Identification of Conserved Amino Acid Residues in Rat Liver Carnitine Palmitoyltransferase I Critical for Malonyl-CoA Inhibition

From the ‡Department of Biochemistry and Molecular Biology, University of Barcelona, School of Pharmacy, E-08028 Barcelona, Spain, the ¶Protein Design Group, National Center for Biotechnology, Consejo Superior de Investigaciones Cientı́ficas, Cantoblanco, E-28049 Madrid, Spain, and the **Department of Biochemistry and Molecular Biology, International University of Catalonia, 08190 Sant Cugat, Spain

The enzyme carnitine palmitoyltransferase (CPT) 1 I catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines, which is the first step in the transport of fatty acyl-CoA groups from the cytosol to mitochondria where they undergo ␤-oxidation. This reaction is inhibited by malonyl-CoA, and so this enzyme could be the most physiologically important regulatory step in mitochondrial fatty acid oxidation (1). This process allows the cell to signal the relative availability of lipid and carbohydrate fuels in liver, heart, skeletal muscle, and pancreatic ␤-cell (2). The mechanism of malonyl-CoA inhibition can be potentially mimicked by pharmacological malonyl-CoA-related agents for the treatment of metabolic disorders such as diabetes, insulin resistance, and coronary heart disease (3).
Mammals express two isoforms of CPT I, a liver isoform (L-CPT I) and a heart/skeletal muscle isoform (M-CPT I), which are the products of two different genes (4,5). The identity in amino acids residues is high (62%) but they are differentially regulated by malonyl-CoA. The L-CPT I isoform is inhibited by malonyl-CoA to a much lesser extent than the M-CPT I isoform (the IC 50 value for M-CPT I is about 2 orders of magnitude lower than for L-CPT I) (6). This property is probably involved in the finer regulation of fatty acid oxidation in heart and skeletal muscle in comparison to liver.
From studies on the pH dependence of the affinity of CPT I for its substrate and from the ability of palmitoyl-CoA to displace [ 14 C]malonyl-CoA bound to skeletal muscle mitochondria it was hypothesized (7) that the palmitoyl-CoA and malonyl-CoA bind at different sites. A number of studies have shown that in rat liver CPT I there are two malonyl-CoA binding sites: one with greater capacity for binding and regulation of the inhibitor and not susceptible to competition from acyl-CoA, which behaves as an allosteric component (8 -12); and a second acyl-CoA binding site, which is located near the catalytic site (13).
Various groups have attempted to establish the basis of the L-CPT I/malonyl-CoA interactions. The probable binding sites of malonyl-CoA in L-CPT I were deduced to be at the C terminus after preparation of several L-CPT I chimeras whose IC 50 values for malonyl-CoA corresponded to the C-terminal region (14) of the chimera. However, the N terminus of L-CPT I was also shown to influence the enzyme/inhibitor interaction. Mutation of Glu 3 , His 5 , or His 140 produced a loss of malonyl-CoA sensitivity (15,16). In addition, the removal of the segment comprised between amino acids 1 and 18 in L-CPT I and 1-28 in M-CPT I produced a decrease in malonyl-CoA sensitivity, which emphasizes the importance of the N terminus before the first transmembrane region as a modulator of the malonyl-CoA inhibition (17,18). On the basis of these results, it was proposed that the two malonyl-CoA inhibitable domains might be located at the C terminus as suggested by several kinetic studies. The development of a CPT I catalytic core model (19) allowed us to assign the low affinity binding site to a domain near the catalytic channel in which palmitoyl-CoA is bound containing the catalytic acyl-CoA binding domain (20) Here we used the SequenceSpace algorithm program to identify five amino acid residues (Thr 314 , Asn 464 , Ala 478 , Met 593 , and Cys 608 ), which may contribute to the sensitivity of CPT I to malonyl-CoA. The proposal is based on the finding that they are present in malonyl-CoA-inhibitable CPT I ((isoforms L-and M-) and COT from various organisms and absent in noninhibitable acyltransferases (CPT II, CAT, and ChAT). Mutation of these amino acids to their counterparts in CPT II showed that mutation of Met 593 by itself, M593S, or the quintuple mutant containing the M593S mutation, T314S/N464D/A478G/M593S/ C608A, or other Met 593 point mutants such as M593A and M593E nearly abolished malonyl-CoA sensitivity of L-CPT I. The remaining mutated amino acids showed slight, varied sensitivity to malonyl-CoA inhibition.

