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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 Jan 22;104(5):1494–1499. doi: 10.1073/pnas.0607621104

Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine

Roberta Leonardi 1, Charles O Rock 1, Suzanne Jackowski 1, Yong-Mei Zhang 1,*
PMCID: PMC1785270  PMID: 17242360

Abstract

The human isoform 2 of pantothenate kinase (PanK2) is localized to the mitochondria, and mutations in this protein are associated with a progressive neurodegenerative disorder. PanK2 inhibition by acetyl-CoA is so stringent (IC50 < 1 μM) that it is unclear how the enzyme functions in the presence of intracellular CoA concentrations. Palmitoylcarnitine was discovered to be a potent activator of PanK2 that functions to competitively antagonize acetyl-CoA inhibition. Acetyl-CoA was a competitive inhibitor of purified PanK2 with respect to ATP. The interaction between PanK2 and acetyl-CoA was stable enough that a significant proportion of the purified protein was isolated as the PanK2·acetyl-CoA complex. The long-chain acylcarnitine activation of PanK2 explains how PanK2 functions in vivo, by providing a positive regulatory mechanism to counteract the negative regulation of PanK2 activity by acetyl-CoA. Our results suggest that PanK2 is located in the mitochondria to sense the levels of palmitoylcarnitine and up-regulate CoA biosynthesis in response to an increased mitochondrial demand for the cofactor to support β-oxidation.

Keywords: carnitine, coenzyme A, β-oxidation, pantothenate kinase-associated neurodegeneration

Pantothenate kinase (PanK) catalyzes the first and rate-controlling step in the biosynthesis of CoA (for review, see ref. 1). In humans, there are three genes that express four isoforms of PanK. PanK1α and 1β arise from the use of alternate initiation exons of the PANK1 gene (2, 3), and the PANK3 gene produces a single polypeptide (4). The PANK2 gene differs from the others in that it encodes a protein that is targeted to the mitochondria (5–7). The PanK2 protein translated from the most 5′ start site is sequentially cleaved at two sites by mitochondrial processing peptidases, generating a long-lived 48-kDa mature protein (5). Two shorter PanK2 isoforms have been described, one of which also localizes to mitochondria (7), whereas the second does not because of the lack of targeting sequences (6). A common feature of all PanK proteins is that they are feedback inhibited by CoA thioesters (1); however, the isoforms are distinguished by their unique sensitivities to the CoA thioester pool (2–4, 8). A fourth gene, PANK4, lacks the essential catalytic glutamate residue present in all other enzymes (9) and may not be a functional PanK.

The PanK2 isoform is the focus of much current research because mutations in the human PANK2 gene give rise to PanK-associated neurodegeneration (PKAN), an inherited autosomal recessive disease (10). PKAN patients constitute a subset of those diagnosed with neurodegeneration with brain iron accumulation, formerly known as Hallervorden–Spatz syndrome (11). PKAN patients have a pathological accumulation of iron in the basal ganglia and a combination of motor symptoms in the forms of dystonia, dysarthria, intellectual impairment, and gait disturbance (11). Early-onset patients have a rapidly progressive disease, and late-onset patients have a slowly progressive, atypical disease, where parkinsonism is common (11, 12). Two hypotheses have been proposed to explain the clinical problems associated with mutations in PANK2: (i) accumulation of cysteine-containing substrates because of inhibition of CoA synthesis, which may undergo rapid autooxidation in the presence of iron, leading to free radical generation and cell damage (10); or (ii) mitochondrial CoA deficiency leading to increased oxidative stress from free radicals (7). Both of these hypotheses posit that the mutated PanK2 proteins are functionally defective (13). Although it is clear that a number of PANK2 mutations give rise to inactive proteins, recent biochemical analyses show that many of the PanK2 missense mutations encode functional proteins that are regulated by acetyl-CoA (5, 8). Disruption of the Pank2 gene in mice does not produce a neurodegenerative phenotype (14), although it does result in retinal degeneration, often linked with the human disease, and azoospermia, which is associated with reduced PanK activity in Drosophila melanogaster (15). Clearly, there is much to be learned regarding the regulatory biochemistry and role of PanK2 in intermediary metabolism.

