From the Department of Biochemistry, Louisiana State University,
Baton Rouge, Louisiana 70803
Acetyl-CoA carboxylase catalyzes the
first committed step in the biosynthesis of fatty acids. The
Escherichia coli form of the enzyme consists of a biotin
carboxylase protein, a biotin carboxyl carrier protein, and a
carboxyltransferase protein. In this report the overexpression of the
genes for the carboxyltransferase component is described. The
steady-state kinetics of the recombinant carboxyltransferase are
characterized in the reverse direction, in which malonyl-CoA reacts
with biocytin to form acetyl-CoA and carboxybiocytin. The initial
velocity patterns indicated that the kinetic mechanism is
equilibrium-ordered with malonyl-CoA binding before biocytin and the
binding of malonyl-CoA to carboxyltransferase at equilibrium. The
biotin analogs, desthiobiotin and 2-imidazolidone, inhibited
carboxyltransferase. Both analogs exhibited parabolic noncompetitive
inhibition, which means that two molecules of inhibitor bind to the
enzyme. The pH dependence for both the maximum velocity (V) and the (V/K)biocytin
parameters decreased at low pH. A single ionizing group on the enzyme
with a pK of 6.2 or lower in the (V/K)biocytin profile and 7.5 in the
V profile must be unprotonated for catalysis.
Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that
a thiol in or near the active site is needed for catalysis. The rate of
inactivation of carboxyltransferase by
N-ethylmaleimide decreased with decreasing pH and
indicated that the pK of the sulfhydryl group had a
pK value of 7.3. It is proposed that the thiolate ion of a
cysteine acts as a catalytic base to remove the N1' proton of
biocytin.
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INTRODUCTION |
The first committed step in fatty acid biosynthesis is catalyzed
by acetyl-CoA carboxylase (1). The enzyme is found in all animals,
plants, and bacteria and catalyzes the biotin-dependent carboxylation of acetyl-CoA to form malonyl-CoA in the two steps of
Reaction 1.
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(Step 1)
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(Step 2)
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In Escherichia coli the enzyme is composed of three
components: biotin carboxylase, carboxyltransferase, and biotin
carboxyl carrier protein, which contains the biotin covalently attached to the
-nitrogen of lysine (2). Biotin carboxylase catalyzes the
first half-reaction in which biotin is carboxylated to form carboxybiotin. Carboxyltransferase catalyzes the second half-reaction where the carboxyl group is transferred from biotin to acetyl-CoA to
form malonyl-CoA. Both biotin carboxylase and the carrier protein are
homodimers, whereas the carboxyltransferase component is an
2
2 tetramer. Each of the three components
can be isolated separately, and the biotin carboxylase and
carboxyltransferase components retain catalytic activity in the absence
of the other two components. Moreover, biotin carboxylase and
carboxyltransferase will utilize free biotin as a substrate, which
makes them ideal model systems for studying mechanistic aspects of
biotin-dependent enzymes.
Most mechanistic studies of acetyl-CoA carboxylase have focused on the
biotin carboxylase component because the gene for the enzyme has been
cloned and overexpressed (3), and a three-dimensional structure of
biotin carboxylase has been determined by x-ray crystallography (4). In
contrast, virtually no mechanistic studies have been done on
carboxyltransferase for more than 20 years because despite cloning of
the genes for carboxyltransferase (5-7), overexpression of the genes
has not been accomplished. As a result, it has been difficult to obtain
sufficient amounts of protein for kinetic, structural, and
site-directed mutagenesis studies. In this report we describe the
overexpression of the genes for carboxyltransferase, purification, and
kinetic characterization of the recombinant enzyme including the
kinetic mechanism, inhibition and chemical modification studies, and
the pH dependence of the reaction. Structural and mechanistic studies
of this enzyme may lend insight into the design of better inhibitors
because the carboxyltransferase component is the site of action in
plant acetyl-CoA carboxylase for several herbicides (8) and in
mammalian acetyl-CoA carboxylase for anti-hyperlipidemic agents
(9).
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MATERIALS AND METHODS |
E. coli strain JM109 and plasmid pGem-11zf were
purchased from Amersham Promega Biotech. The expression vector, pET14b,
and the host strain, E. coli BL21(DE3)pLysS, were from
Novagen. The Wizard kit for plasmid isolation and purification was from
Promega. All restriction enzymes, dNTPs, deep vent polymerase, and T4
DNA ligase were purchased from New England Biolabs. Primers were
synthesized by Life Technologies Inc. A Sequenase Quick-Denature
plasmid sequencing kit with sequenase version 2.0 was from Amersham
Promega Biotech. His-binding resin, columns, and protocol were from
Novagen. All other reagents were from Sigma.