EXPERIMENTAL PROCEDURES
Tree-determinants Analysis-Sequences of proteins from the carnitine-choline acyltransferase family were obtained using BLAST (21). Multiple alignment was performed using ClustalW (22). The analysis of conserved differences (tree-determinants) between malonyl-CoA-regulated (L-CPT I, M-CPT I, and COT) and nonregulated (CPT II, CAT, and ChAT) acyltransferases, using multivariate statistics for low-dimensional representation, was done using the SequenceSpace algorithm (23,24). Graphics of vectors representing protein sequences and individual residues from the multiple alignment were performed using the Sequence Space Java-based viewer (www.industry.ebi.ac.uk/SeqSpace).
Expression of L-CPT I in Saccharomyces cerevisiae-The expression of the constructs containing L-CPT I wild type and mutants (see above) in yeast cells and the preparation of the cell extracts were performed as described in Ref. 19. S. cerevisiae was chosen as an expression system for L-CPT I wild type and the mutants because it does not have endogenous CPT I activity.
Determination of Carnitine Acyltransferase Activity-Carnitine palmitoyltransferase activity was determined by the radiometric method as described in Ref. 19 with minor modifications. The substrates were L-[methyl-3 H]carnitine and palmitoyl-CoA. Enzyme activity was assayed for 4 min at 30°C in a total volume of 200 l. For determination of the K m for carnitine, palmitoyl-CoA was fixed at 135 M (for L-CPT I). For determination of the K m for acyl-CoA, carnitine concentration was fixed at 400 M. When malonyl-CoA inhibition was assayed, increasing concentrations of malonyl-CoA were included. The IC 50 , defined as the malonyl-CoA concentration that produces 50% inhibition of enzyme activity, was determined using 50 M palmitoyl-CoA and 400 M carnitine. K m was estimated by analyzing the data from three experiments using the program Enzifit (Biosoft), and IC 50 was calculated by Excel software using linear regression analysis.
Values reported in the text are the means and standard deviations of three to five determinations. Curve fitting was carried out using Excel software. All protein concentrations were determined using the Bio-Rad protein assay with bovine albumin as standard.
Immunological Techniques-Western blot analysis was performed as described (19). The antibody for rat L-CPT I was kindly given by Dr. V. A. Zammit (Hannah Research Institute, Ayr, Scotland, United Kingdom) and was directed against peptide 428 -441, in the cytosolic catalytic C-terminal domain.

Residues Conserved in Malonyl-CoA Inhibited Versus Noninhibited Carnitine-Choline
Acyltransferases-An exhaustive analysis of the presence of residues shared by all the malonyl-CoA-regulated enzymes of the carnitine-choline acyltransferase family versus the malonyl-CoA nonregulated members of the same family was performed using the algorithm Sequence-Space (23,24). This method uses a vectorial representation of each protein sequence as a point in a multidimensional space (SequenceSpace) and multivariate statistics, principal component analysis, to allow reduction of the number of dimensions. This representation allows us not only to define clusters of proteins according to specific properties by choosing the appropriate axes defined by the highest corresponding eigenvalues (also known as proper values), but also to project the individual residues on the same axes, and thus trace the positions conserved in the subfamilies defined. The main advantage of this method is the possibility of predicting which residues may be responsible for the specific characteristics of each protein subfamily or group of subfamilies as has been reported previously for short-and medium-long substrate specificity for the carnitine-choline acyltransferases protein family (19,20) or effector recognition by some members of the Ras superfamily (25).