One of the puzzling features of PanK2 biochemistry is its extremely high sensitivity to inhibition by acyl-CoA (5, 8). The IC50 for acetyl-CoA is <1 μM at 2.5 mM ATP (8), which is much lower than the cytosolic CoA concentrations estimated at 20–140 μM, and, if PanK2 resides in the mitochondrial matrix compartment, CoA levels are likely between 2.2 and 5 mM (16, 17). These data make it hard to understand how PanK2 functions in vivo because of the high concentration of CoA thioesters. This work describes the reversal of acetyl-CoA inhibition of PanK2 by palmitoylcarnitine. This new biochemical property of PanK2 provides an explanation for how PanK2 is activated in vivo and suggests that its mitochondrial location is important for its function as an acylcarnitine sensor that up-regulates CoA biosynthesis in response to accelerated demand for mitochondrial β-oxidation.

Results

Palmitoylcarnitine Activates PanK2.

One of the conundrums of research on PanK2 is that it binds acetyl-CoA, and other CoA species, with an apparent Kd that is far below the estimated intracellular and intramitochondrial CoA concentrations. Accordingly, PanK2 expressed and partially purified from 293T cells was inhibited by acetyl-CoA with an apparent IC50 of ≈0.3 μM under the conditions tested (Fig. 1A). These data suggested that PanK2 would be inactive in vivo and led us to search for ligands that may regulate PanK2. Palmitoylcarnitine was discovered as an activator of PanK2 activity in 293T cell lysates (Fig. 1B). The enzymatic activity of PanK2 was stimulated to the highest level by 8 μM palmitoylcarnitine. In contrast, carnitine did not have any effect on the PanK2 activity when included in the reactions at the same concentration range (Fig. 1B). We next determined whether palmitoylcarnitine was able to reverse the inhibition of PanK2 by acetyl-CoA (Fig. 1C). Palmitoylcarnitine was capable of partially reversing the inhibition of PanK2 by 0.2 μM acetyl-CoA, whereas carnitine was not. Thus, palmitoylcarnitine was a positive regulator of PanK2 activity.

Fig. 1.

Fig. 1.

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Negative and positive regulators of PanK2 activity. (A) Acetyl-CoA inhibited PanK2 activity in 293T cell lysates with an IC50 value of 0.3 μM. (B) Palmitoylcarnitine (●) activated PanK2 in partially purified 293T cell lysates, whereas carnitine (○) did not affect PanK2 activity. (C) Palmitoylcarnitine (●) reversed the inhibition of PanK2 activity in 293T cell lysates by 0.2 μM acetyl-CoA, whereas carnitine (○) did not.

Analysis of Acetyl-CoA Regulation of the Purified PanK2.

The data in Fig. 1 support palmitoylcarnitine as a positive regulator of PanK2; however, interpreting the mechanism of its activation in partially purified lysates was complicated by the lack of control over the concentrations of endogenous regulatory ligands that might be present. Therefore, the N-terminal His-tagged PanK2 was expressed in Escherichia coli and purified to homogeneity by affinity and size-exclusion chromatography (Fig. 2A). The pure protein exhibited a specific activity of 0.2421 ± 0.006 pmol/ng per min under the standard PanK assay condition. The elution position of PanK2 on the gel filtration column was consistent with previous work in cell lysates that indicated it was a dimer in solution. Acetyl-CoA was a potent inhibitor of purified PanK2 with an IC50 value of ≈60 nM (Fig. 2B). We confirmed that long-chain acyl-CoAs, such as palmitoyl-CoA, also strongly inhibited PanK2 activity (5, 8). The kinetic mechanism of acetyl-CoA inhibition was explored, and the graphical analysis of the data indicated acetyl-CoA inhibition was competitive with ATP (Fig. 2C). This conclusion was confirmed by using a direct binding assay employing the fluorescent ATP analog, TNP-ATP. The binding of TNP-ATP to proteins results in a strong increase in fluorescence that is directly proportional to the extent of binding. The addition of acetyl-CoA to the TNP-ATP binding assay displaced the ATP analog from the protein (Fig. 2D), confirming that PanK2·acetyl-CoA complex formation compromised ATP binding.

