Identification by Mutagenesis of a Conserved Glutamate (Glu487) Residue Important for Catalytic Activity in Rat Liver Carnitine Palmitoyltransferase II*

Mammalian mitochondrial membranes express two active but distinct carnitine palmitoyltransferases: carnitine palmitoyltransferase I (CPTI), which is malonyl coA-sensitive and detergent-labile; and carnitine palmitoyltransferase II (CPTII), which is malonyl coA-insensitive and detergent-stable. To determine the role of the highly conserved C-terminal acidic residues glutamate 487 (Glu487) and glutamate 500 (Glu500) on catalytic activity in rat liver CPTII, we separately mutated these residues to alanine, aspartate, or lysine, and the effect of the mutations on CPTII activity was determined in theEscherichia coli-expressed mutants. Substitution of Glu487 with alanine, aspartate, or lysine resulted in almost complete loss in CPTII activity. Because a conservative substitution mutation of this residue, Glu487 with aspartate (E487D), resulted in a 97% loss in activity, we predicted that Glu487 would be at the active-site pocket of CPTII. The substantial loss in CPTII activity observed with the E487K mutant, along with the previously reported loss in activity observed in a child with a CPTII deficiency disease, establishes that Glu487 is crucial for maintaining the configuration of the liver isoform of the CPTII active site. Substitution of the conserved Glu500 in CPTII with alanine or aspartate reduced theV max for both substrates, suggesting that Glu500 may be important in stabilization of the enzyme-substrate complex. A conservative substitution of Glu500 to aspartate resulted in a significant decrease in the V max for the substrates. Thus, Glu500 may play a role in substrate binding and catalysis. Our site-directed mutagenesis studies demonstrate that Glu487 in the liver isoform of CPTII is essential for catalysis.

and a malonyl-CoA-insensitive, detergent-stable CPTII. CPTI is an integral membrane enzyme located on the outer mitochondrial membrane, and CPTII is a membrane-associated enzyme loosely bound to the matrix side of the inner mitochondrial membrane. A current model for the membrane topology of CPTI predicts exposure of the N-and C-terminal domains crucial for activity and malonyl-CoA sensitivity on the cytosolic side of the outer mitochondrial membrane (3). As an enzyme that catalyzes the rate-limiting step in fatty acid oxidation, CPTI is tightly regulated by its physiologic inhibitor, malonyl-CoA, the first intermediate in fatty acid synthesis (1,2). This is an important regulatory mechanism in fatty acid metabolism and suggests coordinated control of fatty acid oxidation and synthesis. Mammalian tissues express two isoforms of CPTI, a liver isoform of CPTI and a heart/skeletal muscle isoform of CPTI, that are 62% identical in amino acid sequence (4 -7, 9). 2 Rat and human CPTII cDNAs have been cloned and sequenced (10,11). The cDNA sequences predicted proteins of 658 amino acid residues (71 kDa) that had 82 and 85% identity at the amino acid and nucleotide levels, respectively. Because the size of the mRNA in different rat tissues was identical, CPTII appears to be the product of a single gene that is expressed uniformly in every tissue examined thus far (2). CPTII is a distinct, catalytically active, malonyl-CoA-insensitive enzyme, because a rat liver cDNA encoding CPTII synthesizes an active protein when expressed in Escherichia coli, in the yeasts Saccharomyces cerevisiae and Pichia pastoris, or in baculovirus or COS cells (11,(13)(14)(15)(16). Detergent-solubilized yeast-expressed CPTII showed normal saturation kinetics with both substrates, carnitine and palmitoyl-CoA, and the calculated K m and V max were similar to that observed with the rat liver mitochondrial CPTII (15). With the S. cerevisiae-expressed CPTII, mutations of the conserved residues His 372 , Asp 376 , and Asp 464 to alanine resulted in complete loss of CPTII activity, suggesting that these residues may be required for catalysis (14). We and others hypothesize that the reaction catalyzed by CPTI and CPTII in the direction of palmitoylcarnitine formation at the active site pocket involves deprotonation of the hydroxyl group of carnitine by a catalytic base (␣-proton abstraction by His, Glu, or Gln) and attack by the resultant oxyanion at the carbonyl of the thioester of palmitoyl-CoA to generate palmitoylcarnitine and free CoA (14,17,18).
Carnitine palmitoyltransferase deficiencies are common disorders of mitochondrial fatty acid oxidation. Rare human genetic defects in fatty acid oxidation specifically ascribed to CPTI, CPTII, and the carnitine translocase have been reported (19,20). CPTII deficiency, the most common inherited disorder of lipid metabolism affecting skeletal muscle, is an autosomal recessive disorder with three distinct clinical phenotypes (19). The human CPTII gene is 20 kb in length, contains five exons, and is located at chromosome Ip32 (10). More than 25 different mutations and three polymorphisms have been identified in the CPTII gene (18,22,23). One missense mutation, S113L, accounts for ϳ60% of the mutant alleles responsible for the adult myopathic form of the disease (24,25). Recently, an E487K missense mutation in conjunction with S113L was reported in a child with CPTII deficiency characterized by recurrent episodes of myalgia and myoglobinuria triggered by fever (26).
