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Acetyl-CoA Carboxylase from Cyclotella cryptica: Purification and Characterization, Study notes of Biotechnology

This document reports the purification and characterization of acetyl-CoA carboxylase from the diatom Cyclotella cryptica. The study compares the properties of this enzyme with those of higher plant acetyl-CoA carboxylases and investigates the effects of various cellular metabolites and herbicides on its activity.

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Plant
Physiol.
(1990)
92,
73-78
0032-0889/90/92/0073/06/$01
.00/0
Received
for
publication
April
24,
1989
and
in
revised
form
August
29,
1989
Purification
and
Characterization
of
Acetyl-CoA
Carboxylase
from
the
Diatom
Cyclotella
cryptica1
Paul
G.
Roessler2
Biotechnology
Research
Branch,
Solar
Energy
Research
Institute,
Golden,
Colorado
80401
ABSTRACT
Acetyl-CoA
carboxylase
from
the
diatom
Cyclotella
cryptica
has
been
purified
to
near
homogeneity
by
the
use
of
ammonium
sulfate
fractionation,
gel
filtration
chromatography,
and
affinity
chromatography
with
monomeric
avidin-agarose.
The
specific
activity
of
the
final
preparation
was
as
high
as
14.6
micromoles
malonyl-CoA
formed
per
milligram
protein
per
minute,
indicating
a
600-fold
purification.
Native
acetyl-CoA
carboxylase
has
a
mo-
lecular
weight
of
approximately
740
kilodaltons
and
appears
to
be
composed
of
four
identical
biotin-containing
subunits.
The
enzyme
has
maximal
activity
at
pH
8.2,
but
enzyme
stability
is
greater
at
pH
6.5.
Km
values
for
MgATP,
acetyl-CoA,
and
HC03
were
determined
to
be
65,
233,
and
750
micromolar,
respectively.
The
purified
enzyme
is
strongly
inhibited
by
palmitoyl-CoA,
and
is
inhibited
to
a
lesser
extent
by
malonyl-CoA,
ADP,
and
phos-
phate.
Pyruvate
stimulates
enzymatic
activity
to
a
slight
extent.
Acetyl-CoA
carboxylase
from
Cyclotella
cryptica
is
not
inhibited
by
cyclohexanedione
or
aryloxyphenoxypropionic
acid
herbicides
as
strongly
as
monocot
acetyl-CoA
carboxylases;
50%
and
0%
inhibition
was
observed
in
the
presence
of
23
micromolar
cleth-
odim
and
100
micromolar
haloxyfop,
respectively.
taining
subunits
are
commonly
observed,
leading
several
in-
vestigators
to
suggest
that
plant
acetyl-CoA
carboxylases
are
multifunctional
proteins
able
to
catalyze
both
steps
of
the
reaction
(biotin
carboxylation
and
carboxyl
transfer
to
acetyl-
CoA)
(8).
Little
is
known
about
the
allosteric
regulation
of
acetyl-
CoA
carboxylase
activity
in
higher
plants.
Adenylate
nucleo-
tides
(AMP,
ADP,
and
free
ATP)
have
been
shown
to
inhibit
the
enzyme
from
several
higher
plants
(2,
3,
15,
20).
Acetyl-
CoA
carboxylase
from
maize
is
also
inhibited
by
malonyl-
CoA
and
palmitoyl-CoA
(15).
Free
coenzyme
A
has
been
shown
to
inhibit
the
enzyme
from
maize
(15)
but
to
stimulate
the
activity
of
the
enzyme
from
spinach
(1
1).
Citrate,
free
