Involvement of hysteretic effects in the inhibition of carnitine palmitoyltransferase by malonyl-CoA.

Carnitine palmitoyltransferase in its normal mitochondrial environment behaves as a hysteretic enzyme, exhibiting slow changes in reaction rate after the addition of oleoyl-CoA or malonyl-CoA. Reaction rates become constant after a short time, but the sensitivity of the enzyme from fed rats to the inhibition by malonyl-CoA remains much greater than that of starved rats.     

Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity.

In the fed state, hyperthyroidism increased glucose utilization indices (GUIs) of skeletal muscles containing a lower proportion of oxidative fibres. Glycogen concentrations were unchanged, but active pyruvate dehydrogenase (PDHa) activities were decreased. Hyperthyroidism attenuated the effects of 48 h of starvation to decrease muscle GUI. Glycogen concentrations and PDHa activities after 48 h of starvation were low and similar in euthyroid and hyperthyroid rats. The increase in glucose uptake and phosphorylation relative to oxidation and storage in skeletal muscle induced by hyperthyroidism may contribute to increased glucose re-cycling in the fed hyperthyroid state and to glucose turnover in the starved hyperthyroid state.     

Ethanol increases the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA in short-term hepatocyte incubations

The sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA was increased in mitochondria isolated from rat hepatocytes incubated with ethanol. This effect was mimicked by incubation of hepatocytes with acetaldehyde or by preincubation of isolated mitochondria with malonyl-CoA. Both ethanol and acetaldehyde increased the intracellular concentration of malonyl-CoA. Results suggest that the ethanol-induced elevation of intracellular malonyl-CoA levels may be responsible for the enhanced sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA.     

Differences in the sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA are due to differences in Ki values

The hepatic carnitine palmitoyltransferase that is present on the outer surface of the mitochondrial inner membrane demonstrates hyperbolic substrate saturation curves with oleoyl-CoA in both fasted and fed rats. However, the addition of malonyl-CoA resulted in sigmoid substrate saturation curves, suggesting that malonyl-CoA induced the cooperative behavior. There was more of the outer carnitine palmitoyltransferase in liver mitochondria derived from fasted rats and that enzyme had a much greater Ki for malonyl-CoA than the enzyme from fed rats, but the Km values were apparently not different. The Dixon plot with mitochondria from fed rats, but not fasted rats, was curved upward, indicating cooperative inhibition by malonyl-CoA. Carnitine palmitoyltransferase of heart mitochondria had a Ki for malonyl-CoA that was much less than that of the liver enzyme and it did not change on fasting. Furthermore, no evidence for cooperative inhibition was found in the heart. The results of these studies indicate that carnitine palmitoyltransferase is not subject to substrate cooperativity and that malonyl-CoA is not a simple competitive inhibitor of this enzyme but inhibits by a mechanism involving cooperative inhibition. The fasting-feeding cycle induces changes in the liver enzyme that alter its affinity for malonyl-CoA without changing its affinity for its acyl-CoA substrate. Carnitine palmitoyltransferase from heart appears to be different from that of liver and is apparently not subject to the same control mechanisms.     

Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition. Mutation of methionine 593 abolishes malonyl-CoA inhibition

Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E. The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubi, B., Clotet, J., Ari?o, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported.     

Increased sensitivity of carnitine palmitoyltransferase I activity to malonyl-CoA inhibition after preincubation of intact rat liver mitochondria with micromolar concentrations of malonyl-CoA in vitro.

Carnitine palmitoyltransferase I in rat liver mitochondria preincubated with malonyl-CoA was more sensitive to inhibition by malonyl-CoA than was the enzyme in mitochondria preincubated in the absence of malonyl-CoA. For carnitine palmitoyltransferase I in mitochondria from starved animals this increase also resulted in the enzyme becoming significantly more sensitive than that in mitochondria assayed immediately after their isolation. Concentrations of malonyl-CoA that induced half the maximal degree of sensitization observed were 1-3 microM.     

Different sites of inhibition of carnitine palmitoyltransferase by malonyl-CoA, and by acetyl-CoA and CoA, in human skeletal muscle.

The inhibition of carnitine palmitoyltransferase (CPT, EC 2.3.1.21) by malonyl-CoA, acetyl-CoA and free CoA was studied in sonicated skeletal-muscle homogenates from normal human subjects and from five patients with a mutant CPT [Zierz & Engel (1985) Eur. J. Biochem. 149, 207-214]. (1) Malonyl-CoA, acetyl-CoA and CoA were competitive inhibitors of CPT with palmitoyl-CoA. (2) Acetyl-CoA and CoA inhibited normal and mutant CPT to the same degree, whereas malonyl-CoA inhibited mutant CPT more than normal CPT. (3) Triton X-100 abolished the inhibition of normal CPT by malonyl-CoA, but not by acetyl-CoA or CoA. Triton X-100 by itself caused loss of activity of the mutant CPT. (4) In the concentration range 0.1-0.4 mM, the inhibitory effects of any two of the three inhibitors were synergistic. (5) The inhibitory constants (Ki) for acetyl-CoA and CoA were close to 45 microM. The Ki for malonyl-CoA was 200-fold lower, or 0.22 microM. Addition of 40 microM-acetyl-CoA or CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. Addition of 20 microM-CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. (6) The findings indicate that acetyl-CoA and CoA can inhibit CPT at the catalytic site or a nearby site which is different from that at which malonyl-CoA inhibits CPT. (7) The fact that small changes in the concentration of acetyl-CoA and CoA can antagonize the inhibitory effect of malonyl-CoA suggests that these compounds could modulate the inhibition of CPT by malonyl-CoA.     