A comprehensive structure-function study of the CPTII enzyme is necessary for a better understanding of the pathogenesis of human CPTII deficiency diseases and for diagnosis and therapy. In this report, site-directed mutagenesis studies of conserved glutamate residues in the C-terminal domain of a liver isoform of CPTII demonstrate for the first time that the conserved glutamate residue Glu 487 is important for catalysis.
The PCR product obtained by this overlap extension PCR was then purified, digested with SfiI-HindIII, and ligated into SfiI-HindIII-cut wild-type CPTII cDNA in pPROEX-1 to get the mutant CPTII cDNA. Transformants obtained upon transformation of the competent E. coli BL21 cells by the mutant DNAs were screened for the ability to productively serve as templates for PCR using forward primers with 3Ј ends specific to each of the above mutations. For example, the E500A mutant was screened for with the following primer: E500ACK 5Ј-CAAGCACGGCCGCACAGC-3Ј. The mutation-specific 3Ј nucleotides are indicated in bold above and in Table I. The other primers used for screening were E500DCK, E487ACK, E487KCK, and E487DCK. The mutations were confirmed by DNA sequencing. The transformants are designated as EC-CPTII, EC-PPROEX, EC-E487A, EC-E487D, EC-E487K, EC-E500A, and EC-E500D.
Rat Liver CPTII Expression-A single colony of EC-CPTII, EC-PPROEX, EC-E500A, EC-E500D, EC-E487A, EC-E487D, or EC-E487K was inoculated into a 2-ml LB medium containing 100 g/ml ampicillin and incubated at 37°C overnight with shaking (280 rpm). The culture was then transferred to 200 ml of the 2-ml LB medium containing 100 g/ml ampicillin and incubated at 37°C with shaking until the A 600 reached 0.5-0.8 unit. 200 l of 1 M isopropyl-1-thio-␤-D-galactopyrano-side was then added to each culture, and the incubation was continued at 37°C with shaking for 5 h. The cells were harvested by centrifugation at 12,000 ϫ g for 20 min, washed by resuspension in 20 ml of PBS, and finally suspended in 4 ml of PBS. The cells were then lysed by French press at 1500 p.s.i. Whole-cell extracts were obtained after centrifugation of the cell lysates at 12,000 ϫ g for 20 min. Protein was determined using the protein assay kit (Bio-Rad), and the cell extracts were stored at Ϫ80°C.
CPT Assay-CPT activity was assayed by the forward exchange method using L-[ 3 H]carnitine (8,28), and the K m for palmitoyl-CoA was determined by varying the palmitoyl-CoA concentration from 2.8 to 222.2 M at a fixed molar ratio (6.1:1) of palmitoyl-CoA to albumin as described previously (8). The concentration of carnitine was fixed at 200 M. The K m for carnitine was determined by varying the carnitine concentration from 11.1 to 471.7 M at a fixed 111.1 M palmitoyl-CoA.
Western Blot-Proteins in whole-cell extracts were separated by SDS-polyacrylamide gel electrophoresis in a 10% gel and transferred onto nitrocellulose membranes. Immunoblots were developed by incubation with the anti-His tag antibodies (Qiagen, Valencia, CA) as described previously (8). For wild-type and mutant CPTII protein determination in the whole-cell extracts, a quantitative Western blot analysis was employed with a His 6 protein ladder (Qiagen). The fluorescence intensity was measured using the Scion Image software (Scion Corp.).
Sources of other materials and procedures were as described in our previous publication (8).

Expression of Wild-type and Mutant CPTII in E. coli-We
have previously cloned the full-length rat liver CPTI and CPTII cDNA, separately expressed the genes in the yeast P. pastoris, and demonstrated for the first time that CPTI and CPTII are distinct catalytically active enzymes (15). Construction of plasmids carrying the wild-type and mutant CPTIIs was performed as described under "Experimental Procedures." Mutations were confirmed by DNA sequencing (Table I). E. coli was chosen as an expression system for the wild-type and mutant CPTIIs because it has no endogenous CPTII activity, and the E. coli-expressed CPTII is catalytically active (13). We expressed the full-length rat liver CPTII cDNA in E. coli and demonstrated that, unlike CPTI, the expressed CPTII is catalytically active (Table II). Furthermore, no CPTII activity was found in the control E. coli strain with the vector but without the CPTII cDNA insert.
