Subcellular distribution of coenzyme A: Evidence for a separate coenzyme a pool in peroxisomes

186

174

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway in bacteria and is thought to play a similar role in mammalian cells. We examined this hypothesis by identifying and characterizing two murine cDNAs that encoded PanK. The two cDNAs were predicted to arise from alternate splicing of the same gene to yield different mRNAs that encode two isoforms (mPanK1␣ and mPanK1␤) with distinct amino termini. The predicted protein sequence of mPanK1 was not related to bacterial PanK but exhibited significant similarity to Aspergillus nidulans PanK. mPanK1␣ was most highly expressed in heart and kidney, whereas mPanK1␤ mRNA was detected primarily in liver and kidney. Pantothenate was the most abundant pathway component (42.8%) in normal cells providing clear evidence that pantothenate phosphorylation was a ratecontrolling step in CoA biosynthesis. Enhanced mPanK1␤ expression eliminated the intracellular pantothenate pool and triggered a 13-fold increase in intracellular CoA content. mPanK1␤ activity in vitro was stimulated by CoA and strongly inhibited by acetyl-CoA illustrating that differential modulation of mPanK1␤ activity by pathway end products also contributed to the management of CoA levels. These data support the concept that the expression and/or activity of PanK is a determining factor in the physiological regulation of the intracellular CoA concentration.Pantothenate kinase (PanK) 1 (ATP:D-pantothenate 4Ј-phosphotransferase, EC 2.7.1.33) catalyzes the first committed step in the universal biosynthetic pathway leading to CoA (for review see Ref. 1). Phosphopantothenate is rapidly metabolized to CoA, which participates as an acyl group carrier in the tricarboxylic acid cycle, fatty acid metabolism, and numerous other reactions of intermediary metabolism (2). The 4Ј-phosphopantetheine portion of CoA is an essential prosthetic group in a number of enzyme systems including the acyl carrier protein components, bacterial and eukaryotic fatty acid synthases (3), citrate lyase (4), ferrichrome synthetase from Apsergillus quadricinctus (5), and malonate decarboxylase of Malonomonas rubra (6).PanK is the rate-controlling enzyme in CoA biosynthesis in Escherichia coli (1). E. coli is capable of de novo pantothenate biosynthesis, and additionally a sodium-dependent permease actively transports exogenous pantothenate into the cell (7-9). Metabolic labeling experiments established that the utilization, rather than the supply of pantothenate, controls the level of CoA (10). E. coli mutants with temperature-sensitive PanK activity are also temperature-sensitive for CoA biosynthesis and growth (11). The PanK gene of E. coli (coaA) was cloned by functional complementation of this mutant and was identical to a previously sequenced allele called rts (12)(13)(14). E. coli PanK (bPanK) is a homodimer of 36-kDa subunits that exhibit highly positive cooperative ATP binding and mediate sequential ordered catalysis with ATP as the leading substrate (15). CoA and its thioesters inhibit bPanK activity by co...

Section: Discussionmentioning

confidence: 99%

Pantothenate Kinase Regulation of the Intracellular Concentration of Coenzyme A

Rock¹,

Calder²,

Karim³

et al. 2000

186

174

“…Thus, the decreased sensitivity of bPanK to acetyl-CoA allows this pool to expand and accommodate the metabolic demands of rapidly dividing cells. In contrast, mitochondria and peroxisomes are considered to have the highest concentration of CoA and its thioesters in eukaryotes (35)(36)(37)(38)(39). The PanK enzyme, which is rate-limiting also in mammalian CoA biosynthesis (14), is cytosolic (14,22,23).…”

Section: Discussionmentioning

confidence: 99%

Cloning and Characterization of a Eukaryotic Pantothenate Kinase Gene (panK) from Aspergillus nidulans

Calder

Williams

Ramaswamy

et al. 1999

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene from Escherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designated panK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. AcetylCoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart.Pantothenate kinase (PanK)

“…The inhibition of PTE-2 activity by CoASH indicates that this enzyme can "sense" intraperoxisomal CoASH levels, and thus when there is a requirement for CoASH, PTE-2 is active, whereas during times of high free CoASH, PTE-2 can be inhibited. Peroxisomes contain a distinct pool of CoASH (43,44), which has been reported to change during fasting and treatment with peroxisome proliferators (45). The extent of CoASH sequestration may therefore depend on the size of the CoASH pool, the amount and type of lipids being trapped in the ␤-oxidation systems in the peroxisome, as well as activities of acyl-CoA thioesterases and possibly a recently cloned peroxisomal nudix hydrolase, which apparently can hydrolyze CoASH to yield 3Ј,5Ј-ADP and the corresponding 4Ј-phosphopantetheine derivative (46).…”

Section: Fig 3 Pte-2 Is a Peroxisomal Matrix Proteinmentioning

confidence: 99%

Characterization of an Acyl-CoA Thioesterase That Functions as a Major Regulator of Peroxisomal Lipid Metabolism

Hunt

Solaas

Kase

et al. 2002

144

Peroxisomes function in ␤-oxidation of very long and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. Here we have cloned and characterized a peroxisomal acyl-CoA thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA thioesterase 2). PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor ␣. Recombinant PTE-2 showed a broad chain length specificity with acyl-CoAs from short-and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA, and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acidCoA:amino acid N-acyltransferase activity, suggesting that this thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.Peroxisomes are cellular organelles present in all eukaryotic cells. They play an indispensable role in the metabolism of a variety of lipids including very long-chain fatty acids, dicarboxylic fatty acids, bile acids, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic fatty acids (for review, see Refs. 1 and 2). The peroxisomal ␤-oxidation system contains two sets of enzymes, one of which is involved in the oxidation of branched chain fatty acids and intermediates in the hepatic bile acid biosynthetic pathway and consists of one or two branched-chain acyl-CoA oxidase(s), a D-specific bifunctional protein and the sterol carrier-like protein x (SCPx). The second pathway is involved in the oxidation of very long straight-chain fatty acids, CoA esters of prostaglandins, leukotrienes, and thromboxanes (prostanoids) and dicarboxylic acids. This system is composed of a straight-chain acyl-CoA oxidase, an L-specific bifunctional protein and straight-chain 3-ke...