Construction of Expression Vectors--
The steps used to
construct the expression vectors pCZB4 and pCZB5 are summarized in Fig.
1. The gene for the
subunit of carboxyltransferase, designated accA, was contained on
pLS181, which was obtained from Dr. John Cronan, University of
Illinois. The gene for the
subunit of carboxyltransferase,
designated accD, was contained on pCS, which was obtained
from Dr. Dean Tolan, Boston University.
The accA gene was amplified by the polymerase chain reaction
using the primers (5'-CCG CTC GAG TTC TCC TTA CGC GTA ACC GTA GCT CAT
CAG GCG) and (5'-GGA ATT CCA TAT GAG TCT GAA TTT CCT TGA TTT TGA A),
which incorporated the restriction sites EcoRI,
NdeI and XhoI and a ribosome binding site into
the 959-base pair amplified fragment. The accD gene was
amplified using the primers (5'-CGG GAT CCT CAG GCC TCA GGT TCC TGA TCC
GGT AC-3') and (5'-CCG CTC GAG ATG AGC TGG ATT GAA CGA ATT AAA AGC-3'),
which incorporated the restriction sites XhoI and
BamHI into the 899-base pair amplified fragment. Polymerase
chain reaction cycling conditions were as follows: melting, 1 min at
94 °C; annealing, 1 min at 55 °C; and extension, 1.5 min at
72 °C for 40 cycles.
The amplified DNA fragments were digested with EcoRI and
XhoI in the case of accA and XhoI and
BamHI in the case of accD using conditions
recommended by the enzyme supplier (New England Biolabs). The
accA and accD fragments were then ligated to
similarly digested pGem-11zf to generate plasmids pCZB2 and pCZB1,
respectively. pCZB1 was digested with the restriction enzymes
XhoI and BamHI, and the 0.9-kilobase fragment
containing accD was isolated by DNA gel electrophoresis. The
0.9-kilobase fragment containing accD was then ligated to
similarly digested pCZB2 resulting in a new plasmid, pCZB3, containing
both accA and accD. accA and accD were subcloned out of pCZB3 and into pET22b and pET14b
with the restriction enzymes NdeI and BamHI to
produce the vectors pCZB4 and pCZB5, respectively. pCZB5 contains a
His-tag sequence that allows for expression of carboxyltransferase with
a 20-amino acid His-tag (Novagen) fused to the amino terminus of the
subunit. pCZB4 and pCZB5 were transformed into BL21(DE3)pLysS,
which contains a chromosomal copy of the T7 RNA polymerase gene under
the control of the lacUV5 promoter.
Growth Conditions for Overexpression--
Bacteria were grown in
LB medium supplemented with 100 µg/ml ampicillin. A fresh overnight
culture from a single colony was used to inoculate 0.5 liter of medium
(50 ml in 10 125-ml Erlenmeyer flasks). Attempts to do growths in
2-liter flasks resulted in an increase in insoluble protein. The
cultures were grown at 37 °C until A600
reached 0.60-0.70, then lactose was added to a final concentration of
28 mM. The temperature was decreased to 30 °C upon
induction, and the cultures were incubated an additional 2.5 h.
The cells were harvested by centrifugation at 8,000 × g for 10 min at 4 °C.
Purification--
Cell paste from 0.5 liter of culture was
suspended in 20 ml of binding buffer (50 mM imidazole, 500 mM NaCl, 20 mM Tris-Cl, pH 7.9) and lysed by
the freeze-thaw method. His-binding resin was used for rapid one-step
affinity purification of carboxyltransferase containing the His-tag
sequence. The protocol recommended by Novagen for His-tag protein
purification was followed except that carboxyltransferase was eluted
from the 2.5-ml His-tag columns with a solution of 170 mM
imidazole, 500 mM NaCl, 120 mM Tris-Cl, pH 7.9. The protein solution was dialyzed overnight against 0.67 mM
EDTA, 10 mM KHPO4, pH 7.0, and then dialyzed
overnight against 500 mM KCl, 10 mM Hepes, pH
7.0. Carboxyltransferase was concentrated by placing dialysis tubing
containing the enzyme in a container and covering the tubing with
polyvinylpyrrolidone. Freezing of carboxyltransferase was found to
cause irreversible precipitation/denaturation, therefore all protein
solutions were stored at 4 °C.