Mg2+,
K+,
and
glycine
have
also
been
reported
to
stimulate
acetyl-CoA
carboxylase
from
various
plant
sources
(6,
12-
15).
The
research
described
in
this
report
was
carried
out
in
order
to
further
our
understanding
of
acetyl-CoA
carboxylases
from
eukaryotic
microalgae
and
to
compare
the
properties
of
acetyl-CoA
carboxylase
from
the
diatom
Cyclotella
cryptica
with
those
of
higher
plant
acetyl-CoA
carboxylases.
Acetyl-CoA
carboxylase
is
a
biotin-containing
enzyme
that
catalyzes
the
formation
of
malonyl-CoA,
which
is
one
of
the
initial
steps
of
fatty
acid
biosynthesis.
Previous
research
has
indicated
that
changes
in
the
activity
of
this
enzyme
may
play
a
role
in
the
accumulation
of
lipids
when
the
diatom
Cyclo-
tella
cryptica
is
grown
under
silicon-limiting
conditions
(19).
It
was
therefore
of
interest
to
investigate
the
properties
of
acetyl-CoA
carboxylase
from
this
alga.
Although
acetyl-CoA
carboxylase
has
been
purified
from
several
higher
plants,
the
enzyme
has
not
previously
been
purified
from
an
algal
source.
Acetyl-CoA
carboxylases
from
higher
plants
have
several
characteristics
in
common,
including
an
alkaline
pH
opti-
mum
and
an
absolute
requirement
for
ATP
and
divalent
metal
cations.
Early
studies
indicated
that
plant
acetyl-CoA
carboxylases
had
complex
subunit
structures
(three
to
six
peptides
having
different
mol
wt),
but
more
recent
studies
suggest
that
these
earlier
attempts
to
characterize
the
enzyme
were
subject
to
error
due
to
the
effects
of
endogenous
prote-
olytic
activity.
Recent
investigations
have
suggested
simpler
subunit
structures.
High
mol
wt
(200-240
kD),
biotin-con-
'
This
research
was
supported
by
the
Biofuels
and
Municipal
Waste
Division
of
the
U.S.
Department
of
Energy
under
FWP
BF8
1.
2
Present
address:
Department
of
Botany
and
Plant
Pathology,
Michigan
State
University,
East
Lansing,
MI
48824.
MATERIALS
AND
METHODS
Organism
and
Growth
Conditions
Cyclotella
cryptica
Reimann,
Lewin,
and
Guillard
strain
T13L
was
obtained
from
the
Culture
Collection
of
Marine
Phytoplankton
at
the
Bigelow
Laboratory
for
Ocean
Sciences
(W.
Boothbay
Harbor,
ME).
Cells
were
cultured
in
2
L
polycarbonate
bottles
as
described
previously
(18).
Cultures
were
bubbled
with
0.5%
CO2
in
air
(500
mL/min)
and
main-
tained
at
25°C
under
constant
illumination
from
fluorescent
lamps
(photon
flux
density
at
the
vessel
surface
averaged
over
3600
=
85
,umol
quanta-
m-2.
s
').
Materials
Analytical
grade
haloxyfop
and
clethodim
were
kindly
pro-
vided
by
J.
Secor
(Dow
Chemical
Co.,
Walnut
Creek,
CA)
and
A.
Rendina
(Chevron
Chemical
Co.,
Richmond,
CA),
respectively.
Analytical
Methods
Protein
was
quantified
by
the
Coomassie
blue
dye-binding
method
(Bio-Rad)
using
bovine
y-globulin
as
a
standard.
Radioactivity
was
determined
by
liquid
scintillation
counting
in
a
Beckman
model
LS9000
scintillation
counter,
using
the
73
pf3
pf4
pf5

