From the Department of Molecular Biosciences, The University of Kansas, Lawrence, Kansas 66045
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been suggested that the last seven to nine
amino acid residues at the C terminus of the The ATP synthase enzymes of the inner membranes of mitochondria,
chloroplasts and of the bacterial cytoplasmic membrane, couple the
energy of a transmembrane electrochemical proton gradient to the
synthesis of ATP from ADP and inorganic phosphate. The general
structural features of the enzyme are highly conserved from one
organism to another. It is comprised of an integral membrane-spanning H+-translocating segment (F0 or factor O) and a
peripheral membrane segment (F1 or factor 1) which contains
the catalytic sites for ATP synthesis and hydrolysis. The
F1 segment is comprised of five different polypeptide
subunits designated A high resolution crystal structure of the core catalytic portion of
the mitochondrial F1 enzyme was reported recently (2). The
The crystal structure of F1 indicated that the three In this study we have tested the "bearing" hypothesis specifically
suggested by the crystal structure, by selectively deleting amino acid
residues from the extreme C-terminal end of the Materials--
CF1 and CF1 lacking the
ATP (grade I and II) and antibiotics (ampicillin, chloramphenicol, and
tentoxin) were purchased from Sigma. Stock solutions of tentoxin were
prepared by dissolving the inhibitor in ethanol to a final
concentration of 5 mM and stored at Plasmid Construction--
Most of the recombinant DNA methods
used in this study have been described elsewhere (15, 16).
Escherichia coli transformation protocols were as described
by Hanahan (17). Plasmid pSG101 (4), generously supplied by Dr. M. Futai, contains the full-length cDNA for the spinach
(Spinacia oleracea) chloroplast atpC gene encoding the ATP synthase Generation of atpC Gene Mutants--
Eight deletion mutants of
Each mutant gene was isolated from the expression clone by alkaline-SDS
lysis followed by ethanol precipitation after phenol:chloroform extraction (20) and sequenced by an automated fluorescence dideoxy technique (21).
Solubilization and Folding of Overexpressed
The insoluble Assembly of Other Procedures--
ATPase activities were determined by
measuring phosphate release (22) for 5 min at 37 °C. The assay was
carried out in 0.5-ml volumes of assay mixture containing 50 mM Tricine-NaOH, pH 8.0, and 5 mM ATP. The
calcium-dependent ATPase activity was assayed in the
presence of 5 mM CaCl2.
Magnesium-dependent ATPase was carried out in the presence
of 2.5 mM MgCl2 and 25 mM
Na2SO3, and manganese-dependent
ATPase activity was carried out in the presence of 2.5 mM
MnCl2 and 100 mM
Na2SO3. The reaction was started by addition of
1-6 µg of enzyme into the assay mixture and terminated by addition
of 0.5 ml of cold trichloroacetic acid. Protein concentrations were
determined by the Bradford method (23). Absorbance measurements were
obtained using a Beckman DU-70 spectrophotometer. Gel electrophoresis was performed on NOVEX Pre-Cast 10-20% gradient gels.
Overexpression of the Spinach atpC Gene in E. coli--
The
atpC gene encoding the full-length Assembly of the
The results shown in Table I compare the
ATPase activities of protein assemblies reconstituted with the first
two mutants,
Fig. 3 compares the relative rates of ATP
hydrolysis of the remaining mutants,
Activation of the latent Mg-ATPase and Mn-ATPase activities of
CF1 normally requires, in addition to removing the
inhibitory Sensitivity of the Mutant Assemblies to Inhibitors--
The
responses of the different assemblies to the inhibitory
The results of titrating the A cross-sectional view through the structure of the beef heart
mitochondrial F1 is shown in Fig.