Carboxyltransferase Activity--
Carboxyltransferase activity
was measured in the reverse direction with a spectrophotometric assay
in which the production of acetyl-CoA was coupled to the combined
citrate synthase-malate dehydrogenase reaction requiring
NAD+ reduction (10). The reaction mixture (0.5 ml)
contained 100 mM Tris-Cl, pH 8.0, 10 mM
L-malate, 0.6 mg/ml bovine serum albumin, 3.6 units/ml
malic dehydrogenase, 6.8 units/ml citrate synthase. For inhibition
studies with desthiobiotin, the ionic strength was held constant by the
addition of KCl. pH studies were done in a three-component buffer
system of 0.1 M
Mes,1 0.051 M
N-ethylmorpholine, and 0.051 M diethanolamine.
Over the pH range of 6.2-9.6, for which the initial velocities were
measured, the ionic strength of the buffer mixture remained constant at a value of 0.1 M (11). Data were collected using a Uvikon
810 (Kontron Instruments) spectrophotometer interfaced to a PC equipped with a data acquisition program. The temperature was maintained at
25 °C by a circulating water bath with the capacity to heat and cool
the thermospace of the cell compartment. NADH formation was followed
spectrophotometrically at 340 nm. Specific activity is expressed as
µmol/min/mg of carboxyltransferase.
Inactivation Studies--
A diethyl pyrocarbonate (DEPC) stock
solution was prepared in anhydrous ethanol and used within a few hours.
The concentration of the stock solution was determined by measuring the
change in the absorbance at 230 nm when an aliquot was added to a
solution of 10 mM imidazole, pH 7.0, and using an
extinction coefficient of 3000 M
1
cm
1 for the resulting N-carbethoxyimidazole
product (12).
Enzyme modifications by DEPC and N-ethylmaleimide (NEM) were
performed in 10 mM Hepes, 500 mM KCl, pH 7.0. The reactions were initiated by the addition of one of the inactivators
to the carboxyltransferase solution. Aliquots were removed at regular
time intervals and assayed for remaining activity. Aliquots were of
sufficiently small volume compared with the assay volume such that the
inactivating reagent was diluted 100-fold. NEM and DEPC were used at 4 and 1 mM, respectively. The protection by either
malonyl-CoA or biocytin or both was assayed by incubating the
protective agent with the enzyme in 10 mM Hepes, 500 mM KCl, pH 7.0, for 2 min before adding the inactivating
agent. The pH dependence of the rate of inactivation of
carboxyltransferase by NEM was not determined in the tripart buffer
system used for the rate profiles because of inactivation of NEM by
diethanolamine. The buffer system used was Mes, pH 6.2-6.5, Pipes, pH
6.5-7.0, Hepes, pH 7.0-8.0.
Data Analysis--
Data were fitted to the appropriate equation
using either the computer program Enzfitter or the computer programs of
Cleland (13). Initial velocities obtained by varying one of the
substrates (A) were substituted in Equation 1 to yield values for the
maximum velocity (V) and the Michaelis constant for the
substrate (K).
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(Eq. 1)
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When one substrate was varied at fixed levels of the other,
velocity data were fitted to Equation 2, which describes the equilibrium-ordered initial velocity pattern, where v is the
experimentally determined velocity, V is the maximum
velocity, A and B are the substrate concentrations,
Ka and Kb are the respective
Michaelis constants, and Kia is the dissociation constant of A.
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(Eq. 2)
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Data conforming to parabolic noncompetitive inhibition were
fitted to Equation 3,
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(Eq. 3)
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in which I is the inhibitor concentration; Kis1 and
Kis2 are the slope inhibition constants for site 1 and 2, respectively; and Kii1 and Kii2 are the intercept
inhibition constants for site 1 and 2, respectively.
The variation of the values for V, V/K, and
k, the rate of inactivation of carboxyltransferase by NEM as
a function of pH were fitted to the log form of Equation 4. In this
equation y represents the value of V or
V/K at a particular pH value, C represents the
pH-independent value of the parameter; Ka is an acid dissociation constant, and H is the hydrogen ion
concentration.