Fig. 2.

Fig. 2.

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Acetyl-CoA is a competitive inhibitor of PanK2 with respect to ATP. (A) His-tagged PanK2 was overexpressed in E. coli and purified to homogeneity by Ni-affinity and size exclusion chromatography. (Inset) An SDS gel showing the purity of the PanK2 preparation. (B) Acetyl-CoA inhibited purified PanK2 with an IC50 of 60 nM. (C) Lineweaver–Burk plots of PanK2 in the absence (■) or presence of 30 nM (▴) or 60 nM (▾) acetyl-CoA. The lines intercept on the y axis, indicating that acetyl-CoA was a competitive inhibitor of PanK2 with respect to ATP. (D) Acetyl-CoA displaced the ATP analog TNP-ATP from the PanK2 active site. Fluorescent emission spectra of 5 μM TNP-ATP only (dashed line), 5 μM TNP-ATP plus 1 μM PanK2 (dotted line), and 5 μM TNP-ATP plus 1 μM PanK2 and 0.4 μM acetyl-CoA (solid line). The fluorescence of the environmentally sensitive ATP analog increased on binding to PanK2, but the addition of acetyl-CoA displaced TNP-ATP from PanK2.

Palmitoylcarnitine Regulation of PanK2.

Palmitoylcarnitine was able to reverse the inhibition of purified PanK2 by acetyl-CoA (Fig. 3A). In this experiment, 60 nM acetyl-CoA was used, which resulted in ≈50% inhibition of PanK2 activity. However, palmitoylcarnitine stimulated PanK2 activity to a level that was ≈130% of the control PanK2 activity in the absence of acetyl-CoA. Palmitoylcarnitine also reversed the inhibition of PanK2 by palmitoyl-CoA (data not shown). Three PKAN-associated point mutants, which are active and sensitive to acetyl-CoA inhibition (8), were purified and tested for the regulation by palmitoylcarnitine. In the presence of 60 nM acetyl-CoA, all three mutants, PanK2[R286C], PanK2[N404I], and PanK2[T528M], were inhibited by 40–60%. Similar to its effect on the wild-type protein, palmitoylcarnitine stimulated the activities of the mutants to levels higher than those in the absence of acetyl-CoA (Fig. 3A). We tested the effect of palmitic acid and found that it was not effective in the reversal of acetyl-CoA inhibition (data not shown). A kinetic analysis of the interaction between palmitoylcarnitine and acetyl-CoA indicated that palmitoylcarnitine was a competitive antagonist of acetyl-CoA (Fig. 3B). These data demonstrated that the mechanism of palmitoylcarnitine activation was to competitively reverse the inhibition by acetyl-CoA. Paradoxically, palmitoylcarnitine also activated purified PanK2 in the absence of added acetyl-CoA (Fig. 4A). Octanoylcarnitine was not an activator indicating that the activation event is specific for long-chain species of acylcarnitine (Fig. 4A). Fisher et al. (18) reported that carnitine was a nonessential activator of partially purified PanK activity from rat heart. Carnitine did not activate purified PanK2; however, we were able to repeat the observations of Fisher et al. in cell lysates showing that carnitine activated PanK2 at higher concentrations (Fig. 4B). Thus, we attributed the activation of PanK2 by carnitine in cell lysates to the formation of acylcarnitine from carnitine in the presence of the ATP:Mg2+ required for the kinase assay, and we concluded that carnitine was not a regulator of PanK2.

Fig. 3.

Fig. 3.