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Plant Physiol. (1990) 92, 73-

Received for publication (^) April 24, 1989

and in revised form August 29, 1989

Purification and Characterization (^) of Acetyl-CoA

Carboxylase from the Diatom (^) Cyclotella cryptica

Paul G. (^) Roessler Biotechnology Research (^) Branch, Solar Energy Research Institute, Golden, Colorado 80401

ABSTRACT Acetyl-CoA carboxylase from the diatom Cyclotella cryptica has been purified to near homogeneity by the use of ammonium sulfate fractionation, gel filtration chromatography, and affinity chromatography with monomeric avidin-agarose. The specific activity of the final preparation was as high as 14.6 micromoles malonyl-CoA formed per milligram protein per minute, indicating a 600-fold purification. Native acetyl-CoA carboxylase has a mo- lecular weight of approximately 740 kilodaltons and appears to be composed of four identical (^) biotin-containing subunits. The enzyme has maximal activity at pH (^) 8.2, but (^) enzyme stability is greater at pH 6.5. Km values for MgATP, (^) acetyl-CoA, and (^) HC were determined to be 65, 233, and (^750) micromolar, respectively. The purified enzyme is strongly inhibited by (^) palmitoyl-CoA, and is inhibited to a lesser extent by (^) malonyl-CoA, ADP, and (^) phos- phate. Pyruvate stimulates enzymatic activity to a (^) slight extent. Acetyl-CoA carboxylase from Cyclotella cryptica is not inhibited by cyclohexanedione or aryloxyphenoxypropionic acid herbicides as strongly as monocot acetyl-CoA carboxylases; 50% and 0% inhibition was observed in the presence of 23 micromolar cleth- odim and 100 micromolar haloxyfop, respectively.

taining subunits are commonly observed, leading several in-

vestigators to suggest that plant acetyl-CoA carboxylases are

multifunctional proteins able to catalyze both steps of the

reaction (biotin carboxylation and carboxyl transfer to acetyl-

CoA) (8).

Little is known about the allosteric regulation of acetyl-

CoA carboxylase activity in higher plants. Adenylate nucleo-

tides (AMP, ADP, and free ATP) have been shown to inhibit

the enzyme from several higher plants (2, 3, 15, 20). Acetyl-

CoA carboxylase from maize is also inhibited by malonyl-

CoA and palmitoyl-CoA (15). Free coenzyme A has been

shown to inhibit the enzyme from maize (15) but to stimulate

the activity of the enzyme from spinach (1 1). Citrate, free

Mg2+, K+, and glycine have also been reported to stimulate

acetyl-CoA carboxylase from various plant sources (6, 12-

The research described in this (^) report was carried out in

order to further our understanding ofacetyl-CoA carboxylases

from eukaryotic microalgae and to compare the properties of

acetyl-CoA carboxylase from the diatom Cyclotella cryptica

with those of higher plant acetyl-CoA carboxylases.

Acetyl-CoA carboxylase is a biotin-containing enzyme that

catalyzes the formation of malonyl-CoA, which is one of the

initial steps of fatty acid biosynthesis. Previous research has

indicated that changes in the activity ofthis enzyme may play

a role in the (^) accumulation of lipids when the diatom Cyclo- tella cryptica is grown under (^) silicon-limiting conditions (19).

It was therefore of interest to investigate the properties of

acetyl-CoA carboxylase from this alga. Although acetyl-CoA

carboxylase has been purified from several higher plants, the

enzyme has not previously been purified from an algal source.

Acetyl-CoA carboxylases from higher plants have several

characteristics in common, including an alkaline pH opti-

mum and an absolute requirement for ATP and divalent

metal cations. Early studies indicated that plant acetyl-CoA

carboxylases had complex subunit structures (three to six

peptides having different mol wt), but more recent studies

suggest that these earlier attempts to characterize the enzyme

were subject to error due to the effects of endogenous prote-

olytic activity. Recent investigations have suggested simpler

subunit structures. High mol wt (200-240 kD), biotin-con-

' (^) This research was supported by the Biofuels and (^) Municipal Waste Division of the U.S. Department of Energy under FWP BF8 1. (^2) Present address: Department of Botany and Plant Pathology,

Michigan State University, East (^) Lansing, MI 48824.

MATERIALS AND METHODS

Organism and Growth Conditions

Cyclotella cryptica Reimann, Lewin, and Guillard strain

T13L was obtained from the Culture Collection of Marine

Phytoplankton at the Bigelow Laboratory for Ocean Sciences

(W. Boothbay Harbor, ME). Cells were cultured in 2 L

polycarbonate bottles as described previously (18). Cultures

were bubbled with 0.5% CO2 in air (500 mL/min) and main-

tained at 25°C under constant illumination from fluorescent

lamps (photon flux density at the vessel surface averaged over

3600 =^85 ,umol quanta- m-2.s ').