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(Eq. 4)
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RESULTS |
Overexpression and Purification of Carboxyltransferase--
The
genes for the
and
subunits of carboxyltransferase are not
contiguous on the E. coli genome, yet they are presumably expressed stoichiometrically to give the
2
2 tetramer of carboxyltransferase. Therefore, an overexpression system needed to be developed such that
the genes for the
and
subunits were contiguous and expressed stoichiometrically. The gene for the
subunit was expressed using the ribosomal binding site from the pET vector. Thus, what was needed
was an intercistronic region that allowed for efficient expression of
the gene for the
subunit. To accomplish this, a 12-base pair
intercistronic region that was very similar to that found between the
genes for the catalytic and regulatory subunits of E. coli
aspartate transcarbamylase was used (14). Like carboxyltransferase,
aspartate transcarbamylase is composed of two different subunits, a
catalytic subunit and a regulatory subunit. The genes for each subunit
are contiguous on the E. coli genome and are expressed
stoichiometrically so that intact aspartate transcarbamylase is
composed of six catalytic subunits and six regulatory subunits. The
12-base pair intercistronic region that was incorporated between the
genes for the
and
subunits of carboxyltransferase contains a
ribosomal binding site and an XhoI site and allowed the gene
for the
subunit to be co-overexpressed with the
subunit.
When bacteria containing pCZB5 (Fig. 1) were grown at 37 °C after
induction, the genes for both the
and
subunit were expressed very well. Unfortunately, the enzyme was found in inclusion bodies. To
obtain enzyme in the soluble fraction, bacteria were grown at 30 °C
after induction. His-tag carboxyltransferase was purified to apparent
homogeneity using a nickel column (Fig.
2), and control experiments verified that
the native carboxyltransferase did not bind to the nickel column. The
average yield of carboxyltransferase was 6 mg from 3 g of E. coli cells. In contrast, it takes 2 kg of E. coli cells
to obtain 6 mg of carboxyltransferase when the genes are not
overexpressed (2). Attempts to remove the His-tag from
carboxyltransferase by treatment with thrombin resulted in complete
degradation of the
subunit. Because the presence of the His-tag did
not hinder the catalytic ability of the enzyme, the His-tag
carboxyltransferase was left intact. The
and
genes for
carboxyltransferase were sequenced and compared with published sequences to ensure that the polymerase chain reaction did not introduce any mutations (5-7).
The kinetic parameters for carboxyltransferase-catalyzed
transcarboxylation from malonyl-CoA to biotin and two biotin analogs are shown in Table I. Biotin methyl ester
and biocytin had maximal velocities 3 orders of magnitude higher than
biotin. Biocytin is biotin with lysine attached to the carboxyl group
of the valeric acid side chain via an amide linkage with the
-amino
group (Fig. 3). Because biotin reacted
considerably slower than the other two analogs and biotin methyl ester
had to be dissolved in dimethylformamide, all of the subsequent studies
utilized biocytin as substrate.
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Table I
Values of kinetic parameters for biotin and biotin analogs
Transcarboxylation rates were determined as described under
"Materials and Methods"; the Km of each
substrate was determined at a fixed concentration of malonyl-CoA, 0.1 mM. Km is in mM,
Vmax is in µmol/min·mg. DMF
stands for dimethylformamide.
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Fig. 3.
SDS-polyacrylamide gel electrophoresis of
carboxyltransferase. Lane 1, molecular weight markers:
bovine serum albumin, 76,000; ovalbumin, 52,000; carbonic anhydrase,
36,800; soybean trypsin inhibitor, 27,200; lysozyme, 19,000. Lane
2, soluble fraction of cell lysate. Lane 3,
carboxyltransferase after affinity purification on a nickel
column.
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Velocity Studies--
The kinetic mechanism of carboxyltransferase
was investigated by determining the initial velocity pattern. When
malonyl-CoA was varied at several fixed levels of biocytin an
intersecting pattern was obtained, indicating the sequential addition
of malonyl-CoA and biocytin prior to product release (Fig.
4A). When biocytin was varied at several
fixed levels of malonyl-CoA the pattern was intersecting; however, the
intersection point was on the vertical axis (Fig. 4B). A replot of the
slopes of the data versus the reciprocal of the biocytin
concentration when malonyl-CoA was varied went through zero. These data
indicate an ordered addition of substrates to the enzyme with
malonyl-CoA binding before biocytin with the binding of malonyl-CoA to
the enzyme at equilibrium. The data fit best to Equation 2 to yield the
binding constant of malonyl-CoA to the enzyme, Kia
malonyl-CoA = 0.10 ± 0.01 mM, the Michaelis
constant for biocytin, Kbiocytin = 6.3 ± 0.7 mM and the maximum velocity of the reaction
V = 19.6 ± 0.9 µmol/min/mg.