The two-dimensional projection of sequence vectors on the plane defined by the axes corresponding to eigenvalues 2 and 4 showed clustering of the enzyme subfamilies according to their malonyl-CoA inhibition properties (Fig. 1A). Proteins whose activity is not regulated by malonyl-CoA (CPT II, CAT, and ChAT subfamilies) were grouped, whereas the sequences of the proteins regulated by malonyl-CoA (COT, L-CPT I, and M-CPT I) occupy separate, and opposite, zones. The projection of the individual amino acid residues on the same plane ( Fig. 1B) revealed the amino acids responsible for this segregation might be responsible for the susceptibility to malonyl-CoA of the corresponding enzymes. Five of these amino acids (Thr 314 , Asn 464 , Ala 478 , Met 593 , and Cys 608 ) were present in all malonyl-CoA inhibitable carnitine acyltransferases and absent in the nonmalonyl-CoA inhibitable acyltransferases (CPT II, CAT, and ChAT from several species). Fig. 2 shows the sequence alignment of three fragments of the C-terminal region of various acyltransferases. We can also observe that those enzymes that are not inhibitable by malonyl-CoA (CPT II, CAT, and ChAT) show the same amino acids in these positions, which are different from those observed in inhibitable malonyl-CoA acylcarnitines. As an example the positions and amino acids of CPT II are given: Ser 223 , Asp 363 , Gly 377 , Ser 490 , and Ala 505 (Fig. 2).
Expression of Wild Type and Mutants in S. cerevisiae-We  (Table I).
All mutants exhibited standard saturation kinetics when the carnitine concentration was varied relative to a constant concentration of the second substrate, palmitoyl-CoA, and when palmitoyl-CoA concentration was varied relative to a constant carnitine concentration, a property identical to that of the wild type L-CPT I (Fig. 3). The quintuple mutant produced small changes in the kinetic constants for carnitine and palmitoyl-CoA as substrates (Table I). Catalytic efficiency (V max /K m ) was increased by a factor of 2.6 (carnitine) and 2.2 (palmitoyl-CoA). The catalytic efficiency for carnitine as substrate of those point mutants that altered the sensitivity to malonyl-CoA increased (see below). The catalytic efficiency of the methionine mutants increased between 4.2-and 21-fold, C608A increased 8.8-fold, and A478G increased 4.1-fold. T314S, which produced a small change in malonyl-CoA sensitivity (see below), increased the V max /K m value by only 2.8, whereas in N464D, in which the sensitivity to malonyl-CoA was unchanged (see below), the catalytic efficiency was modified by a factor of 5.6.
An analogous tendency was also observed in K m for palmitoyl-CoA but the changes were smaller. K m values for palmitoyl-CoA were 24.3, 15.1, 7.4, 6.1, and 6.3 for mutants C608A, A478G, M593S, M593A, and M593E, respectively (K m value for the wild type was 4.9) (Table I). Catalytic efficiencies for palmitoyl-CoA as substrate increased in all mutants, the values ranging between 2.78 and 4.81 (Table I).
Inhibition of CPT I Wild Type and Mutants by Malonyl-CoA-When inhibitory kinetics versus increasing concentrations of malonyl-CoA was performed, the quintuple mutant practically abolished the sensitivity toward malonyl-CoA (IC 50 of 258 versus 12.3 M of the wild type) (Fig. 4B and Table I).
Even at concentrations as high as 100 M malonyl-CoA the CPT I quintuple mutant maintained 80% of the activity of the control without malonyl-CoA.
We then addressed the individual responsibility of the separate CPT I mutants for the malonyl-CoA sensitivity. Mutants T314S, N464D, M593S, and C608A expressed in S. cerevisiae were incubated with increasing amounts of malonyl-CoA, and CPT I activity was determined. Mutant A478G had been previously studied in Ref. 20 and showed decreased sensitivity to malonyl-CoA (IC 50 of 39.5 versus 12.3 M of the wild type).
The kinetics of inhibition by malonyl-CoA depended on the mutant considered. Whereas mutant M593S (Fig. 4A) showed very low sensitivity at malonyl-CoA inhibition (IC 50 of 319 M), the other mutations produced varied changes in malonyl-CoA sensitivity. L-CPT I C608A slightly modified the sensitivity to malonyl-CoA (IC 50 is 27.5 M), the change in IC 50 of mutant T314S was small, whereas N464D showed similar sensitivity to malonyl-CoA to the wild type ( Fig. 4B and Table I). Because the highest changes in sensitivity to malonyl-CoA and K m values for carnitine were observed in the methionine mutants (point and quintuple mutants), we additionally prepared two new mutants: M593A and M593E to examine whether Met 593 was essential to the malonyl-CoA interaction in L-CPT I. Results show that the sensitivity to malonyl-CoA was also nearly abolished in these mutants (Fig. 4A) (IC 50 values of 155 and 220 M, respectively) as in the M593S mutant, confirming the essential role of Met 593 in this interaction. DISCUSSION We attempted to identify the amino acids in the C-terminal domain of L-CPT I that are responsible for the inhibition of the catalytic activity by malonyl-CoA. Over many years much work has been done to identify the domains in L-CPT I that may bind malonyl-CoA. Different groups have tested different empirical hypotheses and mutated amino acids, mostly in the aminoterminal region of L-CPT I. The results have shown that this domain plays a role in the regulation of CPT I by malonyl-CoA, because in some cases the sensitivity to the inhibitor is impaired.
