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 Científicas,
Cantoblanco, E-28049 Madrid, Spain, and the ** Department of
Biochemistry and Molecular Biology, International University of
Catalonia, 08190 Sant Cugat, Spain
Received for publication, September 30, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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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 (Thr314, Asn464,
Ala478, Met593, and Cys608,
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 Met593
mutants were prepared: M593A and M593E. The catalytic efficiency (Vmax/Km) of CPT I in
mutants A478G and C608A and all Met593 mutants toward
carnitine as substrate was clearly increased. In those CPT I proteins
in which Met593 had been mutated, the malonyl-CoA
sensitivity was nearly abolished. Mutations in Ala478,
Cys608, and Thr314 to their homologous amino
acid residues in CPT II, CAT, and ChAT caused various decreases in
malonyl-CoA sensitivity. Ala478 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., Rubí, B., Clotet, J.,
Ariño, J., Valencia, A., Hegardt, F. G., Serra,
D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met593 may participate in the interaction of
malonyl-CoA in the second affinity site, whose location has not been reported.
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 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 IC50 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 [14C]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 IC50 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 Glu3, His5, or
His140 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 (Thr314, Asn464,
Ala478, Met593, and Cys608), 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 Met593 by
itself, M593S, or the quintuple mutant containing the M593S mutation,
T314S/N464D/A478G/M593S/C608A, or other Met593 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.
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).
Construction of Site-directed Mutants--
Plasmids
pYESLCPTIwt and pYESLCPTA478G were obtained as
previously described (20). Plasmids pYESLCPTT314S,
pYESLCPTN464D, pYESLCPTM593S,
pYESLCPTM593A, pYESLCPTM593E, and
pYESLCPTC608A were constructed using the QuikChange
polymerase chain reaction-based mutagenesis procedure (Stratagene) with
the pYESLCPTwt plasmid as template. The following primers
were used: primer T314S.for
5'-GGGAGCGACTCTTCAATAGTTCCCGGATCCCTGGG-3', primer
T314A.rev 5'-CCCAGGGATCCGGGAACTATTGAAGAGTCGCTCCC-3', primer
N464D.for 5'-CACCTTTGTTGTCTTCAAAGACAGCAAGATAGGC-3', primer
N464D.rev 5'-GCCTATCTTGCTGTCTTTGAAGACACCAAAGGTG-3', primer M593S.for 5'-CCTCACATATGAGGCCTCCAGTACCCGGCTCTTCCG
AGAAGG-3', primer M593S.rev
5'-CCTTCTCGGAAGAGCCGGGTACTGGAGGCCTCATATGTGAGG-3', primer M593A.for
5'-CCTCACATATGAGGCCTCCGCGACCCGGCTCTTCCGAGAAGG-3',
primer M593A.rev
5'-CCTTCTCGGAAGAGCCGGGTCGCGGAGGCCTCATA TGTGAGG-3',
primer M593E.for
5'-CCTCACATATGAGGCCTCCGAGACCCGGCTCTTCCGAGAAGG-3', primer M593E.rev 5'-CCTTCTCGGAAGAGCCGGGTCTCGGAGGCCTCATATGTGAGG-3',
primer C608A.for 5'-GAGACTGTACGCTCCGCCACTATGGAGTCCTGC-3',
and C608A.rev 5'-GCAGGACTCCATAGTGGCGGAGCGTACAGTCTC-3' (the
mutated nucleotides are underlined). The plasmid
pYESLCPTIT314S/N464D/A478G/M593S/C608A was obtained by the
same method, but performing each new mutation stepwise starting on
plasmid pYESLCPTT314S. The appropriate substitutions
as well as the absence of unwanted mutations were confirmed by
sequencing the inserts in both directions with an Applied Biosystems
373 automated DNA sequencer.
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-3H]carnitine and
palmitoyl-CoA. Enzyme activity was assayed for 4 min at 30 °C in a
total volume of 200 µl.
For determination of the Km for carnitine,
palmitoyl-CoA was fixed at 135 µM (for L-CPT I). For
determination of the Km for acyl-CoA, carnitine
concentration was fixed at 400 µM. When malonyl-CoA
inhibition was assayed, increasing concentrations of malonyl-CoA were
included. The IC50, defined as the malonyl-CoA
concentration that produces 50% inhibition of enzyme activity, was
determined using 50 µM palmitoyl-CoA and 400 µM carnitine. Km was estimated by
analyzing the data from three experiments using the program Enzifit
(Biosoft), and IC50 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 SequenceSpace (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
(Thr314, Asn464, Ala478,
Met593, and Cys608) 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: Ser223,
Asp363, Gly377, Ser490, and
Ala505 (Fig. 2).