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Fig. 4.
Panel A, initial velocity patterns
versus malonyl-CoA concentrations at fixed biocytin
concentrations of 15.0 mM ( ), 9.0 mM( ),
6.4 mM( ), 5.0 mM( ), and 4.0 mM( ). Panel B, initial velocity pattern
versus biocytin concentrations at fixed malonyl-CoA
concentrations of 0.35 mM ( ), 0.12 mM ( ),
0.07 mM ( ), 0.05 mM ( ), 0.041 mM ( ) and 0.029 mM ( ).
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Inhibition Studies--
The biotin analogs desthiobiotin and
2-imidazolidone (Fig. 3) were found to inhibit the activity of
carboxyltransferase. Polakis et al. (15) determined that
desthiobiotin and 2-imidazolidone were not alternate substrates for
carboxyltransferase so the observed inhibition is the result of
formation of a dead-end complex between the inhibitor and the enzyme.
Reciprocal plots of the velocity versus biocytin at various
concentrations of desthiobiotin gave noncompetitive inhibition (Fig.
5). Replots of both the slopes and
intercepts versus the desthiobiotin concentration resulted in parabola-shaped curves. Because the replots of the slopes and intercepts versus the desthiobiotin concentration were both
parabolic the data were fitted to an equation that describes
noncompetitive inhibition where both the slopes and intercepts are
parabolic functions of the inhibitor concentration (Equation 3). For
comparison the data were also fitted to equations that describe
noncompetitive inhibition where only the slope or the intercept is a
parabolic function of inhibitor concentration. The data fit best to
Equation 3 based on the fact that the average least squares of the
residuals was the lowest for Equation 3. The inhibition constants for
the two inhibitor binding sites are given in Table
II. The fact that the inhibition data
conform to parabolic noncompetitive inhibition means that two molecules
of desthiobiotin bind to the enzyme simultaneously.

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Fig. 5.
Dead-end inhibitor of the reaction catalyzed
by carboxyltransferase. Inhibition by desthiobiotin at 0 mM ( ), 20 mM ( ), 60 mM ( ),
and 100 mM ( ) with respect to biocytin at concentration
of 0.3 mM malonyl-CoA is shown. Enzyme activity was
measured at 25 °C in 100 mM Tris buffer at pH 8.0. Velocities are expressed as µmol of acetyl-CoA formed/mg of
carboxyltransferase per minute. Data were fitted to Equation 3.
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Table II
Inhibition constants for biotin analogs
The error for the inhibition constants is the standard error. It was
derived from the nonlinear regression analysis and was calculated from
the square root of the variance of the parameter (22).
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The biotin analog 2-imidazolidone also acted as a parabolic
noncompetitive inhibitor of carboxyltransferase, although it was a much
worse inhibitor than desthiobiotin. The inhibition constants are given
in Table II. It should be noted that although 2-imidazolidone is not an
alternate substrate, it was found to stimulate the decarboxylation of
malonyl-CoA (15). Therefore, the rates of decarboxylation of
malonyl-CoA at each level of 2-imidazolidone were subtracted from the
velocities obtained in the presence of biocytin. The rate of
decarboxylation of malonyl-CoA by 2-imidazolidone was 5% or less of
the rate of carboxyl group transfer.
pH Dependence of V and V/K for Biocytin--
The effect of pH on
the carboxyltransferase reaction was determined over the pH range
of 6.2-9.6 by varying the biocytin concentrations at a
saturating fixed level of malonyl-CoA. Under these conditions, the
reaction examined was that of biocytin with the enzyme malonyl-CoA complex. As shown in Fig. 6, both log
V and log (V/K)biocytin decreased
with decreasing pH, although the (V/K)biocytin
only decreased 2-fold. Fitting the data to Equation 4 yielded a
pK of 7.52 ± 0.10 for the V profile and
6.21 ± 0.10 for the V/K profile. However, because data
could not be obtained below pH 6.2 due to enzyme instability, the
pK value in the V/K profile can only be estimated
to be 6.2 or less.

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Fig. 6.