A proportion of rat liver mitochondrial carnitine palmitoyltransferase can be made activatable by malonyl-CoA

Treatment of rat liver mitochondrial membranes with cholate yields a soluble extract containing carnitine palmitoyltransferase (CPT) activity that is insensitive to malonyl-CoA. As found previously (I. Ghadiminejad and D. Saggerson (1990) FEBS Lett. 269, 406-408), addition of polyethylenen glycol 6000 (PEG 6000) to this extract conferred sensitivity to malonyl-CoA on the CPT. It is now shown that a sub-population of the CPT activity which is sedimentable at 7000 x g after addition of PEG 6000 is activated by malonyl-CoA, whereas the remainder is inhibited by malonyl-CoA. The presence of KCl increases the proportion of the activatable form of CPT. Possible physiological significance of this finding is discussed.     

The relationship of rat liver overt carnitine palmitoyltransferase to the mitochondrial malonyl-CoA binding entity and to the latent palmitoyltransferase.

Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.     

Interacting effects of L-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase.

Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.     

Definition by functional and structural analysis of two malonyl-CoA sites in carnitine palmitoyltransferase 1A

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 NH2- 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 NH2- 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 NH2- and COOH-terminal 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 (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 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 Km and malonyl-CoA IC50.     

Restoration of malonyl-CoA sensitivity of soluble rat liver mitochondria carnitine palmitoyltransferase by reconstitution with a partially purified malonyl-CoA binding protein.

Solubilization of rat liver mitochondria in 5% Triton X-100 followed by chromatography on a hydroxylapatite column resulted in the identification of malonyl-CoA binding protein(s) distinct from a major carnitine palmitoyltransferase activity peak. Further purification of the malonyl-CoA binding protein(s) on an acyl-CoA affinity column followed by sodium dodecyl sulfate gel electrophoresis indicated proteins with Mr mass of 90 and 45-33 kDa. A purified liver malonyl-CoA binding fraction, which was devoid of carnitine palmitoyltransferase, and a soluble malonyl-CoA-insensitive carnitine palmitoyltransferase were reconstituted by dialysis in a liposome system. The enzyme activity in the reconstituted system was decreased by 50% in the presence of 100 microM malonyl-CoA. Rat liver mitochondria carnitine palmitoyltransferase may be composed of an easily dissociable catalytic unit and a malonyl-CoA sensitivity conferring regulatory component.     

Alteration of the apparent Ki of carnitine palmitoyltransferase for malonyl-CoA by the diabetic state and reversal by insulin

The effects of streptozotocin-induced diabetes and the subsequent treatment of diabetic animals with insulin were studied using a dose of streptozotocin that produces highly ketotic animals 48 h after injection. Carnitine palmitoyltransferase of diabetic animals had apparent Ki values for malonyl-CoA that were approximately 10 times greater than control animals, indicating a greatly decreased affinity for malonyl-CoA in the diabetic state. Subsequent treatment of diabetic animals with insulin for 5 days produced non-ketotic animals with normal blood glucose, and the affinity of carnitine palmitoyltransferase for malonyl-CoA was increased to the control level. Treatment of other groups of ketotic diabetic animals with insulin produced substantial changes in the carnitine palmitoyltransferase apparent Ki value for malonyl-CoA within 4 h. These results suggest that insulin modulates the ketotic state, at least in part, by increasing the affinity of carnitine palmitoyltransferase for malonyl-CoA to bring about inhibition of fatty acid oxidation and ketogenesis.     