Materials

Analytical grade haloxyfop and clethodim were kindly pro-

vided by J. Secor (Dow Chemical Co., Walnut Creek, CA)

and A. Rendina (Chevron Chemical Co., Richmond, CA),

respectively.

Analytical Methods

Protein was quantified by the Coomassie blue dye-binding

method (Bio-Rad) using bovine y-globulin as a standard.

Radioactivity was determined by liquid scintillation counting

in a Beckman model LS9000 scintillation counter, using the

73

Plant Physiol. Vol. 92, 1990

H# routine for quench correction. Biofluor (Dupont/New

England Nuclear) or Optifluor (Packard) was used as the

scintillation cocktail for all isotope studies.

Electrophoresis

SDS-PAGE was performed as described by Laemmli (10)

with a 7.5% separating slab gel. Proteins were transferred to

nitrocellulose membranes with a semidry electroblotting ap-

paratus (American Bionetics) using the buffer system of

Shafer-Nielsen et al. (23). Proteins were detected by the Bio-

Rad Biotin-Blot procedure, using the manufacturer's proto-

col. Biotin-containing proteins were detected by the same

procedure, except that the protein biotinylation step was

omitted.

Nondenaturing discontinuous PAGE was performed on 5

mm diameter tube gels (5%tota1/5%bis separating gel) with a

Hoefer model DE 102 unit, using the manufacturer's protocol.

Assay for Acetyl-CoA Carboxylase Activity

Acetyl-CoA carboxylase activity was measured by the in-

corporation of ['4C]bicarbonate into acid- and heat-stable

material (malonyl-CoA) using a modification ofthe procedure

of Sauer and Heise (20). The reaction mixture (final volume

= 0.3 mL) contained 100 mm Tricine buffer (pH 8.2), 0.5 mM

acetyl-CoA, 1 mm ATP, 2 mM MgCl2, 10 mm KCI, and 10

mM (^) ['4C]NaHCO3 (specific activity = (^1) 1.1 MBq/mmol). The

reaction was initiated by the addition of enzyme and termi-

nated after 10 min at 3O°C by the addition of 0.3 mL of 2 N

HCI. A^ portion (0.5 mL) of the acidified solution was trans-

ferred to a scintillation vial and heated at 70°C until dry (

h). The residue was dissolved in 0.3 mL of 0.2 N HCI prior to

the addition of scintillation cocktail. Control assays were

carried out in the absence of acetyl-CoA in order to correct

for nonspecific radioactivity, which was typically less than 5%

of the acetyl-CoA-dependent "1C incorporation. One unit of

activity is defined as the amount of enzyme required to

catalyze the formation of 1 ,umol of malonyl-CoA per minute

under standard assay conditions.

The reaction product was analyzed by the procedure de-

scribed by Thomson and Zalik (24), which includes an alka-

line hydrolysis step to cleave the thioester linkage to coenzyme

A. The reaction product comigrated with authentic ["`C]

malonate (Sigma Chemical Co.) on silica gel TLC plates (J.

T. Baker Co., Si250) developed in water-saturated diethyl

ether:formic acid (7:1, v:v) (24) and on Whatman No. 1 paper

developed in^ isobutyric acid:NH40H:H20 (66:1:33, v:v:v)

Preparation of Cell-Free Extracts

Cells were harvested (^) by centrifugation at (^) 5,000g for 5 min and washed with MCD buffer (100 mm Mes containing 10

mM K-citrate and 2 mm DTT, pH 6.5). The cells were then

suspended in^10 to^25 mL^ of^ MCD^ buffer^ and^ passed through a French pressure cell at 15,000 (^) psi. The (^) pressate was centri- fuged at 37,000g for 20 min. The supernatant was diluted

with MCD buffer so that the total protein concentration was

below (^5) mg/mL; this is referred to as the crude extract. Cell

disruption and all subsequent purification steps were carried

out at 4C.

