Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity

Bird, Michael I.; Saggerson, E D

doi:10.1042/bj2220639

Cited by 68 publications

(95 citation statements)

References 21 publications

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“…It is not clearly established whether they bind at the same site, or even whether they bind to the same polypeptide [16][17][18][19][20]. Data presented by Murthy & Pande [12], who used proteinase treatment of intact mitochondria and isolated outer membranes, suggested that the mitochondrial-outer-membrane CPT has its substrate-binding site facing the intermembrane space and the malonyl-CoAbinding site facing the cytosol, clearly suggesting two different binding sites.…”

Section: Introductionmentioning

confidence: 99%

Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors

Kashfi

Cook

1992

Biochemical Journal

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Proteolysis of intact mitochondria by Nagarse (subtilisin BPN') and papain resulted in limited loss of activity of the outermembrane carnitine palmitoyltransferase, but much greater loss of sensitivity to inhibition by malonyl-CoA. In contrast with a previous report Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], we found that trypsin had no effect on malonyl-CoA sensitivity. Even when 80 % of activity was destroyed by trypsin, there was no difference in the malonyl-CoA sensitivity of the enzyme remaining. Trypsin caused release of the intermembrane-space enzyme adenylate kinase, indicating loss of integrity of the mitochondrial outer membrane, whereas Nagarse and papain caused no release of that enzyme. Citrate synthase was not released by any of the three proteinases, indicating no damage to the mitochondrial inner membrane. When we examined the effects of proteolysis on the inhibition of carnitine palmitoyltransferase by a wide variety of inhibitors having different mechanisms of inhibition, we found differential proteolytic effects that were specific for those inhibitors (malonyl-CoA and hydroxyphenylglyoxylate) that have their inhibitory potencies diminished by changes in physiological state. Both of those inhibitors protected carnitine palmitoyltransferase from the effects of proteolysis, but did not inhibit the proteinases directly. Inhibition by two other inhibitors (DL-2-bromopalmitoyl-CoA and N-benzyladriamycin 14-valerate) was not altered by proteinase treatment, even when most of the enzyme activity had been destroyed. Inhibition by glyburide, which is minimally affected by physiological state, was affected only to a slight extent at the highest concentration of trypsin tested. Proteolysis by Nagarse appeared to produce loss of co-operativity in malonyl-CoA inhibition. The effects of proteolysis are discussed and compared with changes in K, occurring with changing physiological states.

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Section: Introductionmentioning

confidence: 99%

Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors

Kashfi

Cook

1992

Biochemical Journal

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“…The palmitoylCoA substrate and the inhibitor would occupy the same position in the "A site." Several authors (7)(8)(9)(10)(11)(12)(13) have reported that malonyl-CoA binds to CPT1 at two separate sites, with low and high affinity. The low affinity binding site is the locus at which palmitoyl-CoA and malonyl-CoA compete (50).…”

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confidence: 99%

Definition by Functional and Structural Analysis of Two Malonyl-CoA Sites in Carnitine Palmitoyltransferase 1A

López-Viñas

Bentebibel

Gurunathan

et al. 2007

Journal of Biological Chemistry

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Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of L-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH 2 -and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH 2 -and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH 2 -and COOHterminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC 50 ) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC 50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine K m and malonyl-CoA IC 50 . Carnitine palmitoyltransferase 1 (CPT1)4 catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine. This is the first step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix, where they undergo ␤-oxidation. CPT1 is tightly regulated by its physiological inhibitor malonyl-CoA. This regulation allows CPT1 to signal the availability of lipid and carbohydrate fuels to the cell (1). Mammalian tissues express three isoforms: CPT1A (liver), CPT1B (muscle and heart), and CPT1C (brain), which are the products of different genes (2-4). CPT1A and -B have 62% amino acid identity, but they are differentially regulated by malonyl-CoA. CPT1A is inhibited to a much lesser extent than CPT1B, which may explain why fatty acid oxidation is more finely regulated in the heart than in the liver. CPT1 is a potential target for the treatment of metabolic disorders involving diabetes and coronary heart disease (5). The interaction between malonyl-CoA and CPT1C may be involved in the "malonyl-CoA signal" in hypothalamic neurons regulating food intake and peripheral energy expenditure (6).It has been postulated that there are two malonyl-CoA binding sites in the molecule of CPT1A (7,8). Kinetic studies indicate that there is a high affinity binding site and a low affinity binding site (9 -13...

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“…The latter site has a greater capacity for regulation by malonyl-CoA and behaves as an allosteric component [9][10][11]. The involvement of His residues in modulation by malonyl-CoA is suggested by the finding that a decrease in pH (which is associated with the protonation of the imidazol group of histidine) increases the affinity of CPT I for malonyl-CoA [12,13].…”

Section: Introductionmentioning

confidence: 99%

Inhibition by etomoxir of rat liver carnitine octanoyltransferase is produced through the co-ordinate interaction with two histidine residues

Morillas

Clotet

Rubı́

et al. 2000

Biochemical Journal

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Rat peroxisomal carnitine octanoyltransferase (COT), which facilitates the transport of medium-chain fatty acids through the peroxisomal membrane, is irreversibly inhibited by the hypoglycaemia-inducing drug etomoxir. To identify the molecular basis of this inhibition, cDNAs encoding full-length wild-type COT, two different variant point mutants and one variant double mutant from rat peroxisomal COT were expressed in Saccharomyces cere isiae, an organism devoid of endogenous COT activity. The recombinant mutated enzymes showed activity towards both carnitine and decanoyl-CoA in the same range as the wild type. Whereas the wild-type version expressed in yeast was inhibited by etomoxir in an identical manner to COT from rat liver peroxisomes, the activity of the enzyme containing the

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Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity

Cited by 68 publications

References 21 publications

Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors

Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors

Definition by Functional and Structural Analysis of Two Malonyl-CoA Sites in Carnitine Palmitoyltransferase 1A

Inhibition by etomoxir of rat liver carnitine octanoyltransferase is produced through the co-ordinate interaction with two histidine residues

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