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INTRODUCTION |
Carnitine palmitoyltransferase I
(CPTI)1 catalyzes the
conversion of long chain acyl-CoA to acylcarnitines in the presence of
L-carnitine, the first reaction in the transport of long
chain fatty acids from the cytoplasm to the mitochondria, a
rate-limiting step in
-oxidation (1, 2). Mammalian tissues express
two isoforms of CPTI, a liver isoform (L-CPTI) and a heart/skeletal muscle isoform (M-CPTI), that are 62% identical in amino acid sequence
(GenBankTM accession number U62317; Refs. 3-7 and 9). As
an enzyme that catalyzes the first rate-limiting step in fatty acid
oxidation, CPTI is regulated by its physiological inhibitor malonyl-CoA
(1, 2), the first intermediate in fatty acid synthesis, suggesting coordinated control of fatty acid oxidation and synthesis.
Understanding the molecular mechanism of the regulation of CPTI by
malonyl-CoA is important in the design of drugs for control of
excessive fatty acid oxidation in diabetes mellitus (10) and in
myocardial ischemia where accumulation of acylcarnitines has been
associated with arrhythmias (11).
We developed a novel high level expression system for rat L-CPTI and
human heart M-CPTI in the yeast Pichia pastoris, an organism devoid of endogenous CPT activity (6, 12, 13). Using this system, we
demonstrated conclusively that L-CPTI and M-CPTI are active, distinct,
malonyl-CoA-sensitive CPTIs that are reversibly inactivated by
detergents. We recently showed that deletion of the conserved first 18 N-terminal amino acid residues of rat L-CPTI abolishes malonyl-CoA
inhibition and high affinity malonyl-CoA binding (14). In this study,
we have constructed and characterized rat L-CPTI deletion mutants of
the first 12 and 6 N-terminal amino acid residues. To identify specific
residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we
also constructed three substitution mutations within the conserved
first 6 N-terminal amino acid residues (Glu3
Ala,
His5
Ala, and Gln6
Ala).
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EXPERIMENTAL PROCEDURES |
Construction of Plasmids for the N-terminal Point and Deletion
Mutants of Rat L-CPTI--
The cDNA used and the construction of
the plasmids for the wild type, deletion, and point mutants for rat
L-CPTI expression in P. pastoris was as described in our
previous publications (12, 14). A HindIII-KpnI
fragment (1-566 base pair; cDNA sequence; Refs. 12 and 15) was
excised from pYGW9, a plasmid containing the full-length rat L-CPTI in
pUC119 to generate the plasmid pYGW12. The
12 mutant was constructed
by polymerase chain reaction amplification of a 662-base pair
HindIII-EcoRI fragment using the plasmid pYGW9 as
a template with the primers RL665 (5'-CCACCAGGATTTTAGCT-3') and RLD12
(5'-CAAGCTAAGCTTGAATTCATGACTGTCACCCCCGATGGCAT-3'). HindIII and EcoRI enzyme restriction sites were
introduced in primer RLD12. An ATG start codon (shown in bold type) was
added immediately after the EcoRI site and before the fourth
amino acid alanine. The polymerase chain reaction product was digested
with HindIII and KpnI and then ligated into
pYGW12 to generate plasmid pYGWD12. An EcoRI fragment of
pYGWD12 containing the mutant
12 was then ligated into the
EcoRI-cut P. pastoris expression vector pHW010
(12, 16). The DNA sequences of the deletion and point mutants were
confirmed by sequencing.
Mutants
6 and
18 and point mutants Glu3
Ala,
His5
Ala, and Gln6
Ala were constructed
in a similar manner as
12, using primer RL665 above and the
following primers for each deletion and point mutant. The new
translation start site or mutated amino acid codon is shown in
bold: RLD6, 5'-GCGATGAAGCTTGAATTCATGGCTGTGGCCTTCCAGTTC-3'; RLD18, 5'-CTTCACAAGCTTGAATTCATGATTGACCTCCGCCTGAGC-3'; RLE3A, 5'-CCCAAGCTTGAATTCATGGCAGCGGCTCACCAAGCTGTGGC-3'; RLH5A, 5'-CCCAAGCTTGAATTCATGGCAGAGGCTGCCCAAGCTGTGGCCTTC-3'; and RLQ6A,
5'-CCCAAGCTTGAATTCATGGCAGAGGCTCACGCAGCTGTGGCCTTCCAGTT-3'. All subsequent procedures were identical to those used for
construction of
12.
