Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis.

Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.

Carnitine palmitoyltransferase (CPT) 1 I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of L-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix (1,2). Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a heart/skeletal muscle isoform (M-CPTI), which are 62% identical in amino acid sequence (3)(4)(5)(6)(7)(8). As an enzyme that catalyzes the first rate-limiting step in fatty acid oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA, the first intermediate in fatty acid synthesis, suggesting a coordinated control of fatty acid synthesis and oxidation (1,2). Previous studies by our laboratory and others have established that M-CPTI is more sensitive to malonyl-CoA inhibition than L-CPTI (3)(4)(5)(6)(7)(8). The molecular/ structural basis for the differences in malonyl-CoA sensitivity between M-CPTI and L-CPTI was established recently by our demonstration that substitution of the conserved Cterminal L-CPTI residue Glu-590 with alanine increased its malonyl-CoA sensitivity close to that observed with M-CPTI (9). Because of its central role in fatty acid metabolism, understanding the catalytic mechanism and regulation of the CPT system is an important first step in the development of treatments for diseases such as myocardial ischemia, diabetes, and obesity and for human-inherited CPTI deficiency diseases (9 -13).
We have expressed human heart M-CPTI, rat liver L-CPTI, and CPTII in the yeast Pichia pastoris, an organism devoid of endogenous CPT activity (6, 14 -16). Our recent deletion and point mutation analyses have demonstrated that Glu-3 and His-5 are necessary for malonyl-CoA inhibition and high-affinity binding of L-CPTI but not for catalysis (17,18). For M-CPTI, our site-directed mutagenesis studies demonstrate that in addition to Glu-3 and His-5, Val-19, Leu-23, and Ser-24 are necessary for malonyl-CoA inhibition and high-affinity binding but not for catalysis (19,20). It has been generally predicted that the catalytic and substrate binding sites in both L-CPTI and M-CPTI reside in the C-terminal region of the enzymes. Recent studies from our laboratory demonstrate that mutations of conserved arginine and tryptophan residues in the C-terminal region of L-CPTI abolish catalytic activity (21). Because the major effect of the mutations was on the V max , we predict that the conserved arginine and tryptophan residues stabilize the enzyme-substrate complex by charge neutralization and hydrophobic interactions (21). Furthermore, our site-directed mutagenesis studies demonstrate that deletion of the conserved C-terminal M-CPTI residue Leu-764 or mutation to Arg inactivates the enzyme (22). CPTI is an active acyltransferase that belongs to the acyltransferase family of enzymes; however, the molecular mechanism by which CPTI transfers the acyl group from the acyl-CoA to carnitine remains to be elucidated. In this study, our cysteine-scanning mutagenesis demonstrates that a single substitution mutation of Cys-305 to alanine abolishes M-CPTI catalytic activity.

EXPERIMENTAL PROCEDURES
Construction of Human Heart M-CPTI Cysteineless Mutants-Mutants were constructed using the QuikChange TM multisite-directed mutagenesis kit (Stratagene, La Jolla, CA) with plasmid p12E (for Nterminal mutagenesis) and p13E (for C-terminal mutagenesis) as the templates. Plasmid p12E is a derivative of pTZ18U, containing wildtype N-terminal human M-CPTI from ATG downstream to 622 bp and a fragment of the vector pHWO10 in the extreme 5Ј-end, whereas plasmid p13E is a derivative of pTZ18U, containing the C-terminal end of M-CPTI from 623 bp to the stop codon. The mutagenesis was performed according to the manufacturer's instructions. The mutants were in the vectors p12E and p13E. Mutations were confirmed by DNA sequencing.
For construction of the cysteineless M-CPTI mutant, a 1.7-kb EcoRI fragment of the mutant C9C containing mutations of all the C-terminal cysteines was used to replace the corresponding EcoRI fragment of the plasmid N6C that contains the N-terminal six cysteine mutants to generate the 15-residue cysteineless M-CPTI mutant in pHW010.
