Carnitine palmitoyltransferase: separation of enzyme activity and malonyl-CoA binding in rat liver mitochondria

We have cloned and sequenced a cDNA encoding human liver carnitine palmitoylransferase (CPTase; pahnitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonudeotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0. A major source for energy production in the cell is the mitochondrial p-oxidation of long-chain fatty acids. In order to cross the mitochondrial membranes, long-chain fatty acids are first activated by coenzyme A and then reversibly conjugated with L-carnitine, a reaction that is catalyzed by the enzyme carnitine palmitoyltransferase (CPTase; palmitoylCoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) (1). One part of CPTase activity, conventionally referred to as CPTase I, is associated with the outer mitochondrial membrane, and another significant pool of CPTase activity, conventionally referred to as CPTase II, is in close relationship with the inner mitochondrial membrane (2). However, all the attempts to purify CPTase led to the identification of a single protein with a subunit of Mr 70 kDa (3)(4)(5)42).An important role in the regulation of CPTase activity is played by malonyl-CoA, an intermediate in fatty acid biosynthesis that inhibits the activity of outer (CPTase I) but not of inner (CPTase II) CPTase (6). The malonyl-CoA binding domain has been proposed to be located either on CPTase I (4) or on a regulatory protein without CPTase activity (7).CPTase activity is also under hormonal control of estrogen (8), insulin, and glucagon. Diabetes causes increased CPTase activity and decreased affinity for malonyl-CoA, both reversed by insulin (9). Glucagon has been reported to stimulate CPTase through phosphorylation by a cAMP-dependent protein kinase (10). Interestingly, hypoglycemic agents, such as sulfonylureas, exercise their pharmacological action by strongly inhibiting CPTase activity (11).CPTase seems also to play a role in the regulation of the hepatic synthesis of very low density lipoproteins (VLDL), since its inhibition increases VLDL production, whereas activation following depletion of malonyl-CoA levels decreases VLDL synthesis (12, 13). Accordingly, the hypolipidemic drug lovastatin causes a 2-fold increase in the activity of hepatic CPTase (14).From these observations it appears that CPTase has a role in the development of metabolic alterations in very common diseases such as diabetes and hyperlipoproteinemias. Furthermore, recessively inherited deficiency of CPTase in man has been described with two distinct phenotypes. In adults, CPTase deficiency causes a muscle disease with weakness, cramps, and myoglobinuria (15). In infants, CPTase deficiency is characterized by hyperammonemia, increased levels of serum transaminases and plasma-free fatty acids, hepatomegaly, nonketotic hypoglycemia, and coma (16). This life-threatening and often fatal d...

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“…The malonyl-CoA binding domain has been proposed to be located either on CPTase I (4) or on a regulatory protein without CPTase activity (7).…”

mentioning

confidence: 99%

cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.

Finocchiaro

Taroni

Rocchi

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

113

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“…The K D and B max values are averages of two independent experiments with different mitochondrial preparations. In intact rat liver mitochondria, values for CPTI activity under similar assay conditions are 1.0 -3.0 nmol/min/mg protein (18,21).…”

Section: Discussionmentioning

confidence: 92%

“…[ 14 C]Malonyl-CoA Binding Assay-[ 14 C]Malonyl-CoA binding was determined by a modified centrifugation assay as described previously (14,21). Isolated mitochondria from wild type and mutants were suspended in 0.5 ml of ice-cold medium composed of 72 mM sorbitol, mM KCl, 25 mM Tris/HCl (pH 6.8), 1.0 mM EDTA, 1.0 mM dithiothreitol, and 1.3 mg/ml fatty acid-free bovine serum albumin (22).…”

Section: Construction Of Plasmids For the N-terminal Point And Deletimentioning

confidence: 99%

A Single Amino Acid Change (Substitution of Glutamate 3 with Alanine) in the N-terminal Region of Rat Liver Carnitine Palmitoyltransferase I Abolishes Malonyl-CoA Inhibition and High Affinity Binding

Shi

Zhu

Arvidson

et al. 1999

Journal of Biological Chemistry

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We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.

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“…Mitochondrial CPT-I is also specifically inhibited by malonyl-CoA, with other short-chain acyl-CoA esters inhibiting to a much lesser extent [19]. There is some evidence that CPT-I malonyl-CoA sensitivity is conferred by a regulatory subunit bearing a specific malonyl-CoA-binding site [20][21][22]. A similar model could account for the malonyl-CoA inhibition of peroxisomal COT.…”

Section: Discussionmentioning

confidence: 99%

Malonyl-CoA inhibition of peroxisomal carnitine octanoyltransferase

a'BHÁIRD

Ramsay

1992

Biochemical Journal

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Although the malonyl-CoA sensitivity of peroxisomal carnitine octanoyltransferase (COT) is reportedly lost on solubilization, we show that malonyl-CoA does inhibit the purified enzyme. Assay conditions such as buffer composition, pH, acyl-CoA substrate and the presence or absence of BSA can affect the observed inhibition. When assayed in the absence of BSA, COT shows simple competitive inhibition by malonyl-CoA. The Ki value for inhibition of purified COT is high (106 microM) compared with physiological concentrations (1-6 microM) and other short-chain acyl-CoA esters inhibit COT to the same degree. However, when COT is assayed in intact peroxisomes, the Ki for malonyl-CoA is almost 20-fold lower than found with the purified enzyme, whereas inhibition by other short-chain acyl-CoA esters does not change significantly. Several features of the inhibition of peroxisomal COT, including the specificity of malonyl-CoA over other short-chain acyl-CoA esters, resemble those of carnitine palmitoyltransferase (CPT)-I, suggesting that the regulation of COT and CPT-I in parallel may be necessary for the control of cellular fatty acid metabolism.

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Carnitine palmitoyltransferase: separation of enzyme activity and malonyl-CoA binding in rat liver mitochondria

Cited by 33 publications

References 19 publications

cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.

cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.

A Single Amino Acid Change (Substitution of Glutamate 3 with Alanine) in the N-terminal Region of Rat Liver Carnitine Palmitoyltransferase I Abolishes Malonyl-CoA Inhibition and High Affinity Binding

Malonyl-CoA inhibition of peroxisomal carnitine octanoyltransferase

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