Purification Procedure

(NH4)2SO4 Fractionation

A saturated solution of ice-cold (NH4)2SO4 was added to

the crude extract with stirring to yield a 30% saturated solu-

tion, which^ was^ then^ centrifuged at 8000g for 5 min. The

precipitate was^ discarded^ and^ the supernatant solution was

subjected to^ further^ fractionation by the addition of

(NH4)2SO4 to 43, 50, and 60% saturation. All precipitates

were discarded except for the one obtained at 60% saturation,

which was dissolved in 5 to 10 mL of MCD buffer and used

for the gel filtration chromatography step.

Gel Filtration Chromatography

The solution obtained after (NH4)2SO4 fractionation was

loaded onto a 90 x 2.2 cm column of Biogel A- 1.5 m (Bio-

Rad) and eluted with MCD buffer at a flow rate of 20 mL/h.

The fractions containing the highest acetyl-CoA carboxylase

activity (numbers 41-57, 4 mL each) were combined and

used in the affinity chromatography step.

Affinity Chromatography

The naturally occurring biotin molecules found in acetyl-

CoA carboxylase allow affinity chromatography through col-

umns containing covalently bound avidin. Monomeric avi-

din-agarose was prepared by incubation of 10 mg tetrameric

chicken egg avidin (Calbiochem) in 4 mL 50 mm Hepes (pH

8.0) with 2 mL ofAffi-gel 10 (Bio-Rad) for 4 h at 4°C, followed

by treatment of the gel with 6 M guanidine-HCl (pH 2.15) for

16 h at 20°C. Noncovalently attached avidin subunits were

removed from the column by washing with several column

volumes of 6 M guanidine-HCl. The column was prepared for

use by passing four column volumes of MKD buffer (100 mm

Mes buffer with 100 mM KCI and 2 mM DTT) containing 0.

mg biotin/mL through the column to saturate the biotin-

binding sites followed by 10 column volumes of 0.1 M glycine

(pH 2) to remove exchangeable biotin (9). Portions of the gel

filtration-purified solution were passed through a 2 mL col-

umn of monomeric avidin-agarose, followed by washing

with 15 mL of MKD buffer. Acetyl-CoA carboxylase was

eluted from the column with MKD buffer containing 0.5 mg

biotin/mL.

RESULTS

Acetyl-CoA carboxylase was purified from Cyclotella cryp-

tica by a simple procedure utilizing (NH4)2S04 precipitation,

gel filtration^ chromatography, and^ affinity chromatography

with monomeric avidin-agarose. This procedure resulted in a

nearly homogeneous preparation having a specific activity of up to 14.6 ,mol malonyl-CoA formed mg protein'. minI', which represented an increase in (^) specific activity of (^) approxi-

mately 600-fold. The results from a typical purification are

shown in Table I.

Gel filtration chromatography of C. cryptica acetyl-CoA

(^74) Roessler

Plant Physiol. Vol. 92, 1990

tion by the use of a desalting column, enzymatic activity was

completely lost and could not be restored by the readdition

of NaCl or KCI. Partially purified acetyl-CoA carboxylase

from C. cryptica could be stored frozen at -20°C for at least 3

weeks with little loss in activity. However, affinity-purified

acetyl-CoA carboxylase was less stable, losing all activity upon

freezing and a substantial portion (30%) ofthe inititial activity

after 24 h at 4°C.

The activity of the enzyme was dependent upon the pres-

ence of divalent metal cations, with Mg2' being the most

effective cation tested. When ATP was included at a concen-

tration of 1 mm, maximal acetyl-CoA carboxylase activity

was observed at a Mg2' concentration of 2 mm (Fig. 3). Above

this concentration, enzyme activity declined^ to a^ slight^ extent.

Mn2+ was able partially to replace the Mg2' requirement, but

2 mm MnCl2 yielded only 20% of the activity observed in the

presence of 2 mm^ MgCl2.^ The enzyme was inactive when

CO2+ (2 mM) was the only divalent metal cation present.