Mutations were confirmed by DNA sequencing. All the primers used for cysteine mutagenesis are listed in Table I, and the mutations are highlighted in bold. All the cysteine residues in human M-CPTI were either altered to alanine (if human L-CPTI has a cysteine residue at the corresponding position) or to the corresponding amino acid residue in human L-CPTI.
Construction of the Single C-terminal M-CPTI Cysteine Mutants C504A, C526A, C548S, C562A, C586A, and C608A-Mutants were constructed by the overlap extension PCR method using the primers shown in Table I with the wild-type plasmid DNA (pGAP M-CPTI) as template (23). For example, to construct the C562A mutant, the primers F2-C562AR and C562AF-R3 were used to generate 840-and 300-bp PCR products, respectively, using the wild-type M-CPTI cDNA as template. The two PCR products were purified, mixed, and used as template for a second-round PCR with the primer F2-R3. The 1.13-kb PCR product was digested with SphI-BglII and ligated into SphI-BglII-cut wild-type M-CPTI cDNA in the pGAP expression vector. The construction of C504A, C526A, C548S, C586A, and C608A mutants was similar to that of C562A using the primers listed in Table I, F2 and R3.
The C305A mutant was constructed by the overlap extension PCR method using the primers F2-C305AR and C305AF-C608AR with the wild-type M-CPTI plasmid DNA as template. The 926 and 940-bp PCR products were purified as described above, mixed, and used as a template for a second-round PCR with the primer F2-C608AR. The 1860-bp PCR product was digested with AflIII-SphI and ligated to AflIII-SphIcut wild-type pGAP-M-CPTI.
Mutants C448A and C659A were constructed using the QuikChange™ multisite-directed mutagenesis kit with plasmid p13E (containing wild-type C-terminal human M-CPTI cDNA in pTZ18U vector) as the template. Primers C448AR and C659AR were each used to separately generate the C448A and C659A mutants. The mutated fragments in p13E were cut with EcoRI and used to replace the corresponding EcoRI fragment in the wild-type plasmid pGAP-M-CPTI.
Construction of the Revertant Mutant A305C-The QuikChange® XL site-directed mutagenesis kit was used with plasmid 13E9C41 (containing C-terminal human M-CPTI with 9 mutated cysteines in vector pTZ18U). The primers used were PAGE-purified A305CF2 and A305CR2. The mutants in the plasmid 13E9C41 were EcoRI-cut and used to replace the corresponding EcoRI fragment in the wild-type plasmid pGAP-M-CPTI.
Bacterial colonies obtained upon transformation of the mutagenesis reactions were screened for the ability to productively serve as templates for PCR using forward primers with 3Ј-end specific to each of the mutants. For example, the C305A mutant was screened with the primer C305ACK. The mutations were confirmed by DNA sequencing. The plasmids were linearized by digestion with the restriction enzyme BspEI and integrated into the His4 locus of P. pastoris GS115 by electroporation (18). Histidine prototrophic transformants were selected on YND (yeast nitrogen base with dextrose) plates and grown on YND medium. Mitochondria were isolated by disrupting the yeast cells with glass beads (14) and used to monitor activity and malonyl-CoA sensitivity.
CPT Assay-CPTI activity was assayed in isolated mitochondria from the yeast strains expressing the wild-type and mutant CPTIs by the forward exchange method using L-[methyl-3 H]carnitine as described previously (6,24). The K m for palmitoyl-CoA was determined by varying the palmitoyl-CoA concentration from 12.5 to 400 M at a fixed molar ratio (6.1:1) of palmitoyl-CoA to albumin as described previously (18,20,25). The concentration of carnitine was fixed at 1.0 mM. The K m for carnitine was determined by varying the carnitine concentration from 100 M to 4.0 mM at a fixed concentration (100 M) of palmitoyl-CoA. 150 g of mitochondrial protein were used, and all incubations were performed at 30°C for 3 min. The malonyl-CoA inhibition was determined by varying the concentration of malonyl-CoA from 50 nM to 10 M at fixed concentrations of carnitine (1.0 mM) and palmitoyl-CoA (100 M). The incubations were performed at 30°C for 5 min.