Western blot analysis of the extract from the E. coli strain expressing the wild-type and mutant CPTIIs using an anti-His tag antibody showed the presence of a 71-kDa protein corresponding to CPTII (Fig. 2). For the wild-type and mutant CPTIIs, proteins of the predicted size were expressed at similar steady-state levels as determined by quantitative immunoblot using a pure His 6 protein as the standard. The quantitative Western blot and the densitometry analysis for the His 6 protein are shown in Fig. 2, B and C. Based on the quantitative immunoblot of the standard protein, the wild-type and mutant CPTII proteins were estimated to contain 7 pmol (0.49 g) of CPTII in 20 g of whole-cell extract protein.
Kinetic Properties of Wild-type and Mutant CPTIIs-Because of the extremely low activity in mutants E487A, E487D, and E487K, it was not possible to determine the K m or V max values for the carnitine or palmitoyl-CoA substrates. Mutants E500A and E500D exhibited normal saturation kinetics when the carnitine concentration was varied relative to a constant second substrate, palmitoyl-CoA (Fig. 3A), a property identical to the wild-type CPTII. For the E500A and E500D, the calculated K m values for carnitine were ϳ2-fold higher than the value for the wild-type CPTII, as shown in Table III, and the V max values were 26 and 60% lower than the wild type, respectively, indicating a major effect of the E500D mutation on catalytic activity. The catalytic efficiency as calculated by the V max /K m for the E500A and E500D was decreased by 55 and 76%, respectively. With respect to the second substrate, palmitoyl-CoA, mutants E500A and E500D exhibited normal saturation kinetics similar to the wild type (Fig. 3B) when the molar ratio of palmitoyl-CoA to albumin was fixed at 6.1:1. The calculated K m values for palmitoyl-CoA for mutants E500A and E500D were 76 and 89% lower, respectively, than the wild type. The V max values for mutants E500A and E500D were 13 and 29% of the wild-type values (Table III), and the catalytic efficiency was similar to the wild type for both mutants E500A and E500D. Thus, substitution of the conserved Glu 500 residue with either alanine or aspartate resulted in substantial loss in catalytic activity, but substitution of the highly conserved Glu 487 with alanine, lysine, or aspartate resulted in nearly complete loss in CPTII activity (Table II). DISCUSSION The site-directed mutagenesis study described here is aimed at elucidating the function of highly conserved acidic residues found at the proximity of the active site of CPTII. Two glutamate residues, Glu 487 and Glu 500 in rat liver CPTII, and the corresponding residues Glu 590 and Glu 603 in a liver isoform of CPTI, are conserved throughout the family of acyltransferases with known primary sequences. Specifically, Glu 487 in CPTII and Glu 590 in CPTI are highly conserved within the family of acyltransferases, but Glu 500 in CPTII and Glu 603 in CPTI are conserved residues within the family of CPT enzymes, whereas other acyltransferases have aspartate at this position (Fig. 1).
To determine the role of the conserved Glu 487 and Glu 500 residues in CPTII in catalysis, we separately changed the highly conserved glutamate residue (Glu 487 ) to alanine (E487A), aspartate (E487D), and lysine (E487K), and the second conserved glutamate residue (Glu 500 ) in CPTII to alanine (E500A) and aspartate (E500D), and we determined the effect of the mutations on CPTII activity in the E. coli-expressed mutant enzyme. Substitution of Glu 487 with alanine, lysine, or aspartate resulted in loss of Ͼ96% CPTII activity. Thus, the presence of this highly conserved Glu 487 residue is probably crucial for maintaining the configuration of the CPTII active site. Mutation of the corresponding highly conserved residue in a liver isoform of CPTI, Glu 590 , to alanine resulted in a 50% loss in CPTI activity (data not shown). The substantial loss in CPTII activity (99%) observed with our E487K mutant along with the loss observed in the child with the CPTII deficiency disease (26) establishes that Glu 487 is probably crucial for  maintaining the configuration of the liver isoform of CPTII active site. All of the Glu 487 mutants had insufficient activity to allow measurement of K m or V max for either carnitine or palmitoyl-CoA. Because a conservative substitution of Glu 487 with aspartate (E467D), a negatively charged amino acid that has only one less methyl group than the glutamate residue in the wild-type enzyme, resulted in 97% loss in activity, we suggest that Glu 487 is at the active-site pocket of CPTII.