5. The tip of the C terminus of the subunit of the ATP
synthase act as a spindle for rotation of the
subunit with respect
to the
subunits during catalysis (Abrahams, J. P., Leslie,
A. G. W., Lutter, R., and Walker, J. E. (1994)
Nature 370, 621-628). To test this hypothesis we
selectively deleted C-terminal residues from the chloroplast
subunit, two at a time starting at the sixth residue from the end and
finishing at the 20th residue from the end. The mutant
genes were
overexpressed in Escherichia coli and assembled with a
native
3
3 complex. All the mutant forms
of
assembled as effectively as the wild-type
. Deletion of the
terminal 6 residues of
resulted in a significant increase (>50%)
in the Ca-dependent ATPase activity when compared with the
wild-type assembly. The increased activity persisted even after
deletion of the C-terminal 14 residues, well beyond the seven residues
proposed to form the spindle. Further deletions resulted in a decreased
activity to ~19% of that of the wild-type enzyme after deleting all
20 C-terminal residues. The results indicate that the tip of the
C
terminus is not essential for catalysis and raise questions about the
role of the C terminus as a spindle for rotation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to
in order of decreasing molecular weight.
The subunit stoichiometry is
3
3
1
1 and
1. Nucleotide binding is associated with the
and
subunits, whereas the
and
subunits play regulatory and/or
structural roles. The
subunit is likely to be involved in binding
the F1 segment to the F0 segment (reviewed in
Ref. 1).
and
subunits alternate with each other to form a hexameric ring
with one nucleotide binding site located at each of the six
subunit interfaces. Part of the structure of the
subunit was also
solved, including well conserved regions of the N and C termini. The C
terminus, from residues 209 to 272 forms a single
helix that
stretches from below the base to the top of the
hexamer (see
Fig. 5). The last nine residues of this remarkably long helix are
predominantly hydrophobic in nature and pass through a greasy sleeve
formed by a ring of hydrophobic residues provided by interacting
N-terminal
barrel domains of all six of the
and
subunits.
On the basis of this unusual asymmetric structure, it was suggested
that the C-terminal helix of the
subunit forms a spindle around
which the
hexamer rotates, rotation being facilitated by the
hydrophobic (greasy) nature of the amino acids involved. That is, the
subunits provide a bearing through which the tip of the
subunit passes and within which the
subunit rotates. Although the
amino acid sequences of
subunits from different organisms show
little overall homology, segments near the N and C termini are quite
well conserved suggesting that they may be involved in forming
important contacts with other F1 subunits (3, 4).
pairs of the
hexamer also make direct contact with other regions of the
subunit to induce different conformational states of the
nucleotide binding sites at the
/
subunit interfaces. During rotation, each nucleotide binding site would sequentially alternate between three different conformational states, each state dictated by a
different type of interaction with the
subunit. Such rotation has
been predicted from kinetic studies (5, 6), has been supported by
several recent experiments (7-9), and is now widely considered to be a
general mechanistic feature of all of the F1 enzymes
(reviewed in Ref. 10).
subunit, which, in
the mitochondrial enzyme, extends through the greasy sleeve of the
hexamer. To do this we utilized an efficient reconstitution
system reported earlier (11) in which the native
subunit isolated
from CF11 was
reconstituted with an isolated
subunit complex. The cloned
subunit could effectively replace the native
subunit in
reconstitution of the core enzyme
complex.2 Eight genetically
engineered
subunits, lacking between 6 and 20 of the C-terminal
amino acids, were tested for assembly with the
subunits, and the
catalytic activities of the assembled complexes were examined. The
results demonstrate that the tip of the C terminus of the
subunit,
from residues 304 to 323 (chloroplast numbering), is not essential for
rapid turnover by CF1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits, CF1(
), were prepared from
fresh market spinach as described previously (12) and stored as
ammonium sulfate precipitates. Prior to use the proteins were desalted
on Sephadex G-50 centrifuge columns (13). The isolated
subunit (14)
was stored in the isolation buffer at 4 °C. An
complex and
the
subunit were isolated from CF1(
) as
described previously (11). The
subunit complex was recycled through the isolation procedure to ensure that trace amounts of contaminating
subunit were removed.
70 °C.