Purified acetyl-CoA carboxylase from^ C.^ cryptica^ exhibited

Michaelis-Menten kinetics with^ respect to MgATP,^ acetyl-

CoA, and NaHCO3. The apparent Km values^ for^ these sub-

strates were determined to be 65, 233, and 750 (^) gM, respec-

tively. Citrate (1 mM) did^ not^ affect^ the Km^ of^ the^ enzyme^ for

acetyl-CoA or the Vma of the reaction using different^ concen-

trations of acetyl-CoA as substrate (data not^ shown).^ Pro-

pionyl-CoA was a poor substrate for^ C.^ cryptica^ acetyl-CoA

carboxylase; 0.5 mM propionyl-CoA was carboxylated at only

one-tenth the rate of 0.5 mM acetyl-CoA.

The effects of various cellular metabolites on the activity of

purified acetyl-CoA carboxylase are shown in Table II. Ma-

lonyl-CoA, ADP, and orthophosphate, which are all products

ofthe reaction, substantially inhibited acetyl-CoA carboxylase

activity. The inclusion of 1 mM ADP and 1 mM^ NaH2PO4 at

me same timc^ LA *w +^ sS1sCA07^ LA_0_I,.:_^ -^ 4-,1^ --A

carboxylase ac

reaction by 84

creasing enzyn

._^ c 0

co

L

I-

0

0 0

5

4

3

2

OE- 0

Figure 3. Effect CoA carboxylas were added per

Table II. Effects of Various Compounds on the Activity of Acetyl- CoA Carboxylase Purified from Cyclotella cryptica The values shown are the mean values obtained from two to four separate experiments. Additions Relative Activity ± SD None looa 1 mM malonyl-CoA 16.0 ±^ 1. (^100) AM malonyl-CoA 59.7 ± 6. 1 mM NaH2PO4 53.0 ± 4. 1 mM ADPb 51.3 ± 2. 1 mM NaH2PO4 + 1 mM ADPb 36.2 ± 0. 1 mM AMP 94.8 ± 1. (^100) yM palmitoyl-CoA 21.8 ± 4. 10 Mm palmitoyl-CoA 64.7 ± 8. 100AMCoA 98.1±3. 1 mM 3-phosphoglycerate 94.4 ± 0. 1 mM phosphoenolpyruvate 84.1 ± 8. 1 mM pyruvate 148.8 ± 10. 1 mm glucose-1-phosphate 103.1 ± 9. 1 mM glucose-6-phosphate 100.1 ± 8. 1 mM fructose-6-phosphate 94.6 ± 7. 1 mm (^) fructose-1,6-bisphosphate 99.7 ± 8. 1 mM citrateb 107.0 ± 8. 1 mm acetate 94.1 ± 6. 1 mM NADH 98.3 ± 5. 1 mM NADPH 87.3 ± 7. 1 mM NAD+ 100.0 ± 0. 1 mM NADP+ 94.5 ± 5.

a 100% relative activity ranged from 6.7 x^1 0-4 to^ 1.2^ x^ 10-3 units/

mL reaction mixture for the different experiments. 2.0 x^ 10-4 to 3.

X 1 0-4 units of affinity-purified enzyme were added per reaction,

depending on the experiment. b^ Additional MgCI2 (1 mM) was also

included in^ these assays to overcome^ the^ effects^ of^ Mg2+^ chelation.

e resuitec in^ a o4,o recuction in^ acetyi-LoA centration of (^100) Mm. Free palmitate was also quite (^) inhibitory; 'tivity, while 1 mM^ malonyl-CoA inhibited^ the^100 AM palmitate (which is approximately three times^ higher

M%. Palmitoyl-CoA was a^ strong inhibitor, de-^ than the solubility limit of palmitate in water)^ inhibited

natic activity by 78% when^ included^ at a^ con-^ malonyl-CoA formation by 44%. The^ photosynthetic/glyco-

lytic intermediates^ 3-phosphoglycerate, phosphoenolpyru-

vate, fructose- 1 ,6-bisphosphate, glucose- 1-phosphate, glucose-

6-phosphate, and^ fructose-6-phosphate had^ little effect^ on

acetyl-CoA carboxylase activity, but 1 mM pyruvate was

shown to consistently stimulate enzymatic activity by approx-

imately 50%. CoA, which stimulates the activity of certain

higher plant acetyl-CoA carboxylases (11) while inhibiting

others (15), had no effect on the enzyme from C. cryptica.