A different approach was employed by our group very recently. This was based on the conservation of two histidine residues, which are present in the inhibitable malonyl-CoA carnitine acyltransferases (CPT I and COT) and absent in noninhibitable enzymes (CPT II and CAT). Mutation of both histidines resulted in the abolition of malonyl-CoA sensitivity in COT (26). Analogous results were observed in CPT I when its concentration at the mitochondrial membranes was not high. Mutation of other amino acids in the domain proximal to the catalytic site (Ala 478 and Pro 479 ) indicated that a malonyl-CoA-  inhibitable domain was probably the low-affinity malonyl-CoA binding site. Our previous studies showed that the location of malonyl-CoA in the structural model was compatible with competition of the inhibitor versus the substrate in the malonyl-CoA low affinity binding site (20).
The site-directed mutagenesis study used here to identify amino acids responsible for malonyl-CoA inhibition is based on the comparison of the sequences in a range of carnitine and choline acyltransferases, taking the positive or negative sensitivity to malonyl-CoA as a discriminatory criterion. The biocomputing study has shown that five amino acids are present in all CPT I (isoforms L-and M-) and in COT from various organisms and that they are absent not only in other nonmalonyl-CoA-inhibitable carnitine acyltransferases but also in ChAT. In rat L-CPT I these amino acids are Thr 314 , Asn 464 , Ala 478 , Met 593 , and Cys 608 . The corresponding positional amino acids in CPT II, CAT, and ChAT are Ser 223 , Asp 363 , Gly 377 , Ser 490 , and Ala 505 , respectively (coordinates of rat CPT II). Therefore, we considered it highly probable that these amino acids were involved in the interaction of malonyl-CoA. Results confirmed in part this supposition. The quintuple mutant reduced malonyl-CoA sensitivity almost completely (80% activity at 100 M malonyl-CoA (which is outside the physiological range)), supporting the initial hypothesis. The results obtained using separate single mutants indicate that not all of these amino acids have the same role in malonyl-CoA inhibition. Whereas M593S nearly abolished the sensitivity to malonyl-CoA like the quintuple mutant, A478G increased the IC 50 from 12 to 39.5 M (20). The other amino acids are less responsible for the inhibition.
The relevance of Met 593 as a critical amino acid for malonyl-CoA sensitivity was confirmed by the results of mutation to other two amino acids, Ala and Glu. The mutants equally showed diminished sensitivity to malonyl-CoA like mutant M593S. Met 593 , when mutated to Ser as it appears in CPT II and CAT, decreased the sensitivity to malonyl-CoA in a stronger fashion than when it was mutated to other amino acids like Ala and Glu, which were unrelated to this position in other carnitine acyltransferases. Therefore, we conclude that the occurrence of Ser in this position has probably been evolutionary conserved in nonmalonyl-CoA-sensitive carnitine acyltransferases because it prevents sensitivity to malonyl-CoA. In any case, it appears that Met 593 is critical in the interaction of malonyl-CoA with L-CPT I.