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Effect of palmitoylcarnitine on the inhibition of PanK2 by acetyl-CoA. (A) Palmitoylcarnitine reversed the inhibition of purified wild-type (□) or mutant ([R286C], ■; [N404I], ●; [T528M], ○) PanK2 by 60 nM acetyl-CoA. (B) PanK2 activity was assayed in the presence of different concentrations of acetyl-CoA in the absence (●) or presence of 2 μM (○) or 4 μM (■) palmitoylcarnitine. Dixon plots of the reciprocal of velocity versus the acetyl-CoA concentration showed that the lines intercepted on the y axis, indicating that palmitoylcarnitine activation was competitive with respect to acetyl-CoA.

Fig. 4.

Fig. 4.

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Activation of PanK2 was specific for palmitoylcarnitine. (A) Palmitoylcarnitine (■) activated PanK2 by using the purified PanK2 preparation, whereas octanoylcarnitine (□) did not. (B) Carnitine activated PanK2 from the 293T cell lysates (■). In contrast, carnitine did not activate purified PanK2 (□).

The PanK2·Acetyl-CoA Complex.

One interpretation of the palmitoylcarnitine activation of PanK2 in the absence of added acetyl-CoA in the cell lysates (Fig. 1B) was the contamination of the sample with endogenous acetyl-CoA, but a similar activation of purified PanK2 that had been subjected to two column chromatography steps and dialysis (Fig. 4A) suggested that a proportion of PanK2 protein was purified as the PanK2·acetyl-CoA complex. Therefore, we analyzed our pure PanK2 preparations for bound acetyl-CoA. The UV spectrum of purified PanK2 exhibited a shoulder at 260 nm compared with the spectrum that was calculated based on the amino acid composition (Fig. 5A). Heat denaturation of PanK2 followed by the removal of the precipitated protein resulted in a supernatant with an absorbance maximum at 260 nm (Fig. 5A), indicating the presence of a bound nucleotide-like molecule. Based on the extinction coefficient of acetyl-CoA and the absorbance at 260 nm, we calculated that between 40% and 60% of the protein in different batches of purified PanK2 contained a bound CoA species. Mass spectrometry clearly identified acetyl-CoA as the nucleotide ligand bound to PanK2 (Fig. 5B). Mass peaks corresponding to doubly charged (m/z = 403.72) and singly charged (m/z = 808.11) acetyl-CoA peaks were detected in the negative-ion scan. The mass spectrum gave no indication for the presence of nonesterified CoA or other CoA species. The bacterial CoA pool consists primarily of acetyl-CoA, but there are also significant concentrations of nonesterified CoA, succinyl-CoA, and malonyl-CoA (19). Thus, we concluded that PanK2 selectively copurified with acetyl-CoA. These data show that a proportion of the total PanK2 protein survived as the PanK2·acetyl-CoA complex during the multistep purification protocol underscoring the tight binding of acetyl-CoA to PanK2. The presence of the PanK2·acetyl-CoA complex explained palmitoylcarnitine activation of PanK2 in the absence of added acetyl-CoA (Fig. 4A).

Fig. 5.

Fig. 5.

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Identification of the PanK2-bound inhibitor by UV-visible spectroscopy and electrospray mass spectrometry (ES-MS). (A) UV-visible spectra of PanK2 (0.9 mg/ml, solid thin line) and the bound inhibitor (solid thick line) released after heat denaturation of the protein sample and removal of the precipitate by centrifugation. The peak at 260 nm was characteristic of a nucleotide-like molecule, and the spectrum of a 25 μM acetyl-CoA standard solution (dashed line) is shown. (B) ES-MS spectrum of the supernatant from a heat-denatured sample of PanK2. The spectrum revealed the presence of peaks at m/z 403.72 and 808.11 corresponding to the acetyl-CoA [M-2H]2− and [M-H]− ions, respectively.