The expression plasmids were linearized and integrated into the HIS4
locus of P. pastoris strain GS115 by electrotransformation (17). Histidine prototrophic transformants were selected on YND plates
and grown on YND medium. Mitochondria were isolated from the wild type
and mutant L-CPTIs as described previously (12).
CPT Assay--
CPT activity was assayed by the forward exchange
method using L-[3H]carnitine as described
previously (12, 18). The Km for palmitoyl-CoA was
determined by varying the palmitoyl-CoA concentration in the presence
of a fixed albumin concentration (1%) or a fixed molar ratio (6.1:1)
of palmitoyl-CoA/albumin (19, 20). The Ki for
malonyl-CoA inhibition of the yeast-expressed wild type and mutant
(E3A) L-CPTIs was determined by assaying CPT activity with varying
concentrations of palmitoyl-CoA (12.5, 25, 50, 75, and 100 µM) in the presence of 0, 1, 2, and 5 µM
malonyl-CoA (wild type), or 0, 2, 25, and 200 µM
malonyl-CoA (E3A), respectively. The concentration of the second
substrate, carnitine, was fixed at 200 µM in all the
assays. 106 µg (wild type) and 118 µg (E3A) of mitochondrial
protein were used, and all incubations were performed at 30 °C for 3 min.
[14C]Malonyl-CoA Binding
Assay--
[14C]Malonyl-CoA binding was determined by a
modified centrifugation assay as described previously (14, 21).
Isolated mitochondria from wild type and mutants were suspended in 0.5 ml of ice-cold medium composed of 72 mM sorbitol, 60 mM KCl, 25 mM Tris/HCl (pH 6.8), 1.0 mM EDTA, 1.0 mM dithiothreitol, and 1.3 mg/ml
fatty acid-free bovine serum albumin (22). This was followed by
addition of 0.1-1000 nM [2-14C]malonyl-CoA,
and the suspension was incubated at 4 °C for 30 min with periodic
vortexing. All subsequent procedures were as described previously (14).
The CPT activity and C50 values are given as the means ± S.D. for at least three independent assays with different
preparations of mitochondria. The KD values are
averages of at least two independent experiments.
Western Blot Analysis--
Proteins were separated by
SDS-polyacrylamide gel electrophoresis in a 7.5% gel and transferred
onto nitrocellulose membranes. Immunoblots were developed by incubation
with the CPTI-specific polyclonal antibody (1:4000 dilution) followed
by an anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000
dilution) as described previously (12). The antigen-antibody complex
was detected using an ECL-enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).
Other Materials and Procedures--
DNA sequencing was performed
at the Oregon Regional Primate Research Center core facility using an
automatic DNA sequencer (23). Protein was determined by the Lowry
procedure (24). All restriction enzymes were from New England Biolabs
(Beverly, MA). L-[3H]Carnitine and
[2-14C]malonyl-coenzyme A were from Amersham Pharmacia
Biotech. Nucleotides were from Amersham Pharmacia Biotech,
palmitoyl-CoA was from Boehringer Mannheim, and malonyl-CoA was from Sigma.
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RESULTS |
Generation of Deletion and Point Mutants and Expression in P. pastoris--
Construction of plasmids carrying the N-terminal
deletions
6 and
12 and point mutants Glu3
Ala,
His5
Ala, and Gln6
Ala of rat L-CPTI
(Fig. 1) was performed as described under "Experimental Procedures." Mutations were confirmed by DNA
sequencing. P. pastoris was chosen as an expression system
for L-CPTI and the mutants, because it does not have endogenous CPT
activity (6, 12-14). The P. pastoris expression plasmids
expressed L-CPTI under control of the P. pastoris
glyceraldehyde-3-phosphate dehydrogenase gene promoter (12, 16). Yeast
transformants with the wild type L-CPTI gene and the mutants were grown
in liquid medium supplemented with glucose. As previously reported
(12), no CPT activity was found in the control yeast strain with the
vector but without the CPTI cDNA insert.

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Fig. 1.
The amino acid sequence of the first 25 N-terminal residues of rat L-CPTI. The position of each of the
deletion and point mutants is shown by an arrow. Sources of
the sequences from the data bank were from Refs. 12 and 15 as indicated
in the text.