Expression of Wild Type and Mutants in S. cerevisiae--
We
prepared a quintuple mutant, T314S/N464D/A478G/M593S/C608A, and
separately, the point mutants T314S, N464D, A478G, M593S, and C608A and
all were expressed in S. cerevisiae. After we observed that
mutant M593S nearly abolished the sensitivity to malonyl-CoA (see
below), new point Met mutants were prepared: M593A and M593E. All
transformed yeast cells expressed a protein with the same molecular
mass (88 kDa) and the mutant enzymes were expressed in roughly the same
proportion per milligram of protein as the wild type L-CPT I as deduced
from immunoblot analysis (data not shown).
Kinetic Properties of CPT I Wild Type and Mutants--
L-CPT I
activities of the wild type, quintuple mutant variant
T314S/N464D/A478G/M593S/C608S, and point mutants were similar (values
ranged between 14 and 20 nmol min
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
(Vmax/Km) 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 Vmax/Km
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 Km for
palmitoyl-CoA but the changes were smaller. Km
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
(Km 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 (IC50 of 258 versus 12.3 µM of the wild type) (Fig.
4B and Table I). Even at
concentrations as high as 100 µM 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 (IC50 of 39.5 versus 12.3 µM 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 (IC50 of 319 µM), the other mutations produced varied changes in
malonyl-CoA sensitivity. L-CPT I C608A slightly modified the
sensitivity to malonyl-CoA (IC50 is 27.5 µM),
the change in IC50 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 Km 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 Met593 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)
(IC50 values of 155 and 220 µM, respectively) as in the M593S mutant, confirming the essential role of
Met593 in this interaction.
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 amino-terminal 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
(Ala478 and Pro479) 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 Thr314,
Asn464, Ala478, Met593, and
Cys608. The corresponding positional amino acids in CPT II,
CAT, and ChAT are Ser223, Asp363,
Gly377, Ser490, and Ala505,
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 µM 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 IC50 from 12 to 39.5 µM (20). The other amino acids are less responsible for
the inhibition.
The relevance of Met593 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.
Met593, 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 Met593 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 Km or
Vmax for carnitine. Whereas the
Km for C608A was half of the wild type, its Vmax was 3.6-fold higher. The mutant M593S had
the same Km value for carnitine as the wild type but
its Vmax increased 20-fold. The mutant A478G
increased both the Km value and the Vmax 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
Vmax/Km 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 IC50 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 Met593 to the site of carnitine binding to perform the
catalytic event. However, Met593 is very near the
tripeptide TET602-604, 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 Met593 and TET602-604 would explain
the inverse correlation observed between the catalytic efficiency for
carnitine and the IC50 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 Vmax/Km
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
IC50 values for malonyl-CoA increased up to 17-fold and the binding of [2-14-C]malonyl-CoA was reduced by 35% at 50 µM 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. Ala478 is one of
the amino acids present in the low affinity site of malonyl-CoA
interaction. This amino acid together with Pro479 and
His483 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, Asn464
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 Asn464 to Asp464 does not alter sensitivity to
the inhibitor. As a corollary of this study, we conclude that the
occurrence of the five other amino acids (Ser223,
Asp363, Gly377, Ser490, and
Ala505) 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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence space analysis of the
carnitine-choline acyltransferase family. A, protein
sequences projected onto the plane defined by principle axes 2 and 4. This two-dimensional space allows separation of protein subfamilies
according to their malonyl-CoA regulatory characteristics; CPT II, CAT,
and ChAT (CACP) enzymes (malonyl-CoA insensitive) are
clustered to the lower left corner of the panel, whereas CPT
I (L- and M-isoforms) and COT (malonyl-CoA inhibitable enzymes) are
projected on the upper and right areas of the
vertical and horizontal axes, respectively. B, the sequence
of each subfamily is represented as a vector point in a
multidimensional space (sequence space), with residue positions and
types as the basic dimensions. Single residues completely conserved in
CPT I or COT subfamilies are projected in the same position as their
corresponding protein sequences. Residues conserved in both groups of
malonyl-CoA-regulated enzymes occupy the upper right corner,
whereas the residues conserved in the nonregulated cluster of
acyltransferases (CPT II, CAT, and ChAT) occupy the opposite
one. Residues located in alignment positions present in both opposite
corners of the two-dimensional plot are responsible for protein cluster
segregation and are predicted to be involved in malonyl-CoA
sensitivity.