Discussion

The positive and negative regulation of PanK2 by intermediates in mitochondrial metabolism provides a mechanistic basis for understanding the biological function of the enzyme (Fig. 6). The mature form of human PanK2 resides in the mitochondria where it phosphorylates pantothenate to 4′-phosphopantothenate. Because the enzymes catalyzing the subsequent reactions in the CoA biosynthetic pathway are cytosolic, the product of PanK2 exits the mitochondria and is converted to CoASH. To put PanK2 in the proper physiological context with its activator and inhibitors, one has to understand where PanK2 localizes within the mitochondria: the intermembrane space (IMS) or the matrix. Because of the presence of an abundant outer mitochondrial membrane (OMM) protein named porin, the OMM is highly permeable to small molecules with molecular masses <5 kDa. Thus, the cytosolic concentrations of the CoA and carnitine species equilibrate with the IMS. Experimental data are not available to pinpoint in which mitochondrial compartment PanK2 is located. However, we propose that PanK2 is localized to the IMS because: (i) like known IMS proteins such as cytochrome b2, cytochrome c1, and cytochrome peroxidase, PanK2 possesses two cleavable mitochondrial targeting signals (20, 21), predicting an IMS localization; and (ii) the IMS localization enables ready transport of pantothenate into IMS for PanK2 activity and product release across the OMM to the cytosol where the downstream reactions in the CoA biosynthetic pathway are located. In animal tissues, the cytosolic concentrations of total CoA range from 20 to 140 μM (16, 17, 22). Approximately 30% (6–40 μM) of the total CoA pool are acyl-CoAs including both short- and long-chain species. Carnitine is more abundant than CoA. The concentration of total carnitine is 2 mM in liver and 4 mM in heart (23). Long-chain acylcarnitine species constitute 10% of the carnitine pool, leading to a concentration ranging from 200 to 400 μM. Thus, the IMS compartment contains more activators of PanK2 than inhibitors. The concentrations of the effectors used in our assays are achievable in vivo. We also tested and confirmed the ability of palmitoylcarnitine to reverse the acetyl-CoA inhibition at a higher acetyl-CoA concentration (1 or 10 μM).

Fig. 6.

Fig. 6.

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A model illustrating the positive and negative regulation of PanK2 activity. Human PanK2 is proposed to localize in the IMS of mitochondria, where it phosphorylates pantothenate (Pan) to form 4′-phosphopantothenate (P-Pan). P-Pan translocates to the cytosol and is converted to CoA by the CoA biosynthetic pathway. Fatty acid β-oxidation in the matrix requires the carnitine shuttle system, which converts acyl-CoA to acylcarnitine in the IMS by the action of carnitine palmitoyltransferase I (CPTI); acylcarnitine is shuttled into the matrix by the carnitine:acylcarnitine translocase (CT) and converted back to acyl-CoA by carnitine palmitoyltransferase II (CPTII). Acyl-CoAs, including acetyl-CoA (Ac-CoA), potently inhibit PanK2 activity by competing with the ATP substrate. Palmitoylcarnitine reverses the acetyl-CoA inhibition on PanK2 by competing with acetyl-CoA for the enzyme. The existence of both negative and positive regulation of PanK2 activity provides a mechanism to ensure ample supply of CoA for β-oxidation by controlling the activity of the mitochondria-localized PanK2, a rate-limiting step in the CoA biosynthetic pathway. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane.

The carnitine shuttle system transports acyl groups into the mitochondrial matrix and is composed of three proteins: the OMM carnitine palmitoyltransferase I that releases acylcarnitine into the IMS, the inner membrane acylcarnitine:carnitine translocase, and the inner membrane carnitine palmitoyltransferase II that transfers the acyl group from carnitine to CoASH in the matrix (24). Thus, matrix CoASH is required to initiate mitochondrial fatty acid β-oxidation, and a deficiency in CoASH would lead to the accumulation of long-chain acylcarnitine. The potent inhibition of PanK2 by acyl-CoAs maintains the enzyme in a normally off position by preventing the binding of ATP:Mg2+; however, the presence of long-chain acylcarnitine antagonizes the inhibitory action of acyl-CoA and activates PanK2. This regulatory effect on the rate-controlling enzyme in CoA biosynthesis boosts the cytosolic concentration of CoASH, which is actively transported into the mitochondrial matrix by the Leu5p protein, an inner membrane carrier (25). The mitochondrial localization of PanK2 places it in an ideal subcellular location to sense the levels of long-chain acylcarnitine and the status of mitochondrial β-oxidation.