Variation of pH of log V and log
(V/K) biocytin for the reaction catalyzed by
carboxyltransferase. Initial velocity data were obtained by
varying biocytin at a fixed malonyl-CoA concentration of 4 mM. The curves for V ( ) and V/K
( ) represent the best fits of the data to Equation 4.
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Inactivation Studies--
DEPC reacts preferentially with
histidine residues to yield an N-carbethoxyhistidyl
derivative (12). Incubation of carboxyltransferase with 1 mM DEPC resulted in a time-dependent loss in
activity with a pseudo-first order rate constant of 0.09 min
1. However, neither biocytin nor malonyl-CoA provided
significant protection against DEPC inactivation, indicating that the
histidine is probably not in the active site. Modification of the
poly-His-tag by DEPC is not occurring because nickel sulfate did not
protect against the modification.
Carboxyltransferase was found to be susceptible to inactivation by the
sulfhydryl-modifying reagent NEM. NEM (4 mM) inactivated carboxyltransferase with a pseudo-first order rate constant of 0.06 min
1. In the presence of 330 µM
malonyl-CoA, almost complete protection (90%) against inactivation by
NEM was observed. Biocytin provided no protection against inactivation
by NEM. The rate of inactivation of carboxyltransferase by NEM
decreased with decreasing pH (Fig. 7).
Fitting the data to Equation 4 yielded a pK value of
7.32 ± 0.16.

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Fig. 7.
Effect of pH on the rate of inactivation of
carboxyltransferase by NEM. Carboxyltransferase (0.06 mg) was
incubated with 4 mM NEM at pH values ranging from 6.35 to
7.8. Residual enzyme activity was measured at various times, and the
pseudo-first order rate constant for inactivation was determined.
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DISCUSSION |
Acetyl-CoA carboxylase plays a critical role in metabolism in that
it catalyzes the committed and regulated reaction in the synthesis of
long chain fatty acids. The metabolic significance of this enzyme is
evident from the fact that inhibitors directed against the plant or
mammalian form of the enzyme can be either herbicides or plasma
lipid-lowering drugs. These agents work by inhibiting the
carboxyltransferase component of acetyl-CoA carboxylase. Despite the
apparent importance of acetyl-CoA carboxylase to agriculture and
medicine, very little work has been done on the carboxyltransferase component, presumably because there has never been an adequate source
of the enzyme. With the overexpression system described here
significant amounts of protein have been obtained, allowing for a more
rigorous characterization of carboxyltransferase than has been done
previously (15).
A major experimental problem with site-directed mutagenesis studies of
E. coli enzymes is contamination of the mutant enzyme with
wild-type enzyme derived from the chromosomal copy of the gene. In most
cases this problem can be overcome by removing the chromosomal copy of
the gene. If the enzyme is involved in an essential metabolic pathway,
then for mutant enzymes with very little to no activity, a metabolic
intermediate subsequent to the step in question can be added to the
culture medium to sustain bacterial growth. Unfortunately, none of the
metabolic intermediates after the step catalyzed by acetyl-CoA
carboxylase are transported into E. coli. Because fatty acid
synthesis is essential for bacterial growth, it would be impossible to
remove the chromosomal copy of carboxyltransferase and sustain
bacterial growth while overexpressing a mutant form of
carboxyltransferase with very little to no activity. Because one of the
uses of the overexpression system for carboxyltransferase will be for
production of mutant enzymes, the genes for the
and
subunits of
carboxyltransferase were subcloned into pET14b to produce the
expression vector pCZB5 (Fig. 1). Using this expression vector
carboxyltransferase is produced with a His-tag sequence fused to the
NH2 terminus of the
subunit which allows the enzyme to
be purified by affinity chromatography using a nickel column. The
chromosomal copy of the enzyme, which lacks the His-tag sequence, does
not bind to the nickel column. Therefore, the His-tag version of
carboxyltransferase was purified and characterized to lay the groundwork for future site-directed mutagenesis studies.
The intersecting initial velocity pattern when malonyl-CoA and biocytin
are varied indicates that the kinetic mechanism of carboxyltransferase
is sequential. That the lines intersected on the 1/v axis
when biocytin was varied at several fixed levels of malonyl-CoA (Fig.
4B) and that a replot of the slopes of the data
versus the reciprocal of the biocytin concentration went through zero suggest that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA at equilibrium. An equilibrium-ordered pattern means that the biocytin
does not form a binary complex with the enzyme but only combines with
the enzyme-malonyl-CoA complex.
