold induction of the acyl coenzyme A (CoA) synthetase and a fivefold increase in the activity of the 3-hydroxyacyl-CoA dehydrogenase. There was a slight induction of the enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase, but no apparent induction of the flavin-linked acyl-CoA dehydrogenase. These noncoordinate changes in the fatty acid degradation enzymes suggest that they are not organized into a multienzyme complex as is found in bacteria.     

A Dictyostelium long chain fatty acyl coenzyme A-synthetase mediates fatty acid retrieval from endosomes

We have identified a subset of Dictyostelium endosomes that carry a long chain fatty acyl coenzyme A-synthetase (LC-FACS 1) on their cytosolic surface. Immunofluorescence studies and observations using GFP-fusion proteins collectively suggest that LC-FACS 1 associates with endosomes a few minutes after their formation, remains bound through the acidic phase of endocytic maturation and dissociates early in the phase where the endosomal content is neutralised prior to exocytosis. Mutants in the fcsA gene, encoding the LC-FACS 1 protein, were constructed by homologous recombination. These cells show a strong defect in the intracellular accumulation of fatty acids, either taken up together with the liquid medium or bound to the surface of particles. Because the mutant cells are otherwise fully competent for macropinocytosis and phagocytosis, we conclude that the LC-FACS 1 protein mediates the retrieval of fatty acids from the lumen of endosomes into the cytoplasm.     

Acetyl coenzyme A binding by chloramphenicol acetyltransferase: long-range electrostatic determinants of coenzyme A recognition.

The possible involvement of arginyl and lysyl side chains of chloramphenicol acetyltransferase (CAT) in binding coenzyme A (CoA) was studied by means of chemical modification, site-directed mutagenesis, variation in ionic strength, use of competitive inhibitors or substrate analogues, and X-ray crystallography. Unlike a number of enzymes, including citrate synthase, CAT does not employ specific ion pairs with the phosphoanionic centers of CoA to bind the acetyl donor, and arginyl residues play no role in recognition of the coenzyme. Although phenylglyoxal inactivates CAT reversibly, it does so by the formation of an unstable adduct with a thiol group, that of Cys-31 in the chloramphenicol binding site. The inhibitory effect of increasing ionic strength on kcat/Km(acetyl-CoA) can be explained by long-range electrostatic interactions between CoA and the epsilon-amino groups of Lys-54 and Lys-177, both of which are solvent-accessible. The epsilon-amino group of Lys-54 contributes 1.3 kcal.mol-1 to the binding of acetyl-CoA via interactions with both the 3'- and 5'-phosphoanions of CoA. Lys-177 contributes only 0.4 kcal.mol-1 to the productive binding of acetyl-CoA, mediated by long-range (approximately 14 A) interactions with the 5'-alpha- and -beta-phosphoanions of CoA. The combined energetic contribution of Lys-54 and Lys-177 to acetyl-CoA binding (1.7 kcal.mol-1) is less than that previously demonstrated (2.4 kcal.mol-1) for a simple hydrophobic interaction between Tyr-178 and the adenine ring of CoA (Day & Shaw, 1992).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic characterization of long chain fatty acyl coenzyme A ligase from rat liver mitochondria.

Long chain fatty acyl coenzyme A ligase (EC 6.2.1.3) purified from rat liver mitochondria has been characterized with respect to several kinetic parameters. Many of the kinetic properties of the mitochondrial enzyme are similar to those of the purified microsomal enzyme with respect to palmitoyl-CoA formation, but there are distinct differences. The fatty acid and nucleotide specificities of the mitochondrial enzyme are similar to those of the microsomal enzyme, as are the apparent Km values for ATP and coenzyme A. On the other hand, the mitochondrial enzyme differs from the microsomal enzyme in that it has a lower pH optimum, is different in molecular weight, and does not show simple saturation kinetics with palmitate as substrate. Of particular interest is the evidence presented which indicates that the mitochondrial long chain fatty acyl-CoA ligase, unlike short and medium chain ligases, does not utilize an acyladenylate as an intermediate in the formation of fatty acyl-CoA.