Labeling of Wild-type and Mutant M-CPTI with [U-14 C]Palmitic
Acid-Isolated mitochondria (100 g of protein) from the yeast strains expressing the wild-type and mutant M-CPTI were incubated with 50 M [U-14 C]palmitic acid (specific activity, 824 mCi/mmol; Amersham Biosciences) and 250 M CoA and 5 mM ATP for 3 h at 30°C (42). At the end of the incubation, the samples were divided into two fractions of equal volume, and one fraction was treated with 2.0 M hydroxylamine (adjusted to pH 7.5 with NaOH) at 30°C for 3 h. Hydroxylamine-treated and untreated samples were heat-denatured and subjected to SDS-PAGE followed by electrophoretic transfer onto nitrocellulose membranes. The blots were dried and exposed to screens of Amersham Biosciences phosphorimaging plates for 1-7 days, and signals were analyzed in a Amersham Biosciences Phos-phorImager SI.
Western Blot-Proteins were separated by SDS-PAGE in a 10% gel and transferred onto nitrocellulose membranes. Immunoblots were developed by incubation with the M-CPTI-specific polyclonal antibodies as described previously (6,14,15). Sources of other materials and procedures were as described in our previous publications (19,22,26).

RESULTS
Preincubation of isolated mitochondria from the yeast strain expressing human heart M-CPTI with 250 M 5,5Ј-dithiobis(2nitrobenzoic acid), an -SH-specific modifying reagent, at 4°C for 30 min caused a 75% loss in CPTI activity, indicating that there are cysteine residues in M-CPTI that are important for catalysis and/or substrate binding. These preliminary chemical modification studies with 5,5Ј-dithiobis(2-nitrobenzoic acid) by us and others provided evidence that CPTI may contain a cysteine residue that is important for catalysis (27).
Generation of Mutations and Expression in P. pastoris-Construction of plasmids carrying the N-terminal cysteineless M-CPTI (N6C), the C-terminal cysteineless M-CPTI (C9C), the cysteineless M-CPTI (NC15C), and all the single substitution mutations was performed as described under "Experimental Procedures." P. pastoris was chosen as an expression system for M-CPTI and the mutants because it does not have endogenous CPT activity (6, 14 -18). The P. pastoris expression plasmids expressed M-CPTI under the control of the P. pastoris glyceraldehyde-3-phosphate dehydrogenase gene promoter (28). Yeast transformants with the wild-type M-CPTI gene and the mutants were grown in liquid medium supplemented with glucose (14).
Western blot analysis of wild-type and M-CPTI (80 KDa) and the mutants using a polyclonal antibody directed against a maltose-binding protein-M-CPTI fusion protein (6,19) showed proteins of predicted sizes with similar steady-state levels of expression.
Effect of Mutations on M-CPTI Activity and Malonyl-CoA Sensitivity-The C-terminal 9-residue cysteineless M-CPTI and the complete 15-residue cysteineless M-CPTI were inactive, despite the high level of protein expression observed for the mutants on the Western blot. The N-terminal 6-residue cysteineless mutant M-CPTI had activity and malonyl-CoA sensitivity similar to that of the wild type as shown in Table II, suggesting that 1 or more of the 9 cysteine residues in the C-terminal region of M-CPTI may be important for activity. Separate substitution mutation of C305A, C448A, C504A, C526A, C548S, C562A, C586A, C608A, and C659A showed that of the nine cysteine mutants, only the substitution mutant C305A was inactive, demonstrating that a single change of Cys-305 to Ala (or serine) 2 resulted in complete loss in M-CPTI activity. As shown in Table II, a change of cysteine to either alanine or serine of the other 8 C-terminal cysteines had no effect on activity and malonyl-CoA sensitivity. A single change of Ala-305 to Cys, A305C, in the 9-residue C-terminal cysteineless mutant resulted in a mutant enzyme with similar activity and malonyl-CoA sensitivity (Table II) as the wild-type M-CPTI, demonstrating that Cys-305 (but not the other 8 C-terminal cysteine residues) is essential for M-CPTI activity. In short, our studies identify for the first time that Cys-305 in the C-terminal region of M-CPTI is essential for catalytic activity because mutation of this residue to Ala inactivated M-CPTI.