It was of interest to measure the kinetic constants of all CPT I mutants. Several authors reported the competition between malonyl-CoA and carnitine (27,28). The tissues in which the sensitivity of CPT I to malonyl-CoA is highest are those that require the highest concentration of carnitine to drive the reaction and the requirement for carnitine and sensitivity to malonyl-CoA appears to be inversely related. The authors concluded that the sites to which the two metabolites bind are closely associated (27,7). Studies by Bird and Saggerson (28) showed on the one hand that malonyl-CoA reduced the effectiveness of carnitine as substrate, and on the other hand, that carnitine might diminish the regulatory effect of malonyl-CoA (29). Although a clear mechanism for this competition could not be established, the data strongly supported this idea. In the present study the various CPT I mutants have altered K m or V max for carnitine. Whereas the K m for C608A was half of the wild type, its V max was 3.6-fold higher. The mutant M593S had the same K m value for carnitine as the wild type but its V max increased 20-fold. The mutant A478G increased both the K m value and the V max with respect to the wild type values. The relationship between these values and catalysis is best revealed in the term catalytic efficiency. This term as calculated by the V max /K m ratio varies considerably among different mutants. Carnitine catalytic efficiencies for mutants M593S, M593A, M593E, C608A, and A478G increased 21-, 12-, 4.2-, 8.8-, and 4.1-fold with respect to the wild type. This means that mutations designed to decrease malonyl-CoA sensitivity strongly modified the catalytic efficiency of CPT I mutants measured in the absence of malonyl-CoA. Interestingly, the increase in catalytic efficiency appears to be roughly proportional to the extent of the alteration in malonyl-CoA sensitivity. The IC 50 values for malonyl-CoA run in the same direction to the catalytic efficiency of the mutants. This indicates that those mutants that can locate carnitine better at the catalytic site might displace malonyl-CoA from its site, preventing the binding of the metabolite and thus the inhibition of CPT I.
Because L-CPT I has not been crystallized, we do not know the proximity of Met 593 to the site of carnitine binding to perform the catalytic event. However, Met 593 is very near the tripeptide TET 602-604 , which has been reported to play an important role in the accommodation of carnitine in catalysis. Cronin (30) showed that mutation of the homologous tripeptide VDN in choline acetyltransferases to TET greatly increased the catalytic efficiency of the reaction (137-fold) using carnitine as substrate. This proximity between Met 593 and TET 602-604 would explain the inverse correlation observed between the catalytic efficiency for carnitine and the IC 50 for malonyl-CoA values of the mutants assayed. A new scenario appears in the mutual interaction between carnitine and malonyl-CoA in CPT I. The domain comprised, at least, between amino acid residues 593 and 604 is probably the site of interaction between carnitine and malonyl-CoA, which exclude each other. Higher catalytic efficiencies for carnitine in the mutants are followed by decreases in the inhibitory sensitivity to malonyl-CoA.
It is equally interesting to note that all mutants tested show higher catalytic efficiency for palmitoyl-CoA as substrate than the wild type. The increase in V max /K m ranges from 2-to 3-fold. Previous work with a partially purified preparation of CPT I had indicated that the kinetics of the reaction with respect to carnitine concentration could be highly dependent on the concentration of the second substrate, palmitoyl-CoA (29). Experiments carried out by Bird and Saggerson (28) showed that in fasted animals, in which carnitine concentration was decreased, the IC 50 values for malonyl-CoA increased up to 17fold and the binding of [2-14 -C]malonyl-CoA was reduced by 35% at 50 M palmitoyl-CoA and to even lower values at increasing palmitoyl-CoA concentrations.
Only two of these mutated amino acids are located in the three-dimensional CPT I structural model. Ala 478 is one of the amino acids present in the low affinity site of malonyl-CoA interaction. This amino acid together with Pro 479 and His 483 conform a domain to which malonyl-CoA appears to bind (20). Mutation of this amino acid would explain a decrease in sensitivity to malonyl-CoA, and therefore it would also explain the increase in catalytic efficiency. On the other hand, Asn 464 is also present in the catalytic core of the structural model of CPT I (20), but its location does not permit any conclusions about a participation in the malonyl-CoA inhibitory effect. In fact it is located on the opposite site to malonyl-CoA (data not shown). Therefore, it is not surprising that its mutation from Asn 464 to Asp 464 does not alter sensitivity to the inhibitor. As a corollary of this study, we conclude that the occurrence of the five other amino acids (Ser 223 , Asp 363 , Gly 377 , Ser 490 , and Ala 505 ) at the positions, respectively, identical to those amino acids seen in CPT I may be sufficient to prevent the sensitivity to malonyl-CoA not only to carnitine acyltransferases such as CPT II and CAT but also to ChAT.
The use of either the quintuple mutant or the methionine point mutants may allow studies on the influence of these negative dominant CPT Is, which are expected to be independent of malonyl-CoA concentration in a range of tissues such as liver, muscle, and the ␤-cell, in which the metabolism of fatty acids plays important roles in ketone body synthesis, resistance to insulin, and glucose-stimulated insulin secretion, respectively. Some of these topics are the subject of current investigations in our laboratory.