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Western blot analysis of wild type L-CPTI (88 kDa) and the mutants
using a polyclonal antibody directed against a maltose-binding protein-L-CPTI fusion protein (12) is shown in Fig.
2. For the wild type and all the deletion
and point mutants (
18,
12,
6, Glu3
Ala,
His5
Ala, and Gln6
Ala), proteins of
predicted sizes were synthesized and were expressed at similar
steady-state levels.

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Fig. 2.
Immunoblot showing expression of wild type,
deletion, and point mutant L-CPTIs in the yeast P. pastoris. Mitochondria (40 µg of protein) from the
wild type yeast strain and the strains expressing each of the deletion
and the point mutants were separated on a 7.5% SDS-polyacrylamide gel
electrophoresis and blotted onto a nitrocellulose membrane; the
immunoblot was developed as described under "Experimental
Procedures." Lane 1, wild type L-CPTI; lanes
2-6, 12, 6, Glu3 Ala, His5 Ala, and Gln6 Ala, respectively.
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Effect of Mutations on L-CPTI Activity and Malonyl-CoA
Inhibition--
All of the mutants retained significant CPT activity,
which was 60-80% of that observed with the wild type yeast strain
expressing L-CPTI (Table I). The
IC50 for malonyl-CoA inhibition of the wild type strain
expressing L-CPTI was 2.0 µM, in agreement with our
previous report (12), whereas the IC50 for
6,
12, and the point mutant Glu3
Ala was 200 µM,
representing a 100-fold decrease in malonyl-CoA sensitivity compared
with the wild type strain (Table I). The Ki for
malonyl-CoA inhibition of the Glu3
Ala mutant L-CPTI
was approximately 10-fold higher than that of the wild type (32 µM versus 2.7 µM), a trend
similar to the increase in IC50 for malonyl-CoA inhibition
observed in the Glu3
Ala mutant. Mutation of histidine
residue 5 to alanine increased the IC50 for malonyl-CoA
inhibition to 25 µM compared with the 2.0 µM value of the wild type strain, representing a mild
12.5-fold decrease in malonyl-CoA sensitivity, whereas mutation of
glutamine residue 6 to alanine had no effect on malonyl-CoA sensitivity (Table I).
6 and point mutant Glu3
Ala showed
decreased malonyl-CoA sensitivity at all levels of the inhibitor tested
compared with the wild type (Fig. 3), whereas only minor reduction in malonyl-CoA sensitivity was observed in
the point mutant His5
Ala compared with the
control.
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Table I
Activity, malonyl-CoA sensitivity, and malonyl-CoA binding in yeast
strains expressing wild type L-CPTI, N-terminal deletion, and point
mutants
Mitochondria were isolated from the yeast strains separately expressing
L-CPTI, and the deletion and point mutants were assayed for CPT
activity, malonyl-CoA sensitivity, and binding, as described under
"Experimental Procedures." The IC50 is the
concentration of malonyl-CoA needed to inhibit 50% of the activity of
the yeast-expressed L-CPTI, and the results are the means ± S.D.
of at least three independent experiments with different mitochondrial
preparations. The KD and Bmax
values are averages of two independent experiments with different
mitochondrial preparations.
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Fig. 3.
Effect of increasing concentrations of
malonyl-CoA on the activities of yeast-expressed wild type and mutant
L-CPTI. Approximately 150 µg of mitochondrial protein were used
for the assay. , wild type; , 6; , Glu3 Ala; , His5 Ala.
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Kinetic Properties of Wild type and Mutant L-CPTIs--
The
Glu3
Ala mutant exhibited normal saturation kinetics
when the carnitine concentration was varied relative to a constant second substrate, palmitoyl-CoA, a property identical to that of wild
type L-CPTI (Fig. 4A) and
similar to that previously reported from our laboratory (12). For the
Glu3
Ala mutant, the calculated Km
for carnitine was 74.5 µM and the
Vmax was 8.3 nmol/min/mg protein, which is
similar to the 45 µM and 12.6 nmol/min/mg for wild type
L-CPTI (12). With respect to the second substrate, palmitoyl-CoA, the
wild type, but not the Glu3
Ala mutant, showed
non-Michaelis-Menten saturation kinetics at a fixed concentration of
albumin (1% W/V) (Fig. 4B), characteristics similar to our
previous report (12). However, both the wild type and the
Glu3
Ala mutant exhibited normal saturation kinetics
when the molar ratio of palmitoyl-CoA:albumin was fixed at 6.1:1 (Fig.