In addition to its essential role in transport described in Fig. 6, carnitine is also involved in modulating the acyl-CoA:CoASH ratio to release CoASH to support β-oxidation, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase activities, and the tricarboxylic acid cycle (26, 27). The cellular carnitine concentration can approach 3 mM, and ≈90% of the total carnitine is located in the cytosol (28). Like CoA, carnitine exists as free carnitine and short- and long-chain carnitine esters, and the ratio of free to esterified carnitine varies depending on the availability of oxidative substrates (26, 29). In human disorders that disrupt mitochondrial β-oxidation, acylcarnitines accumulate and are secreted in the urine (30, 31), consistent with the role of carnitine in releasing CoASH to support intermediary metabolism. Our model for the regulation of PanK2 by palmitoylcarnitine suggests that the absence of this enzyme activity in PKAN patients may lead to a defect in the regulation of mitochondrial matrix CoASH and the impairment of fatty acid β-oxidation. The presence of other PanK isoforms ensures that PKAN patients are not severely deficient in total CoA; thus, the mitochondrial dysfunction in this disease would be milder than in severe mitochondrial β-oxidation disorders, perhaps accounting for the variability of symptoms among PKAN patients (12). The severity of symptoms in β-oxidation disorders is highly dependent on diet, and the seriousness of the clinical symptoms of the human β-oxidation disorders can be ameliorated, in some cases, by a high-carbohydrate, low-fat diet supplemented with carnitine (32). This diet effect suggests that a similar strategy should be considered in the management of PKAN disease; however, the failure of PanK2 knockout mice to accurately recapitulate the properties of the human disease (14) makes the experimental verification of this idea problematical in an animal model.

Materials and Methods

Transfection of HEK 293T Cells and Preparation of Cell Lysates.

The coding sequence for the mature form of human PanK2 protein (from residue 141 of the full-length protein) was cloned into pcDNA3.1-HisA (Invitrogen, Carlsbad, CA) to yield pKM56 (8), which was used to transfect HEK 293T cells. The cells were cultured in DMEM and 10% FCS (Atlanta Biologicals, Lawrenceville, GA) after transfection with FuGENE 6 according to the manufacturer's recommendation (Roche, Basel, Switzerland). Cells were collected and lysed 48 h after transfection, and Western blotting was used to confirm the expression of the mature form of human PanK2 in the cell lysate as described (8).

Purification of His-Tagged PanK2.

The PANK2 gene fragments encoding the mature form (amino acids 141–570) was subcloned into expression plasmid pET-28a (Novagen, Darmstadt, Germany) between the NheI and BamHI restriction sites. The resultant plasmid pKM44 was transformed into the E. coli BL21(DE3) (Stratagene, La Jolla, CA) strain to express the mature form of PanK2 with an N-terminal His-tag. Transformed cells were grown at 37°C in terrific broth in the presence of 30 μg/ml kanamycin to an A600 of 2 and induced with 1 mM IPTG at 18°C for 20 h. The cells were harvested by centrifugation, resuspended in MCAC-20 (20 mM Tris·HCl, pH 8/500 mM NaCl/20 mM imidazole/10% glycerol) buffer containing 1 mM PMSF, and lysed with a Microfluidizer high-pressure fluids processor. The cell-free extract was precipitated with 50% saturated ammonium sulfate, and the protein pellet was resuspended in MCAC-20 buffer containing 50 mM KSCN before being loaded onto a 5-ml HiTrap Chelating HP column. The his-tagged PanK2 protein was eluted by using a linear gradient of imidazole from 60 to 300 mM. The fractions containing PanK2 protein were pooled, concentrated, and further purified to homogeneity by a Superdex 200 column (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in buffer (20 mM Tris·HCl, pH 7.5) containing 300 mM NaCl. Three PKAN disease-associated point mutations, [R286C], [N404I], and [T528M] (numbering was based on the full-length protein), were introduced into pKM44 by using the Quikchange mutagenesis kit (Stratagene) according to the manufacturer's protocol. The His-tagged recombinant mutant PanK2 proteins were expressed and purified as the wild-type enzyme.