Regulation of carnitine palmitoyltransferase by insulin results in decreased activity and decreased apparent Ki values for malonyl-CoA

Administration to normal rats of 100 mg of streptozotocin/kg body weight produced ketotic diabetic rats in which the affinity of carnitine palmitoyltransferase for malonyl-CoA was decreased by 10-fold and its activity was increased by 30%, but the injection of insulin brought the affinity and the activity back to normal within 4 h. Administration of 60 mg of streptozotocin/kg produced non-ketotic diabetic rats and caused a less substantial change in the affinity of carnitine palmitoyltransferase for malonyl-CoA. In the BB Wistar diabetic rat, the onset of diabetes also increased the activity of carnitine palmitoyltransferase and decreased its affinity for malonyl-CoA. Injection of insulin brought both of these values back to normal within 2 h. The total activity of mitochondrial carnitine palmitoyltransferase (outer + inner activities) was 40% greater in the BB Wistar diabetic rat, but treatment with insulin did not decrease the total activity to normal values within 2 h. The elevated activity and decreased affinity for malonyl-CoA found in fasting rats did not respond to short-term insulin treatment. The evaluation of a previous report that cycloheximide blocks the effects of starvation indicated that cycloheximide did not act by inhibiting protein synthesis, but produced its effect by preventing gastric emptying. Current data suggest that diabetes increases the activity of carnitine palmitoyltransferase and greatly diminishes the affinity of the enzyme for malonyl-CoA and that the severity of diabetes is associated with differences in the affinity of the enzyme for its inhibitor. Insulin acts on the outer carnitine palmitoyltransferase to reverse these effects very rapidly, but diabetes produces some change in the total activity that is not reversed by short-term treatment with insulin.     

Pig liver carnitine palmitoyltransferase I, with low Km for carnitine and high sensitivity to malonyl-CoA inhibition, is a natural chimera of rat liver and muscle enzymes

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids. The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI. When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI. However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes. Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.     

Reversible sensitization and desensitization of carnitine palmitoyltransferase I to inhibition by malonyl-CoA in isolated rat liver mitochondria. Significance for the mechanism of malonyl-CoA-induced sensitization.

Preincubation of rat liver mitochondria with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) followed by removal of excess reagent by washing the mitochondria with 0.5 mM-reduced glutathione resulted in a desensitization of carnitine palmitoyltransferase (CPT) I activity to malonyl-CoA inhibition. The effect was not observed if mitochondria were washed with 0.5 mM-dithiothreitol. The desensitization effect of Nbs2 could be reversed by a second incubation in the presence of 8 microM-malonyl-CoA. In addition, malonyl-CoA, when present simultaneously with Nbs2, protected CPT I activity against the desensitization effect of the thiol-group reagent. These results suggest that malonyl-CoA exerts an effect on one or more thiol groups of the enzyme, and that this effect is related to the ability of the metabolite to sensitize CPT I to malonyl-CoA inhibition.     

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.

[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18nM, KD(2) = 30 microM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 microM, KD(2) = 5.6 microM, N1 = 11pmol/mg of protein, N2 = 165pmol/mg of protein. In the presence of 40 microM-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 microM, with no change in N1 and for liver KD(1) = approx. 2 microM, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBS Lett. 129, 225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5'-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Extraction of rat liver mitochondria twice with 0.5% Triton X-100 in a salt-free medium leaves less than 10% of the carnitine palmitoyltransferase membrane bound. The remaining membrane-bound enzyme is inhibited virtually completely by 10 microM malonyl-CoA. Preincubation of the extracted membranes with palmitoyl-CoA and salts (KCI) for several minutes activates the enzyme and makes it increasingly insensitive to malonyl-CoA. Addition of malonyl-CoA to the preincubation reverses this desensitization. In albumin-containing media salts also decrease the binding of palmitoyl-CoA to albumin and stimulate carnitine palmitoyltransferase by increasing substrate availability in free solution. The reverse reaction shows accelerated desensitization by palmitoylcarnitine and resensitization by malonyl-CoA.     

Biosynthesis of carnitine octanoyltransferase and carnitine palmitoyltransferase

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine octanoyltransferase (COT) in the liver was increased 23.5-fold in rats given DEHP. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rate of synthesis of COT was 14.1-fold higher and that of its degradation was 1.5-fold lower in the DEHP group. COT was translated much more effectively in free polysomes than in membrane-bound polysomes. The molecular size of the in vitro product was the same as that of the mature enzyme. The translation activity of mRNA coding for COT measured with total hepatic RNA was 16.6-fold higher in the DEHP group. Carnitine palmitoyltransferase (CPT) was increased 5.9-fold after administration of DEHP. The rate of synthesis of CPT measured in the in vivo experiment was 5.0-fold higher in the DEHP group. The rate of its degradation was the same in the two groups. CPT was also translated much more effectively in free polysomes. The size of the preenzyme was larger than that of the subunit of the mature enzyme by about 2,400 daltons. In contrast to COT, the increase in the translation activity of mRNA for CPT by administration of DEHP was markedly higher than the increase in the rate of its synthesis measured in the in vivo experiment.     

Structural model of a malonyl-CoA-binding site of carnitine octanoyltransferase and carnitine palmitoyltransferase I: mutational analysis of a malonyl-CoA affinity domain.

Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an "in silico" macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001-45008). The putative malonyl-CoA domain contained His(340), implicated together with His(131) in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Ari?o, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183-186). When we mutated COT His(131) the IC(50) increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala(332), present in the domain 8 amino acids away from His(340), decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His(277), His(483), and Ala(478) were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro(479), which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.