4C). The calculated Km for palmitoyl-CoA
for the point mutant Glu3
Ala was 69.4 µM
and the Vmax was 23.3 nmol/min/mg, which is similar to the wild type values of 104 µM and 41.5 nmol/min/mg. Thus, deletion of the first 6 N-terminal amino acid
residues or a substitution mutation of Glu residue 3 to Ala in L-CPTI
abolishes malonyl-CoA sensitivity, but not catalytic activity. Mutation of glutamine residue 6 to alanine had no effect on malonyl-CoA inhibition or catalytic activity.

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Fig. 4.
Kinetic analysis of wild type and
Glu3 Ala mutant L-CPTI activities. Isolated
mitochondria (150 µg protein) from the yeast strains expressing the
wild type ( ) and the Glu3 Ala ( ) mutant L-CPTI
were assayed for CPTI activity in the presence of increasing
concentrations of carnitine and palmitoyl-CoA as described under
"Experimental Procedures." The figure shows the resulting
dose-response curves for L-CPTI. A, carnitine. B,
palmitoyl-CoA with fixed albumin concentration (1% W/V). C,
palmitoyl-CoA with fixed molar ratio of palmitoyl-CoA:albumin
(6.1:1).
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[14C]Malonyl-CoA Binding in Yeast-expressed Mutant
LCPTIs--
Malonyl-CoA binding to the mitochondria from the yeast
strains expressing
6 and point mutants Glu3
Ala and
His5
Ala was significantly lower compared with that
observed in the mitochondria from the wild type strain but was
saturable (Fig. 5). Malonyl-CoA binding
clearly resolved into a high affinity and a low affinity site in the
mitochondria from the wild type and point mutant His5
Ala as shown by the Scatchard plots in Fig.
6 (A and B), but only very low affinity binding was observed in the mitochondria from
point mutant Glu3
Ala and
6 (Fig. 6B).
Deletion of the first 6 N-terminal residues or substitution of glutamic
acid 3 with alanine completely abolished high affinity malonyl-CoA
binding (KD1) and further decreased the
low affinity malonyl-CoA binding (KD2)
by 100-fold (Table I). Furthermore, the loss of high affinity
malonyl-CoA binding observed with the Glu3
Ala mutant
correlates with the increase in IC50 and
Ki for inhibition of L-CPTI by malonyl-CoA. Although
the mutations increased the KD2 for the
low affinity malonyl-CoA-binding site (
6 and Glu3
Ala), there was no change in the calculated
Bmax2 (which was 10-fold higher than the
Bmax1 of the wild type), suggesting that the
observed loss in malonyl-CoA sensitivity and binding is not due to
decreased abundance of the second malonyl-CoA-binding entity of
L-CPTI.

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Fig. 5.
Binding of [14C]malonyl-CoA to
mitochondria isolated from the yeast strain expressing the wild type
L-CPTI, 6, and point mutants Glu3
Ala and His5 Ala. Approximately 200 µg of
protein were used for the binding assay. Malonyl-CoA binding values for
the wild type and deletion mutants were corrected for malonyl-CoA
binding to the mitochondria from the yeast strain with the vector but
no insert. , wild type; , 6; , Glu3 Ala;
, His5 Ala.
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Fig. 6.
A, Scatchard plot for binding of
[14C]malonyl-CoA to mitochondria from yeast strains
expressing wild type L-CPTI. B, Scatchard plot for binding
of [14C]malonyl-CoA to mitochondria from yeast strains
expressing mutant L-CPTIs 6 ( ), Glu3 Ala ( ),
and His5 Ala ( ).
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Substitution of histidine 5 with alanine showed a moderate loss in
malonyl-CoA sensitivity of L-CPTI (12.5-fold), resulting in loss of the
high affinity and a significant decrease in low affinity malonyl-CoA
binding (Table I). The low affinity binding site in mutant
His5
Ala resolved into two classes of binding sites
(Fig. 6B), with the KD1 and
KD2 for the mutant being 100- and 16-fold higher, respectively, than the corresponding wild type values
(Table I). The calculated Bmax1 for mutant
His5
Ala was close to the wild type value, but
Bmax2 was 3-fold lower, indicating a decrease in
the malonyl-CoA-binding entity of L-CPTI. These studies suggest that
histidine 5 in L-CPTI is one of the residues involved in malonyl-CoA
binding and inhibition of the enzyme.
