Feedback Regulation of Murine Pantothenate Kinase 3 by Coenzyme A and Coenzyme A Thioesters

Abstract

Pantothenate kinase catalyzes a key regulatory step in coenzyme A biosynthesis, and there are four mammalian genes that encode isoforms of this enzyme. Pantothenate kinase isoform PanK3 is highly related to the previously characterized PanK1␤ isoform (79% identical, 91% similar), and these two almost identical proteins are expressed most highly in the same tissues. PanK1␤ and PanK3 had very similar molecular sizes, oligomeric form, cytoplasmic cellular location, and kinetic constants for ATP and pantothenate. However, these two PanK isoforms possessed distinct regulatory properties. PanK3 was significantly more sensitive to feedback regulation by acetyl-CoA (IC 50 ‫؍‬ 1 M) than PanK1␤ (IC 50 ‫؍‬ 10 M), and PanK3 was stringently regulated by long-chain acyl-CoA (IC 50 ‫؍‬ 2 M), whereas PanK1␤ was not. Domain swapping experiments localized the difference in the two proteins to a 48-amino-acid domain, where they are the most divergent. Consistent with these more stringent regulatory properties, metabolic labeling experiments showed that coenzyme A (CoA) levels in cells overexpressing PanK3 were lower than in cells overexpressing an equivalent amount of PanK1␤. Thus, the distinct regulatory properties exhibited by the family of the pantothenate kinases allowed the rate of CoA biosynthesis to be controlled by regulatory signals from CoA thioesters involved in different branches of intermediary metabolism.Pantothenate is the essential precursor for CoA, 2 which is a cofactor for a multitude of metabolic reactions including the oxidation of fatty acids, carbohydrates, pyruvate, lactate, ketone bodies, and amino acids, as well as many synthetic reactions. PanK catalyzes the first committed step and is the rate-controlling enzyme in CoA biosynthesis in bacteria (1) and mammals (2). PanK expression levels define the upper threshold of the cellular CoA content (3, 4), and PanK biochemical activity is feedback-regulated by CoA and/or CoA thioesters (3, 5-8), providing a mechanism to coordinate the rate of CoA synthesis with metabolic demand. Loss of feedback regulation by mutation at a single residue results in runaway CoA production (9). A notable exception to this rule is the PanK from Staphylococcus aureus, which lacks CoA feedback regulation (10), consistent with a role for CoA as the primary thiol in the disulfide redox system in this organism, as well as being the major acylgroup carrier (11-13). In mammals, PanK activity and/or CoA content are altered in response to metabolic state (14 -18), insulin (2), glucagon or glucocorticoids (15), fibrates (14, 19 -22), or diabetes (23, 24). CoA is found in all cellular compartments, but the highest concentrations of CoA exist in mitochondria and peroxisomes (25,26). Mitochondrial CoA is used as a cofactor in the tricarboxylic acid cycle and fatty acid ␤-oxidation, and the concentrations of CoA and its thioesters regulate the rates of these processes (27). Peroxisomes play a major role in very long chain fatty acid ␤-oxidation and also have high concentrations of CoA (28, 29),...

Localization and regulation of mouse pantothenate kinase 2

“…Thus, the regulation of PanK2 in both species was similar. The difference in the acetyl-CoA IC 50 values between mPanK2 and hPanK2 (0.06 lM vs. 0.12 lM) is not considered to be significant in comparison with the IC 50 values determined for the other PanK isoforms which are 10-fold and 100-fold higher, e.g., PanK3, 1 lM; PanK1b, 10 lM [6].…”

“…Briefly, the reaction mixtures contained 45 lM D D- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]pantothenate (specific activity 27.5 mCi/mmol), 250 lM ATP pH 7.0, 10 mM MgCl 2 , 0.1 M Tris-HCl pH 7.5, 5 lg of protein and varying concentrations of acetyl-CoA or palmitoylcarnitine.…”

Allosteric Regulation of Mammalian Pantothenate Kinase

“…The principal mechanism for controlling mammalian pantothenate kinase activity is through feedback inhibition by acetyl-CoA, and the isoforms differ in their sensitivity to this regulator ( Fig. 1) (4,8,9). The 1␣ and 1␤ isoforms are least sensitive to inhibition, whereas isoforms 2 and 3 are more potently inhibited by acetyl-CoA.…”

“…In mammals, there are four distinct kinases that exhibit tissue-specific expression as follows: PANK1␣ and PANK1␤ are splice variants of the PANK1 gene, and PANK2 and PANK3 are encoded by the PANK2 and PANK3 genes, respectively (4,5). The kinase isoforms possess almost identical catalytic cores but differ in their amino termini that direct the enzymes to different subcellular compartments (6).…”

Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine

“…In humans, there are three genes that express four isoforms of PanK. PanK1␣ and 1␤ 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).…”