Pfu DNA polymerase and its reaction buffer were purchased
from Strategene. T4 DNA ligase and its reaction buffer were obtained
from Promega. DNase I was from Roche Molecular Biochemicals. Tryptone
and yeast extract were obtained from Difco. Urea (ultra pure) was
purchased from Fluka and hydroxylapatite from Bio-Rad. All other
chemicals were of the highest quality reagent grade available.
subunit. A 1.1-kilobase pair
BsaI-BamHI fragment of pSG101 was subcloned into the
NcoI and BamHI cleaved expression vector pET8c
(18) via an NcoI-BsaI adaptor.2 The resulting
plasmid pET8cgam bb1 was transformed into the expression host E. coli BL21(DE3)/pLysS (19). Plasmid DNA for sequencing was prepared
by alkaline-SDS lysis and polyethylene glycol precipitation (20).
were generated by "inverse" PCR with a forward primer that was
complementary to the termination codon of the atpC gene and
the downstream sequence of the pET8cgam bb1 plasmid. The reverse primer
was complementary to the required C-terminal amino acid and its
adjacent upstream sequence. PCR primers were 24-31 base pairs long and
were 5'-phosphorylated. Oligonucleotides were synthesized by
Macromolecular Resources, Colorado State University. Plasmid DNA for
PCR was prepared by ethanol precipitation after phenol:chloroform
extraction (17). PCR was carried out in 50 µl of cloned
Pfu DNA polymerase reaction buffer, which also contained 60 ng of the pET8cgambb1 plasmid, 4 mM total
MgSO4, 22 pmol of each primer, 0.4 mM dNTPs,
and 2.5 units of cloned Pfu DNA polymerase. The components
were mixed on ice and placed in a GenAmp PCR System 2400 (Perkin-Elmer)
prewarmed to 94 °C. Cycling parameters were: 94 °C for 1 min,
56 °C for 1 min, 72 °C for 12 min, for 20 cycles. The PCR product
was purified by agarose gel electrophoresis followed by electroelution
into an ISCO micro-trap. The eluted DNA was precipitated with ethanol and circularized (14). For this 100-200 ng of the DNA was incubated with 3 units of T4 DNA ligase in the T4 DNA ligase buffer overnight at
room temperature (~22 °C). The resulting plasmid was transformed into E. coli XL1-Blue cells for amplification. The amplified
plasmid was isolated using boiling lysis followed by isopropanol and
ethanol precipitation and transformed into the expression host E. coli BL21(DE3)/pLysS (19).
Mutants--
E. coli cells containing the atpC
gene were grown at 37 °C in LB medium containing
L-ampicillin (100 mg/ml) and chloramphenicol (34 mg/ml). In
mid-exponential phase growth, cells were induced with 0.1 mM isopropyl-
-D-thiogalactopyranoside for up
to 5 h. Cells were harvested by centrifugation at 4000 × g for 10 min, washed once with TE50/2 buffer (50 mM Tris-HCl, 2 mM EDTA, pH 8.0), and
resuspended in a small volume (10-15 ml) of TE50/2. Cells were lysed
by one to three cycles of freezing (at
70 °C or in a dry
ice/ethanol bath) and thawing (15). 10 mM MgCl2 and 10 mg of DNase I were added to the lysed cells, which were incubated on ice for 20 min. DNA was then sheared by sonication with a
Branson 250 sonifier for 2 × 15 s at an output of 4 and a
duty cycle of 10. After the sonication cells were kept on ice for
additional 20 min. Inclusion bodies, together with some cell debris,
were sedimented at 4000 × g for 10 min. The pellet,
containing mostly insoluble
polypeptide, was washed three times
with 25 ml of TE50/2 before solubilization.
polypeptide was dissolved in a solution containing 4 M urea, 50 mM NaHCO3-NaOH, pH 9.5, 1 mM EDTA, 5 mM dithiothreitol, 20% (v/v)
glycerol, and slowly dialyzed for 24 h against a solution containing 0.3 M LiCl, 50 mM
NaHCO3-NaOH, pH 9.5, 1 mM EDTA, 5 mM dithiothreitol, 20% (v/v) glycerol. The final
concentration of protein was approximately 1 mg/ml. The protein was
stored at
70 °C.