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DISCUSSION |
To determine the role of the first 130 N-terminal amino acid
residues of rat L-CPTI in malonyl-CoA sensitivity and binding, we
previously constructed a series of deletion mutants and demonstrated that a mutant lacking the first conserved 18 N-terminal amino acid
residues had activity and kinetic properties similar to those of wild
type L-CPTI but had completely lost malonyl-CoA sensitivity and high
affinity binding (14). Based on these previous studies (14), we report
here on deletion mutations of the conserved first 12 and 6 N-terminal
residues of L-CPTI. Like
18,
12 and
6 had 60-70% of the wild
type activity and showed loss of both malonyl-CoA sensitivity and high
affinity malonyl-CoA binding, indicating that residue(s) essential for
malonyl-CoA binding and sensitivity reside within the conserved first 6 N-terminal amino acids. Of these conserved first 6 N-terminal amino
acids, including the start codon Met, residues 2 and 4 are Ala, residue
3 is Glu, residue 5 is His, and residue 6 is Gln. Therefore, we
constructed mutants with substitutions of Glu3 with Ala,
His5 with Ala, and Gln6 with Ala of L-CPTI.
The mutant L-CPTI with a replacement of Glu3 with Ala had a
phenotype similar to that of the N-terminal deletion mutants. The mutation resulted in complete loss of malonyl-CoA sensitivity and high
affinity malonyl-CoA binding and a decrease in the low affinity
malonyl-CoA binding. In contrast, substitution of Glu3 with
Ala did not have a significant effect on the kinetic properties of the
enzyme, because there was no change in the Km value
for palmitoyl-CoA and only a slight increase in the
Km value for carnitine. The 29-40% loss in
catalytic activity observed with the deletion and point mutants
compared with the wild type could be due to a reduction in the
expression level or lack of interaction of the N-terminal domain with
the catalytic domain as a result of the N-terminal mutations. A protein
of the expected size (88 kDa) was detected in the mitochondria of the
Glu3
Ala mutant strain on immunoblotting with L-CPTI
specific antibodies. These results demonstrate clearly that
Glu3 in the wild type L-CPTI is essential for malonyl-CoA
inhibition and binding but not for catalysis, because the kinetic
properties of the mutant enzyme are virtually indistinguishable from
those of the wild type. This is the first report to demonstrate the critical role of Glu3 residue of L-CPTI for malonyl-CoA
sensitivity and binding.
The high affinity site (KD1,
Bmax1) for binding of malonyl-CoA to L-CPTI was
completely abolished in the Glu3
Ala,
6, and
18
mutants, suggesting that the >100-fold decrease in malonyl-CoA
sensitivity observed in these mutants was due to the loss of the high
affinity binding entity of the enzyme. Although low affinity
malonyl-CoA binding was weakened, there was no change in the
Bmax2 value between wild type L-CPTI and mutants
Glu3
Ala,
6, and
18, suggesting that the residual
malonyl-CoA sensitivity observed in the mutants was due to the low
affinity malonyl-CoA-binding entity of the enzyme. The results of this study provide strong evidence implicating Glu3 as one of
the residues involved in high affinity malonyl-CoA binding. We
hypothesize that the Glu3
Ala substitution may disrupt
a hydrogen bonding network or a salt bridge, perhaps to a residue near
the active site of CPTI. As the high affinity site is abolished and
binding to the low affinity site is weakened, the two sites may
partially overlap. Alternatively, the possible loss of a salt bridge
may weaken KD2 indirectly.
Replacement of His5 with Ala had a much less drastic effect
on the IC50 for malonyl-CoA inhibition of L-CPTI but
severely diminished both high and low affinity malonyl-CoA binding. The
Bmax1 for this mutant showed a slight increase,
but Bmax2 showed a significant decrease compared
with the wild type value, suggesting that the 10-fold lower
IC50 for malonyl-CoA inhibition observed with this mutant,
compared with mutants Glu3
Ala,
6, and
18, may be
due to a slight increase in abundance of the high affinity binding
entity with a lowered (100-fold) affinity for malonyl-CoA. The decrease
in low affinity malonyl-CoA binding observed for the His5
Ala mutant (~15-fold increase in K
D2) may be due, in part, to the
decreased abundance of the low affinity binding entity of the enzyme
(~3-fold decrease in Bmax2). Because mutation of His5
Ala reduced the malonyl-CoA sensitivity and
binding, L-CPTI may be affected by pH. A pH-induced shift in
malonyl-CoA sensitivity has been reported for CPTI (25, 26).