Cys-305 in M-CPTI Is Important for Catalysis
Kinetic Characteristics of Wild-type and Mutant M-CPTIs-Mutant A305C exhibited normal saturation kinetics when the carnitine concentration was varied relative to a constant second substrate, palmitoyl-CoA, a property identical to that of the wild-type M-CPTI. For the mutant A305C, the calculated K m value for carnitine was only 12% higher and the V max was 15% lower compared with the wild-type M-CPTI, as shown in Table III, indicating almost complete restoration of catalytic activity and no major effect of substitution of the 8 C-terminal cysteine residues with Ala or Ser on M-CPTI activity. The catalytic efficiency as estimated by V max /K m for mutant A305C decreased by 24% compared with the wild type. With respect to the second substrate, palmitoyl-CoA, mutant A305C exhibited normal saturation kinetics similar to that of the wild type when the molar ratio of palmitoyl-CoA to albumin was fixed at 6.1:1. The calculated K m and V max values for the A305C mutant were only 10% lower than those for the wild type, and the catalytic efficiency was similar to that of the wild type. Thus, substitution of Ala-305 with cysteine in the inactive 9-residue C-terminal cysteineless M-CPTI resulted in a mutant, A305C, that had activity, malonyl-CoA sensitivity, and kinetic properties similar to those of wild-type M-CPTI, demonstrating for the first time that of the 9 C-terminal cysteine residues present in M-CPTI, only Cys-305 is essential for catalytic activity and malonyl-CoA sensitivity.
Acylation of Wild-type and Mutant M-CPTI-The loss in CPTI activity in the C305A mutant and the recovery of activity in the A305C revertant suggested that an acyl-enzyme intermediate may be involved in the catalytic mechanism of M-CPTI. We therefore investigated whether the loss in activity in the C305A mutant was due to the failure of an acylenzyme intermediate formation in the catalytic mechanism of M-CPTI by incubation of the wild-type and mutant M-CPTI with [U-14 C]palmitic acid in the presence of CoA and ATP, but in the absence of carnitine and measurement of covalent incorporation of labeled palmitate into the enzyme. Both the wild-type and A305C revertant were strongly labeled with radioactive palmitate (Fig. 1) but the C305A mutant was not. Furthermore, the [U-14 C]palmitate was covalently bound because the labeling could be removed by treatment with neutral hydroxylamine (Fig. 1). These results provide strong support for an acyl-enzyme intermediate in the catalytic mechanism of M-CPTI.

DISCUSSION
Our cysteine-scanning mutagenesis of M-CPTI demonstrated that both the 9-residue C-terminal and the complete 15-residue cysteineless mutant enzymes are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. These results suggested that 1 or more of the 9 Cterminal cysteine residues was important for catalysis. Separate substitution mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be essential for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type. Cys-305 is a conserved residue within the family of enzymes that includes CPTI, carnitine acetyltransferase, and choline acetyltransferase from different species, but it is not conserved in CPTII or carnitine octanoyltransferase, in which the corresponding Cys-305 is replaced by Asp as shown in Fig. 2. Of the remaining 8 C-terminal cysteine residues in M-CPTI, 7 are conserved in both M-CPTI and L-CPTI but not in the other acyltransferase-family enzymes. The 8th residue, Cys-548, is only present in M-CPTI, and Ser replaces Cys in wild-type L-CPTI. The M-CPTI mutant C548S had activity and malonyl-CoA sensitivity similar to those of the wild type. Human heart M-CPTI has 6 Nterminal cysteine residues that are absent in L-CPTI . Because M-CPTI is much more sensitive to malonyl-CoA inhibition than L-CPTI , we hypothesized that the differences in malonyl-CoA sensitivity observed between the two isoforms could be due to the presence of the 6 additional N-terminal cysteine residues in M-CPTI compared with L-CPTI. However, our cysteine-scanning mutagenesis demonstrated that the N-terminal cysteineless M-CPTI had activity and malo-   nyl-CoA sensitivity similar to those of the wild-type enzyme, suggesting that these residues do not play a role in M-CPTI activity and inhibitor sensitivity.