PanK Activity Assay.

Briefly, the standard PanK assays (3, 33) contained 45 μM d-[1-14C]pantothenate (specific activity, 55 mCi/mmol; Amersham Biosciences), 250 μM ATP (pH 7.0), 10 mM MgCl2, 0.1 M Tris·HCl (pH 7.5), and the indicated amount of protein from the cell extracts containing PanK2 or the purified PanK2 proteins in a total volume of 40 μl. The mixture was incubated at 37°C for 10 min. The radioactive product was quantitated by scintillation counting as described (8). The effect of different CoA or carnitine species on the enzymatic activity was determined by including the indicated concentrations of CoA or carnitine species in the assay mix before the addition of PanK2. To determine the inhibitory mechanism of acetyl-CoA on the mature form of PanK2, the Km of PanK2 for ATP in the presence or absence of acetyl-CoA was determined in standard reaction mixtures containing 180 μM d-[1-14C]pantothenate and increasing concentrations of both ATP and the CoA thioester, as indicated.

UV-Visible Spectra of PanK2 and Mass Spectrometry Analysis.

UV-visible spectra of PanK2 [0.9 mg/ml in 20 mM Tris·HCl (pH 7.4), 300 mM NaCl, 1 mM EDTA, 1 mM DTT] were recorded on an Agilent 8453 spectrophotometer before and after removal of the enzyme by heat denaturation and centrifugation. The extinction coefficient at 260 nm for acetyl-CoA was determined to be 11,500 M−1·cm−1 from the absorbance of standard solutions and used to estimate the amount bound to PanK2.

The supernatant (100 μl) from a heat-denatured sample of PanK2 [1.15 mg/ml in 50 mM ammonium acetate (pH 7.0)] was desalted on a C-8 MicroTip Column (Harvard Apparatus) for mass spectrometry (MS) analysis. The column was washed with water, and the protein-bound small molecules were eluted with methanol (100 μl), dried under N2 flow, and resuspended in methanol:water (1:1 vol/vol, 10 μl). MS analysis was performed by using a Finnigan TSQ Quantum (Thermo Electron Corporation, San Jose, CA) triple quadrupole mass spectrometer equipped with the Nanospray Ion Source. Samples were introduced via static nanoelectrospray by using EconTips (New Ojective, Woburn, MA). The instrument was operated in the negative ion mode by using single MS (Q1) scanning. Ion source parameters were spray voltage 1,600 V, capillary temperature 270°C, and capillary offset −35 V, and tube lens offset was set by infusion of the polytyrosine tuning and calibration solution (Thermo Electron Corporation) in electrospray mode. MS acquisition parameters for Q1 scanning were as follows: scan range 250–1,100 m/z; scan time, 0.85 s; peak width Q1, 0.7 full width, half maximum. Instrument control and data acquisition were performed with the Finnigan Xcalibur (version 1.4 SR1) software (Thermo Electron Corporation).

TNP-ATP Fluorescence Spectra.

The ATP analog TNP-ATP (Molecular Probes, Eugene, OR) was excited at 410 nm, and the fluorescence emission spectra were recorded at 25°C on a FluoroLog spectrofluorimeter (Horiba Jobin Yvon, Edison, NJ) equipped with a circulating water bath. Binding of TNP-ATP to PanK2 and subsequent displacement by acetyl-CoA were detected by the change in fluorescence of a 5 μM TNP-ATP solution in 100 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 5% glycerol, after the addition of 1 μM protein and 400 nM acetyl-CoA.

Acknowledgments

We thank Ruobing Zhou and Karen Miller for their expert technical assistance and Phil Poston (Hartwell Center of St. Jude Children's Research Hospital) for the mass spectrometry experiments. This work was supported by National Institutes of Health Grants GM 62896 (to S.J.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities.

Abbreviations

IMS

intermembrane space

OMM

outer mitochondrial membrane

PanK

pantothenate kinase

PKAN

pantothenate kinase-associated neurodegeneration.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission.

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