Citrate, acetate, AMP, NADH, NADPH, NAD+, and^ NADP+

(1 mM) likewise did^ not^ alter^ acetyl-CoA carboxylase activity

substantially.

Since acetyl-CoA carboxylase has recently been shown to

be the site of action of monocot-specific aryloxyphenoxypro-

pionic acid and cyclohexanedione herbicides^ (1, 16, 21), it

was of interest to determine the^ effects^ of^ these^ herbicides^ on

the activity of^ purified C.^ cryptica^ acetyl-CoA carboxylase.

2 4 6 8 10 12 The cyclohexanedione herbicide clethodim inhibited C. cryp-

tica acetyl-CoA carboxylase activity in a dose-dependent man-

[MgCI2 ]^ (mM) (^) ner, with 50% inhibition occurring at a concentration of 23

t of MgC12 on the activity of purified C. cryptica acetyl- AM (Fig. 4). Conversely, the aryloxyphenoxypropionic acid

e. A total of 6.1 x^1 0 4units^ of^ affinity-purified enzyme herbicide^ haloxyfop did not affect^ enzymatic^ activity^ at^ con-

reaction. centrations^ up^ to^100 MM.

76 Roessler

ACETYL-CoA CARBOXYLASE FROM CYCLOTELLA CRYPTICA

100

80

0

z 0

60

40

20

0 I 10 100

[CLETHODIM] (MM)

Figure 4. Inhibition of purified C. cryptica acetyl-CoA carboxylase by clethodim. A freshly prepared 20 mm clethodim stock solution (in ethanol) was diluted into 50 mm Tricine (pH 8.2) immediately prior to the assay. The mean values (±SD) from two separate experiments are shown. The^ mean^ value^ for^ 100% relative^ activity^ for^ the^ two experiments was^ 6.5^ units/mg protein.^ An^ average^ of 6.3^ x^1 04 units of (^) affinity-purified enzyme were added per reaction.

DISCUSSION

Many properties of acetyl-CoA carboxylase from the^ dia-

tom Cyclotella cryptica are similar to those of acetyl-CoA

carboxylases isolated from various higher plants. Like the enzyme from^ higher plants, C.^ cryptica acetyl-CoA carboxyl- ase is a large (about 740 kD) protein composed of^ several subunits. The molecular mass ofacetyl-CoA carboxylase from wheat germ (4), avocado (12), castor seed (6), maize (15), and cultured parsley cells^ (5) was^ reported to be^ 700, 650,^ 528, 500, and 420 kD, respectively. C.^ cryptica acetyl-CoA carbox- ylase appears to be a tetrameric protein composed of four identical biotin-containing subunits, each having a molecular mass of about 185 kD. This suggests that the subunits are multifunctional (^) peptides containing domains responsible for both biotin carboxylation and^ subsequent carboxyl transfer to acetyl-CoA. Acetyl-CoA carboxylase from maize^ is also composed of multiple identical subunits (15), but in this case the subunits are (^) only 60 to 61 kD. Acetyl-CoA carboxylase from cultured parsley cells is^ composed oftwo^ equal subunits, each having a molecular mass of 220 kD (5). Although the mol wt of native acetyl-CoA carboxylase from soybean and oil seed rape are not known, these^ enzymes have^ been^ shown to be composed of identical subunits having molecular mass

of 240 and 220 kD, respectively (2, 8). Another variety of

soybean ("Wayne")^ exhibited^ a more^ complex^ subunit^ struc- ture, however (2). Acetyl-CoA carboxylase from C. cryptica and the (^) majority of acetyl-CoA carboxylases from higher plants therefore appear to be similar in that they are composed of multiple, but identical, subunits. The number of subunits in the various holoenzymes can vary substantially, however. It is rather surprising that so much diversity in the structure of this^ ubiquitous enzyme exists^ among different^ plants. It is interesting to^ note that^ the^ subunit^ structure^ of C.^ cryptica acetyl-CoA carboxylase is^ more^ similar^ to^ acetyl-CoA carbox-