As the terminal enzyme that transports long chain fatty acids from the cytosol to the mitochondrial matrix, CPTII in the presence of CoA-SH reversibly catalyzes the conversion of long chain acylcarnitines to long chain acyl-CoAs. Similar to other acyltransferases, CPTII contains a general acid/base, His 372 , a highly conserved residue that may form a hydrogen-bonding network or a salt bridge to a nearby conserved glutamate residue such as Glu 487 or Glu 500 . Substitution of Glu 487 with alanine, lysine, or aspartate resulted in an inactive enzyme. Glu 487 may thus be involved in facilitating catalysis by orienting the imidazole ring of His 372 for optimum productive interaction with the substrate (12). We hypothesize that the Glu 487 to alanine or lysine substitution may disrupt a hydrogen-bonding network or a salt bridge, perhaps to a residue like His 372 at the active site of CPTII. However, a change of Glu 487 to aspartate may result in the carboxylate being outside the hydrogen bond distance of His 372 , the predicted general acid/base at the active site (12).
The characterization of E487K, E487A, and E487D described here is consistent with previously reported studies of a child with a novel CPTII deficiency disease that exhibited clinical episodes of myalgia and myoglobinuria induced by intercurrent febrile illnesses (26). The patient was heterozygous for a G-to-A substitution at codon 487, changing the highly conserved Glu 487 to lysine (E487K), whereas the other allele carried the common benign S113L missense mutation. However, for the rat liver CPTII, a single substitution mutation of Glu 487 to lysine resulted in almost complete loss (99%) in activity, and even a single conservative change of Glu 487 to aspartate (E487D) or alanine (E487A) caused Ͼ96% loss in catalytic activity, further supporting a pathogenic role for a mutation of this residue. Thus, substitution mutation of the highly conserved negatively charged Glu 487 residue located in the C-terminal region inactivates CPTII, suggesting a critical role of this residue in catalysis. CPTII deficiency, inherited as an autosomal recessive trait, is the most common disorder of lipid metabolism affecting muscle and is the most frequent cause of hereditary myoglobinuria in adults that is triggered by prolonged exercise or fasting (or both) and by cold, stress, or fever, conditions associated with increased dependence of muscle on lipid metabolism (19,24,25). A hepatic form of CPTII deficiency associated with hypoketotic hypoglycemia, hepatopathy, cardiopathy, and sudden death has also been reported in infants (19).
To investigate the role of the second conserved glutamate residue in CPTII (Glu 500 ) on catalytic function, Glu 500 was changed to alanine and aspartate and the mutant enzyme was characterized. Substitution of the conserved Glu 500 with alanine and aspartate resulted in partial loss of activity and a decrease in the V max for both substrates. In addition, a change of the conserved Glu 500 to aspartate caused a significant decrease in the V max for both substrates with a 2-fold increase in the K m for carnitine. For both substrates, a lower but similar decrease in the V max and a 2-fold increase in the K m for carnitine was observed with the E500A mutant, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. However, the mutations could lead to misfolding in 90% of the molecules, producing a lower K cat and unchanged K m values. Because the E500A and E500D mutations caused a substantial decrease in the V max , this could be related to the alteration of intrinsic CPTII stability. Furthermore, Glu 500 appears to be critical for the structural stability of CPTII. A conservative substitution of Glu 500 to aspartate resulted in Ͼ7and 2.5-fold decreases in the V max for the FIG. 3. A, kinetic analysis of wild-type and mutant CPTII activities. Proteins (150 g) of whole-cell extract from wild type (squares), EC500A (circles), and EC500D (triangles) were assayed for CPT activity in the presence of increasing concentration of carnitine. B, same as A, except CPT activity was measured in the presence of an increasing concentration of palmitoyl-CoA. substrates palmitoyl-CoA and carnitine, respectively, with a relatively minor change in carnitine binding but a significant increase in the affinity for palmitoyl-CoA. It is suggested that the Glu 500 mutation exists within a region containing a possible adenine binding loop, because the affinity of the enzyme for palmitoyl-CoA that has an adenine group is greatly increased, but additional experimental evidence is needed to substantiate this hypothesis. Hence, Glu 500 may help orient the loop containing the active site residue by hydrogen bonding of a backbone residue and may play a role in substrate binding and catalysis (12).
Sequence alignment of various acyltransferases shows the presence of highly conserved histidine and glutamate residues in CPTII. Mutation of the conserved His 372 residue in CPTII and the corresponding residue in CPTI, His 473 to alanine, inactivates both enzymes (14,21). In this report, we have demonstrated that the conserved Glu 487 of rat liver CPTII is required for catalysis. Despite its similar negative charge and potential for hydrogen bonding, an aspartate residue cannot fulfill this requirement, suggesting that the extra methyl group in glutamate is needed for optimal catalysis and maintenance of active site integrity. A change of Glu 487 to lysine or alanine also resulted in an inactive enzyme. The loss in activity observed with the E487K is consistent with previously reported studies of a child with a novel CPTII deficiency disease (26). Substitution of the conserved Glu 500 in CPTII with alanine or aspartate reduced the catalytic efficiency, suggesting that this residue may be important in stabilization of the enzyme-substrate complex.