More recently, in a patient with CPTI deficiency disease, a complete loss in L-CPTI activity was reported, which was due to substitution of Cys-305 with Trp, the conserved cysteine residue corresponding to that of human L-CPTI and M-CPTI that we demonstrated to be essential for human heart M-CPTI activity by our cysteine-scanning mutagenesis (29). Members of the acyltransferase family of enzymes, including M-CPTI and L-CPTI, contain a highly conserved His residue (Fig. 2) at the active site pocket, His-473, a general acid/base that, when mutated to Ala in CPTI, was shown to cause complete loss in activity (30). As a general acid/base, His-473 may form a hydrogen-bonding network or a salt bridge to a nearby conserved aspartate residue. There are also three highly conserved Asp residues within the family of acyltransferases, namely, Asp-323, Asp-454, and Asp-567, which are present in both CPTI and CPTII, as shown in Fig. 2. In a patient with hepatic CPTI deficiency disease who was homozygous for a D454G missense mutation, the yeast-expressed mutant L-CPTI exhibited only 2% of the wild-type L-CPTI activity, demonstrating that a change of D454 to Gly in CPTI was the cause for the disease (31). Because the conserved Asp-454 is closer to the conserved CPTI active site residue (His-473) that may form a hydrogenbonding network or a salt bridge to Asp-454, the loss in activity observed in the D454G mutant may be due to disruption of the hydrogen-bonding network or salt bridge to His-473. Asp-323 and Asp-567 may be too far from the conserved His-473 at the active site pocket and thus may not be ideally positioned for such an interaction due to their location. Furthermore, the recent three-dimensional structure of carnitine acetyltransferase, a membrane-associated enzyme that belongs to the acyltransferase family of enzymes, suggests that Asp-567 may hydrogen-bond with Glu-590 (32), and more recent data from our laboratory 3 show that a change of Asp-567 to Ala or His but not Glu resulted in a significant loss in CPTI activity. Our cysteine-scanning mutagenesis and the His-473 and Asp-454 mutation studies with CPTI suggest that there are at least two conserved residues, namely, Cys-305 and His-473, at the active site pocket of CPTI that are essential for catalysis because separate mutation of these residues to Ala inactivates CPTI. In addition, Asp-454 may interact with His-473 and indirectly facilitate catalysis because a mutation of this conserved residue that is located close to the active site

Cys-305 in M-CPTI Is Important for Catalysis 4529
His-473 caused a significant loss in CPTI activity (33)(34)(35)(36)(37)(38). As a rate-limiting enzyme that transports long-chain fatty acids from the cytosol to the mitochondrial matrix, CPTI in the presence of carnitine catalyzes the conversion of long-chain acyl-CoAs to acylcarnitines. However, the molecular mechanism by which CPTI transfers the acyl group from the acyl-CoA to carnitine remains to be elucidated. We have hypothesized previously that the reaction catalyzed by CPTI at the active site, conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of L-carnitine, involves deprotonation of the hydroxyl group of carnitine by the catalytic base, His-473, and attack by the resultant oxyanion at the carbonyl of the thioester of palmitoyl-CoA to generate palmitoylcarnitine and free CoA (39). Based on our cysteine-scanning mutagenesis and acylation studies with CPTI and studies by others, we now propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of residues Cys-305, His-473, and Asp-454 as shown in Fig. 3. The cysteine residue forms a more stable acyl-enzyme intermediate that may allow the acceptor molecule, carnitine, to act as a second nucleophile and complete the acyl transfer reaction. We propose a