Our data clearly demonstrate that there are two classes of
malonyl-CoA-binding sites in L-CPTI, namely, a high affinity and a low
affinity binding site, similar to earlier studies in isolated rat liver
and heart mitochondria (26, 27). A previous attempt to express a mutant
L-CPTI that lacked the first 82 N-terminal residues was described by
Brown et al. (28), but results were inconclusive due to
extremely low expression levels (12). The residual malonyl-CoA
sensitivity shown by the deletion mutants is similar to that observed
with yeast-expressed CPTII (12), suggesting that for these mutants
malonyl-CoA may inhibit via direct interaction with the active site.
Additional studies are needed to determine whether the active site acts
as a low affinity malonyl-CoA-binding site, but our data suggest that
there may be some overlap between the malonyl-CoA and palmitoyl-CoA
binding sites. In the absence of malonyl-CoA, free CoA (50 µM) and acetyl-CoA (500 µM) inhibited the
activities of both the wild type and the Glu3
Ala
mutant L-CPTI by 50%.2
Because a total loss of the high affinity malonyl-CoA binding site was
observed in the Glu3
Ala mutant, the results suggest
that CoA and acetyl-CoA inhibit by binding to the active site or the
low affinity malonyl-CoA-binding site. At high concentrations, both CoA
and the substrate palmitoyl-CoA reduce the inhibition of L-CPTI by
malonyl-CoA (18, 29), suggesting partial overlap between the
malonyl-CoA and the substrate binding sites.
Based on limited proteolysis studies of intact and outer membrane rat
liver mitochondria, a model for the membrane topology of L-CPTI has
been proposed that predicts exposure of 90% of L-CPTI, including N and
C termini domains crucial for activity and malonyl-CoA sensitivity of
the enzyme on the cytosolic side of the outer mitochondrial membrane
(30). A more recent detailed deletion mutation analysis study of the
129 N-terminal amino acid residues of the yeast-expressed L-CPTI from
our laboratory clearly demonstrated that residues critical for
malonyl-CoA inhibition and binding of L-CPTI are located within the
conserved first 18 N-terminal amino acid residues of the enzyme (14).
In this study, we demonstrate that glutamic acid residue 3 and
histidine 5 are essential for malonyl-CoA binding and inhibition.
Limited proteolysis of intact and outer membrane preparations of rat
liver mitochondria result in a marked loss in L-CPTI activity and
malonyl-CoA sensitivity (30, 31), accompanied by the cleavage of the
extreme N terminus (<1 kDa) of L-CPTI (30). Mitochondria isolated from
fasted and diabetic rat livers, metabolic conditions with increased
fatty acid oxidation, exhibit increased L-CPTI activity and decreased
malonyl-CoA sensitivity (32). Furthermore, insulin reverses the effects
of diabetes on L-CPTI activity and malonyl-CoA sensitivity (32). Thus,
fasting and diabetes, metabolic conditions that enhance protein
degradation, reduce the sensitivity of CPTI to malonyl-CoA inhibition
(32-34). The L-CPTI gene in INS-1 cells may be an early response gene
like c-fos (35), suggesting that the enzyme may be subject
to metabolic regulation by proteolysis (36), employing the cytosolic
ubiquitin-proteasome system (36). Diabetes is a pathophysiologic
condition associated with increased protein degradation, fatty acid
oxidation, CPTI activity, and decreased malonyl-CoA inhibition of CPTI
(33). Thus limited in vivo proteolysis of L-CPTI induced by
diabetes, such as cleavage of the first 6 N-terminal residues, may
decrease malonyl-CoA sensitivity and alter the normal control of
hepatic fatty acid oxidation. Insulin inhibits proteasome activity
resulting in decreased cellular protein degradation and controlled
fatty acid oxidation (8). We are currently conducting in
vitro partial proteolysis studies with yeast-expressed L-CPTI to
determine the role of protein degradation on malonyl-CoA sensitivity.