View larger version (50K):
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Fig. 2.
Alignment of representative sequences of
mammalian carnitine-choline acyltransferases. Amino acid sequence
of 18 representative members of the malonyl-CoA-insensitive enzymes,
CPT II (CPT2) from rat, mouse, and human; CAT (CACP) from
human and mouse; ChAT (CLAT) from human, pig, rat, and
mouse; and malonyl-CoA inhibitable enzymes L-CPT I (CPT1) from rat,
mouse, and human; M-CPT I (CPTM) from human, rat, and mouse; and COT
(OCTC) from human, rat, and bovine, were obtained from the
SwissProt data bank and aligned using ClustalW (22). A,
schematic representation of the position of the tree-determinant
residues obtained using the SequenceSpace algorithm (23, 24) on the rat
CPT II and L-CPTI proteins: Ser223/Thr314,
Asp363/Asn464,
Gly377/Ala478,
Ser490/Met593,
Ala505/Cys608. Transmembrane regions of L-CPT I
are also represented (tm1 and tm2). Position of
the catalytic histidine (His372/His473) as well
as the previously three-dimensional modeled core of the proteins, 2dub,
(amino acids 368-567 of L-CPT I) (19, 20), are indicated.
B, selected regions of the multiple alignment of the protein
family. Subfamily conserved residues according to malonyl-CoA
regulation are shadowed. Position of catalytic histidine
(arrowhead) is also indicated.
1 mg
protein
1) when the protein was overexpressed 20 h
after galactose induction, showing that the various mutations assayed
produce small changes in L-CPT I activity (Table
I).
Enzyme activity, malonyl-CoA sensitivity and kinetic parameters of
carnitine palmitoyltransferase I in Saccharomyces cerevisiac cells
expressing CPI I wild type and point mutants, T314S, N464D, A478G,
C608A, M593S, M593A, M593E and quintuple mutant
T314S/N464D/A478G/M593S/C608A
(QM)
View larger version (28K):
[in a new window]
Fig. 3.
Kinetic analysis of wild type and different
mutants of L-CPT I. Yeast extracts (10 µg of protein) of
(A and C) wild type (open circles) and
mutants M593S (open triangles), M593A (black
rhombus), M593E (black squares), and (C and
D) T314S (open rhombus), N464D (open
squares), A478G (black squares), C608A (black
triangles), and quintuple mutant T314S/N464D/A478G/M593S/C608A
(black circles) were incubated at increasing concentrations
of carnitine (A and B) and palmitoyl-CoA
(C and D).
View larger version (19K):
[in a new window]
Fig. 4.
Effect of malonyl-CoA on the activity of
yeast overexpressed L-CPT I (wild type) and several mutants.
A, L-CPT I wild type (open circles) and point
methionine mutants M593S (black circles), M593A (black
rhombus), M593E (black squares), and B,
quintuple mutant (QM) (black circles) and point
mutants T314S (open circles), N464D (open
rhombus, broken line), A478G (open
triangles, broken line), and C608A (open
squares) overexpressed in yeast were incubated with increasing
concentrations of malonyl-CoA and the enzyme activity was measured.
Data are expressed relative to control values in the absence of
inhibitor (100%) as the mean of three independent measurements.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
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ACKNOWLEDGEMENT |
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We are grateful to Robin Rycroft of the Language Service for valuable assistance in the preparation of the manuscript.
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FOOTNOTES |
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* This work was supported in part by Dirección General de Investigación Científica y Técnica, Spain, Grant BMC2001-3048 and Ajuts de Suport als Grups de Recerca de Catalunya Grant 2001SGR-00129 (to F. G. H.) and the Marató de TV3.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Contributed equally to the results of this study.
Recipient of a fellowship from the Ministerio de Ciencia y
Tecnología, Spain.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, School of Pharmacy, Diagonal 643, E-08028 Barcelona, Spain. Tel.: 34-93-402-4523; Fax: 34-93-402-4520; E-mail: hegardt@farmacia.far.ub.es.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M209999200
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ABBREVIATIONS |
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The abbreviations used are: CPT, carnitine palmitoyltransferase; L-CPT I, liver isoform of carnitine palmitoyltransferase I; M-CPT I, muscle isoform of carnitine palmitoyltransferase I; CAT, carnitine acetyltransferase; COT, carnitine octanoyltransferase; ChAT, choline acetyltransferase.
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REFERENCES |
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