mechanism for the catalysis whereby the active site catalytic base His-473 aided by Asp-454 abstracts a proton from the -SH group of Cys-305 to generate a reactive thiolate anion that carries out a nucleophilic attack on the substrate acyl-CoA, yielding a tetrahedral intermediate that is subsequently converted to a thioacyl-enzyme covalent intermediate (acyl-S-enzyme). It is envisaged that the negative charge from the carboxyl ion of Asp-454 is transferred to His-473 and then to Cys-305 to enhance the power of the nucleophile (33)(34)(35)(36)(37)(38). Deacylation and transfer of the acyl group to carnitine occur by a nucleophilic attack of the thioester bond of the acyl-S-enzyme intermediate with the reactive carnitine oxyanion generated by abstraction of a proton from the hydroxyl group of carnitine by the general base catalyst at the active site, His-473, assisted by Asp-454, resulting in the formation of a second tetrahedral intermediate that breaks down to yield the product acylcarnitine and regenerate the enzyme as shown in Fig. 3.
CPTII and carnitine octanoyltransferase, membrane-associated enzymes located on the matrix face of the inner mitochondrial membrane, contain Asp instead of Cys at the corresponding position for Cys-305, as shown in Fig. 2. This is probably due to the differences in the reaction mechanisms catalyzed by CPTI and CPTII, generally referred to as the forward and reverse reactions, respectively. In vivo, CPTII catalyzes the transfer of an acyl group from acylcarnitine to CoA-SH, which is the reverse of the reaction catalyzed by CPTI resulting in the regeneration of acyl-CoA on the matrix side, thus completing the transport of long-chain acyl-CoAs from the cytosol to the mitochondrial matrix (40,41). The conserved active site His-373 in CPTII shown in Fig. 3 probably abstracts a proton from the CoA-SH to generate a nucleophile that attacks the acylcarnitine, resulting in the transfer of the acyl group to CoA and formation of the acyl-CoA.
Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank TM led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. This catalytic triad was found to be conserved in human, rat, and mouse CPTI and other acyltransferases, with the exception of CPTII and carnitine octanoyltransferase. Mutation of Cys-305, Asp-454, and His-473 results in loss of CPTI activity, which strongly suggests that CPTI is an enzyme containing a Cys-His-Asp catalytic triad, providing a possible mechanism for the acyltransferase activity. Sitedirected mutagenesis of His-473 and Asp-454 to alanine resulted in inactivation of CPTI, suggesting that these two resi-dues are critical for CPTI activity. Because the Asp and His residues are responsible for providing the nucleophile negative charge and relaying it to the cysteine residue, their mutation would be expected to destroy the charge relay system, thereby inactivating the enzyme. Whereas Cys-305 is important for CPTI activity, it is clear that none of the remaining 8 Cterminal cysteine residues are important for CPTI activity, and the disulfide bond formation involving these residues also must not be important for catalytic activity, although they may be involved in mediating the interaction of CPTI with other proteins. In short, our studies demonstrate that CPTI is a thiol acyltransferase and that Cys-305 is the essential nucleophilic residue critical for catalysis. In the presence of both substrates, carnitine and palmitoyl-CoA, CPTI acts primarily as an acyltransferase, but in the absence of either carnitine or CoA, CPTI may exhibit low hydrolase activity, resulting in the breakdown of palmitoyl-CoA or palmitoylcarnitine by a mechanism similar to that of the cysteine proteases and hydrolases, which utilize a Cys-His-Asp catalytic triad (33)(34)(35)(36)(37)(38).