Mutants--
The purified
mixture was
diluted to about 100 µg/ml with a solution containing 20% glycerol,
50 mM Hepes-NaOH, pH 7.0, 2 mM
MgCl2, 2 mM ATP, and 2 mM
dithiothreitol and kept on ice. The
subunit preparation was added
dropwise to the
mixture to give a final molar ratio of
3
:1
. The mixture was gently mixed and left to sit at room
temperature (~22 °C) for 2 h. Unreconstituted subunits were
separated from the reconstituted
by anion exchange chromatography as described previously (11).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of the
spinach chloroplast ATP synthase was inserted into the pET8c expression vector and overexpressed at high levels (>100 mg/liter of cells at the
end of log-phase growth). The overexpressed protein was solubilized
from insoluble inclusion bodies into 4 M urea and recovered
by slow dialysis. The cloned protein was identical to the native
protein (11) in its ability to reconstitute with native
subunits
to form a fully active core enzyme complex.2 Eight deletion
mutants of the atpC gene were prepared and transformed into
the overexpression host and the deletions verified by sequencing each
entire gene. The truncated polypeptides were designated
D6 to
D20, according to the number of
amino acid residues missing from the C terminus. Amino acid sequences
of the C-terminal fragment of the full-length
subunit and the eight
deletion mutants are shown in Fig. 1.
Also shown in Fig. 1 is the corresponding sequence at the C terminus of
the bovine mitochondrial F1
subunit. The sequence
underlined corresponds to that part of the
subunit that
is in the immediate vicinity of the hydrophobic sleeve, the last seven
residues (267-273) actually passing through the sleeve (2). Deletion
of all ten C-terminal residues would arguably be sufficient to test the
bearing hypothesis. The C-terminal segment of
shown in Fig. 1 is
one of the most highly conserved regions among
subunits from
different species. This is evident from the more than 50% direct
sequence identity between the bovine mitochondrial and chloroplast
subunits (Fig. 1).
View larger version (34K):
[in a new window]
Fig. 1.
Amino acid sequences of the C-terminal
fragments of the subunits of the ATP
synthases from bovine heart mitochondria
(MF1-
; Ref. 14), wild-type spinach
chloroplast (CF1-
WT),
and C terminus deletion mutants of spinach chloroplast
subunit beginning with deletion of 6 residues from
the C terminus (
D6) continuing
with successive deletion of 2 residues up to 20 residues
(
D20).
Mutants--
Each of the
constructs was
tested for its ability to organize the
subunits into a stable
core enzyme complex. For this, folded
polypeptide was
incubated with the isolated
complex, and the resulting
assembly was purified by DEAE-cellulose column chromatography as
described earlier for purifying
assembled using the native
F1 subunits (11). Incubation of each of the
constructs
with the
subunits resulted in formation of an
complex,
which is eluted from DEAE-cellulose at the same salt concentration as
the native complex and which is significantly higher than that required
to elute unassembled subunits (11). The polypeptide profiles of all of
the assemblies were very similar to each other as indicated for the
D12 and
D20 assemblies, which
are compared with the
WT assembly in Fig.
2. This suggests that all of the
mutants were capable of assembling with the
subunits.
View larger version (116K):
[in a new window]
Fig. 2.
Gel electrophoresis profile of the
reconstituted, purified
assemblies and isolated
CF1(
).
Wild-type and mutant
subunits were reconstituted with the native
subunits and the protein assemblies purified by ion exchange
chromatography as described previously (9). Electrophoresis was
performed on a 10-20% Tris-glycine gel, and proteins were stained
with Coomassie Brilliant Blue R. Each lane contained 4 µg of protein.
A, isolated CF1 lacking the two small subunits,
and
(CF1(
)); WT, D12 and D20,
the
assemblies containing the wild-type
,
D12, and
D20 mutants, respectively.
D6 and
D8 with the wild-type
. Remarkably, both mutant assemblies were significantly more active
than the wild-type assembly in calcium-dependent ATP
hydrolysis. The magnesium-dependent activities of the two
mutants, however, were significantly reduced. The apparent Km and Kcat for Ca-ATP
hydrolysis of the
D6 mutant were measured and compared
with the wild-type assembly (Table I). Only the Kcat exhibited a measurable change in
the mutant.
