dephospho-CoA Kinase Research Articles

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA

Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.

A bifunctional enzyme complex in coenzyme A biosynthesis: purification of pantetheine phosphate adenylyltransferase and dephospho-CoA kinase

Pantetheine phosphate adenylyltransferase (EC 2.7.7.3) and dephospho-CoA kinase (EC 2.7.1.24) were purified to near homogeneity from pig liver. The purification steps included the use of Sepharose-linked triazine dyes and affinity elution by CoA. Both activities co-purified at every stage of the 18 000-fold purification. An Mr of 115 000 was obtained by gel filtration on Sephadex G-150, and the final preparation yielded one major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with a subunit Mr of 57 000. It is concluded that pantetheine phosphate adenylyltransferase and dephospho-CoA kinase exist as a bifunctional dimeric protein, which could be designated CoA synthetase.

Mitochondrial pantetheinephosphate adenylyltransferase and dephospho-CoA kinase.

Pantetheinephosphate adenylyltransferase and dephospho‐CoA kinase, which are last in the sequence leading to the biosynthesis of CoA, are localized in mitochondria (about 30% of the activity in the cell) and cytosol. The first three enzymes in the pathway are only found in cytosol [Skrede, S. and Halvorsen, O. (1979) Biochem. Biophys. Res. Commun. 91, 1536–1542]. Both enzymes are present in the mitochondrial matrix, but dephospho‐CoA kinase also has a more ‘external’ pool and is less easily removed from the membrane fractions of the mitochondria. In accordance with the submitochondrial fractionation studies, pantetheinephosphate adenylyltransferase is to a larger extent activated by detergents or mechanical disruption of the mitochondria than is dephospho‐CoA kinase. In intact mitochondria, the biosynthesis of CoA from 4‐phosphopantetheine was stimulated by uncoupling agents, probably because of facilitated penetration of the substrate through the inner mitochondrial membrane and or of leakage of a regulator. Intact mitochondria could increase the synthesis of CoA 5–10 times, when conditions were chosen to promote biosynthesis from 4‐phosphopantetheine and to remove CoA by acylation and/or ‘leakage’ out of the mitochondria. The kinetic properties of both mitochondrial enzymes are quite similar to the cytosolic ones (but our reinvestigation of the latter enzymes only partially confirm previous studies). Km for 4‐phosphopantetheine and dephosplio‐CoA were 0.015 and 0.01 mmol/1, respectively, for the mitochondrial enzymes. Mitochondrial biosynthesis of CoA was strongly inhibited by dephospho‐CoA at the pantetheinephosphate adenylyltransferase level and more slightly by CoA at the dephospho‐CoA kinase level

Mitochondrial biosynthesis of coenzyme A

All enzymes required for the biosynthesis of CoA from pantothenic acid are present in the particle-free supernatant fraction from rat liver. We now report that also mitochondria have the capacity for biosynthesis of CoA, with 4′-phosphopantetheine as the initial precursor. Rat liver mitochondria do not contain pantothenate kinase, 4′-phosphopantothenoyl-1-synthetase or 4′-phosphopantothenoyl-1-cysteine decarboxylase. Dephospho-CoA pyrophosphorylase and dephospho-CoA kinase are present in the inner mitochondrial membrane, however, at specific activities as high as in cytosol. K m of mitochondrial dephospho-CoA kinase for dephospho-CoA is about 0.01 mmol/1, which is one order of magnitude lower than reported for the kinase from cytosol.

Formation of palmityl-[3′- 32P]coenzyme a from [γ- 32P]ATP in mitochondrial extracts of guinea pig liver

Investigations of the incorporation of 32P into acyl-coenzyme A (CoA) in incubation mixtures containing a soluble protein preparation derived from mitochondria, [γ- 32P]ATP, and palmityl-CoA have led to the discovery of an enzymatic activity which catalyzes the exchange of palmityl groups between molecules of CoA: CoA ∗ + palmityl-CoA ↔ palmityl-CoA ∗ + CoA. The preparation also contains dephospho-CoA kinase and palmityl-CoA thiolester hydrolase activities. The initial detection of the exchange reaction resulted from the formation of [3′- 32P]CoA via the dephospho-CoA kinase reaction with exogenous [γ- 32P]ATP. The described preparation of palmityl-[3′- 32P]CoA and palmityl-[ 35S]CoA facilitated demonstration of the reversibility of the reaction and ruled out the possibility that the exchange of fragments of the CoA molecule mediated the observed incorporation. The reversible palmityl group exchange does not appear to be catalyzed by a previously described enzyme. None of the possible acyl group acceptors considered in these studies participated in the reaction as efficiently as CoA itself. The possibility is discussed that the exchange reaction may explain reports of an unknown lipid formed by an oligomycin-sensitive mitochondrial ATPase preparation.

Incorporation of pantothenate-1- 14C into pigeon liver fatty acid synthetase: 4′-phosphopantetheine, a prosthetic group of the multienzyme complex

Pantothenate-l- 14C was injected into starved and refed pigeons. 14C-Labeled soluble fatty acid synthetase was then isolated from the liver, and the protein bound radioactivity was released from the enzyme complex by mild alkaline hydrolysis. The liberated moiety was shown to be 4′-phosphopantetheine by co-chromatography with the authentic compound on a DEAE-cellulose column and by conversion to CoA in the presence of dephospho-CoA pyrophosphorylase, dephospho-CoA kinase, and ATP. The product was then converted to acetyl-CoA to further confirm its identity. These results show that the pigeon liver fatty acid synthetase contains covalently bound 4′-phosphopantetheine.

Investigations on pantothenic acid and its related compounds. XII. Biochemical studies (7). Dephospho-coA pyrophosphorylase and dephospho-coA kinase as a possible bifunctional enzyme complex.

Investigations on pantothenic acid and its related compounds. XII. Biochemical studies (7). Dephospho-coA pyrophosphorylase and dephospho-coA kinase as a possible bifunctional enzyme complex.

Investigations on pantothenic acid and its related compounds. XI. Biochemical studies. 6. A final stage in the biosynthesis of CoA.

1. The final steps of CoA biosynthesis in rat liver were investigated using purified dephospho-CoA pyrophosphorylase [EC 2.7.7.3] and dephospho-CoA kinase [EC 2.7.1.24] and phospho pantothenoylcysteine decarboxylase [EC 4.1.1.36]. 2. It was concluded that the pathway involving the following metabolic conversions is the only operative one: phosphopantetheine→dephospho-CoA→CoA. 3. The possibilities of other reaction sequences were also examined and excluded.