Dead-end inhibition studies would be extremely helpful to define
further the kinetic mechanism. However, no suitable inhibitor was
found. Because the assay for carboxyltransferase involves detecting the
production of acetyl-CoA using citrate synthase, the inhibition studies
precluded the use of CoA analogs and thus were limited to analogs of
biotin. Although not very informative mechanistically, inhibition by
two analogs of biotin proved very interesting and worth noting. Both
desthiobiotin and 2-imidazolidone were parabolic noncompetitive
inhibitors of the carboxyltransferase reaction when either biocytin or
malonyl-CoA was varied. In the case of dead-end inhibitors like
desthiobiotin and 2-imidazolidone, parabolic noncompetitive inhibition
occurs only when two molecules of the inhibitor bind to the enzyme(16).
One inhibitor site (site 1) may be at or near the binding site for
biocytin, and the second inhibitor is probably at another location on
the enzyme (site 2) because saturating amounts of substrate do not
relieve inhibition totally. This interpretation would help to explain
why the error for the inhibition constant for site 1 at saturating
levels of substrate (Kii1) is greater than the
parameter. Also, it is not clear why there is a second inhibitor
binding site. It should be noted, however, that the carboxyltransferase
component of E. coli acetyl-CoA carboxylase is inhibited by
guanosine 5'-diphosphate-3'-diphosphate (ppGpp) and guanosine
5'-triphosphate-3'-diphosphate (pppGpp) (17). These nucleotides have
been shown to inhibit RNA synthesis during amino acid starvation in
what is referred to as the stringent response (18). Fatty acid
synthesis is also reduced during amino acid starvation. The decrease in
fatty acid synthesis is thought to be mediated by inhibition of
carboxyltransferase by ppGpp and pppGpp. The second inhibitor site on
carboxyltransferase may be the same site at which ppGpp and pppGpp bind
to regulate fatty acid synthesis.
The pH dependence of the carboxyltransferase reaction suggests that an
enzymic residue acts as a base in catalysis. The activity for both the
log V and the log (V/K)biocytin
parameters decreased at low pH indicating that a group must be
deprotonated for activity. The group has a pK value of 7.5 in the V profile. The V profile indicates the
pK values of groups in the enzyme substrate complex that are
required for catalysis. Thus, it is very probable that the observed
base acts in a catalytic role because it is found in the V
profile.
The inactivation of carboxyltransferase by NEM and the protection
against inactivation by malonyl-CoA suggest that an active site
cysteine residue is needed for catalysis. Like the pH dependence of the
V and V/K parameters, the pH dependence of the
rate of inactivation of carboxyltransferase by NEM decreased with
decreasing pH. The pK value derived from the pH dependence
of inactivation (7.32 ± 0.16) was, within error, the same as the
pK value seen in the V profile (7.52 ± 0.10). Thus, given the similarity of the pH rate profiles and the pH
dependence of the rate of inactivation of carboxyltransferase by NEM it
is plausible that the residue that must be deprotonated for activity is
a cysteine. The reverse reaction of carboxyltransferase requires an
activated biocytin molecule that has the proton removed from the
1'-nitrogen prior to carboxylation. The thiolate ion of the cysteine
suggested above may be the catalytic base that abstracts the N1' proton
of biocytin. It has been proposed that the thiolate ion of cysteine
abstracts the N1' proton of biotin in biotin carboxylase (19) and
pyruvate carboxylase (20, 21).
Verification of this mechanism in terms of which cysteine residue is
involved would be aided greatly by a three-dimensional structure of
carboxyltransferase followed by site-directed mutagenesis studies. To
this end, overexpression of the gene for carboxyltransferase now
provides us with ample amounts of enzyme for structure/function studies
such as crystallography and site-directed mutagenesis experiments. The
His-tag carboxyltransferase allows mutant enzyme to be separated from
the wild-type chromosomal copy so that there are no ambiguities in the
interpretation of the kinetics of the mutant enzyme. And finally,
crystals of the carboxyltransferase have been obtained and the crystal
structure determination is currently underway.
We thank Dr. Frank Raushel of Texas A&M
University for providing the computer program for collecting enzyme
kinetic data on a PC. We thank Dr. John Cronan of the University of
Illinois, Dr. Dean Tolan of Boston University, and Dr. Barry Shane of
the University of California at Berkeley for plasmids containing the genes for the
and
subunits of carboxyltransferase. We also thank Dr. W. W. Cleland of the University of Wisconsin for helpful comments on the manuscript.