ylase from brewer's yeast^ (which^ is^ composed^ of four^ identical 150 kD subunits [22]) than to^ the acetyl-CoA^ carboxylases described thus far^ from^ higher plants. The activity of all acetyl-CoA carboxylases studied to date has been shown to be dependent upon the presence ofdivalent metal cations. C. cryptica acetyl-CoA carboxylase also exhib- ited this characteristic (Fig. 3). It has been demonstrated by several researchers (6, 12, 13, 20) that MgATP is the actual substrate for acetyl-CoA carboxylase, and it is assumed that this is also the^ case^ for^ acetyl-CoA^ carboxylase^ from^ C. cryptica. In^ addition, free^ Mg2+ stimulates^ acetyl-CoA^ carbox- ylase activity^ in^ several higher plants (6, 12,^ 13,^ 20).^ Based^ on the increase in^ activity of C. cryptica acetyl-CoA carboxylase

due to concentrations of^ Mg2> that^ exceeded the^ ATP^ concen-

tration in^ the assay mixture^ (Fig. 3), it^ appears^ that free^ Mg2+ also stimulates the activity of the C.^ cryptica enzyme. The Km values obtained for C.^ cryptica acetyl-CoA carbox- ylase for acetyl-CoA, MgATP, and^ bicarbonate (233,^ 65,^ and 750 uM, respectively) are similar to the^ Km values^ reported for higher plant acetyl-CoA carboxylases. A^ survey of Km values for acetyl-CoA carboxylases from several higher plants, (in- cluding spinach [12, 20], avocado [12], maize [15], wheat [7], parsley [5], soybean [2], barley [16], and castor seed [6]) yielded ranges of 26 to (^320) tLM for acetyl-CoA, 21 to 460 uM for (^) MgATP, and 0.86 to 8 mm for bicarbonate. Unlike the case for acetyl-CoA carboxylase from^ avocado^ and^ spinach (12), citrate did not affect the Vma of the reaction when the enzyme was supplied with^ different concentrations^ of^ acetyl- CoA. The activity of C. cryptica acetyl-CoA carboxylase can^ be modulated by several metabolites. As is the case with^ acetyl- CoA carboxylase from several higher plants (2, 3, 15, 20), the diatom enzyme is^ inhibited^ by ADP. For the^ higher plants examined, this^ inhibition^ appears to^ be^ competitive^ with respect to ATP.^ C.^ cryptica acetyl-CoA carboxylase^ is^ also inhibited by orthophosphate and^ malonyl-CoA. It is^ not^ clear whether the inhibitory effects of ADP, phosphate, and^ ma- lonyl-CoA are due to true allosteric mechanisms or^ simply to shifts in the thermodynamic equilibrium of the reaction. Nonetheless, it is clear that acetyl-CoA carboxylase activity would be higher during periods of photosynthesis due to the combined effects of increased pH and Mg" levels and de- creased ADP and orthophosphate levels within the chloroplast (the presumed location of^ the^ enzyme). The strongest inhibitor of C. cryptica acetyl-CoA carbox- ylase activity tested^ was^ palmitoyl-CoA,^ which^ inhibited^ the reaction by 35 and 78% at concentrations of^ 10 and 100 ,uM, respectively. It appears that the acyl component is at^ least partially responsible for this inhibition since free palmitate also inhibited enzymatic activity quite strongly. Palmitoyl- CoA was also reported to inhibit maize leaf^ acetyl-CoA car- boxylase activity (15), with^ nearly complete inhibition^ occur- ring at a concentration of 37.5 (^) gM. The low concentration of acyl-CoA (or free fatty acids) required to inhibit acetyl-CoA carboxylase suggests that this inhibition may be physiologi- cally relevant under conditions when acyl chains are not incorporated into membrane lipids or exported from the chloroplast at rates comparable to their rates of synthesis. The only metabolite tested that stimulated the activity of

10 - 23.3 (^) pM x

77