ATPase activities of wild-type and mutant assemblies
Stimulation by sodium sulfite of the ATPase activities of the
assemblies
D10 through
D20, in the presence of either Ca2+,
Mg2+, or Mn2+ as the divalent cation substrate.
The
D10,
D12, and
D14
mutant assemblies all showed similar responses to those of the
D6 and
D8 mutant assemblies in that their
Ca-ATPase activities were significantly higher than that of the
wild-type enzyme. The maximum activity was obtained with the
D14 mutant, which had a specific activity of 55 µmol·min
1·mg
1, which is the highest
rate of Ca-ATP hydrolysis that we have ever observed with the
chloroplast enzyme. However, deletion of 16 residues from the
C
terminus resulted in a sharp decrease in Ca-ATPase activity, which
continued upon deletion of additional residues ending with an activity
that was ~19% of the wild-type control at
D20. In
contrast to the Ca-ATPase activity, the Mg-ATPase and Mn-ATPase
activities declined continuously with each additional pair of residues
deleted. Nevertheless, even after deleting 20 residues from the C
terminus, the enzyme exhibited significant rates of catalysis: 17% of
the wild-type Mg-ATPase activity and 20% of the wild-type Mn-ATPase
activity.
View larger version (35K):
[in a new window]
Fig. 3.
Relative ATPase activity of the
reconstituted, purified
constructs. All of the assays were carried out as described
under "Experimental Procedures." The columns represent the relative
ATPase activities of the different
assemblies in the presence
of calcium chloride: white, magnesium chloride;
gray, manganese chloride; black, D10 to
D20 are designations for
D10 to
D20 assemblies. Activities of the
WT (100% controls) were: Ca-ATPase, 35.5 ± 1.1; Mg-ATPase, 47.5 ± 1.5; and Mn-ATPase, 67.9 ± 5.4 µmol·min
1·mg
1. Values shown are the
averages and S.D. for five separate determinations.
subunit (14), the presence of oxyanions such as ethanol,
carbonate, or sulfite, which overcome a strong inhibition caused by
free metal ions binding to and stabilizing bound ADP at the catalytic site(s) (24). The degree of stimulation by oxyanions usually varies
between 10- and 100-fold depending on the divalent cation and the
oxyanion concentrations. The Ca-ATPase activity, however, is already
high once
is removed and is slightly inhibited by oxyanions (25).
So the magnesium- and manganese-dependent ATPase activities
listed in Table I and shown in Fig. 3 were measured in the presence of
high concentrations (25 mM) of sulfite ions. It was of
interest to examine the affects of the
deletions on the Mg-ATPase
activities in the absence of the stimulatory oxyanions. The results of
this study are shown in Table II. The Mg-ATPase activity in the absence
of sulfite was, like the Ca-ATPase activity, stimulated by deletion of
residues from the
C terminus and was highest in the
D14 mutant. The activity of this mutant was almost 4-fold that of the wild-type enzyme, and in parallel to the Ca-ATPase activity, it decreased markedly upon deletion of 16 or more residues. The
D20 mutant still retained a readily measurable
activity, which was ~45% of that of the wild-type enzyme (Table
II).
subunit and
to the fungal inhibitor tentoxin were examined, in part to evaluate the
effect of the deletions on the ability of the two inhibitors to block
activity and in part to verify that the observed activities are
representative of the normal activity of CF1, which
responds to these inhibitors with absolute specificity. The inhibitory
responses of the Ca-ATPase activities of the different constructs to a
fixed concentration (10-fold molar excess) of added
subunit are
summarized in Table III. All of the
enzyme assemblies, including the enzyme assembled with the
D20 mutant, were strongly inhibited by
, although
there was a significant variation (between 64 and 83%) in the extent of inhibition observed, and all were less inhibited than the wild-type assembly (91%). The
D14 mutant, which exhibited the
highest activity, was the least inhibited in the presence of a 10-fold
molar excess of
. However, in the presence of a 30-fold molar excess
of the
subunit, the
D14 mutant was inhibited by the
same extent as the wild-type enzyme (results not shown), indicating
that the deletion had reduced the apparent affinity of the enzyme for
but not the maximal extent of inhibition.
Inhibition by the subunit of the activity of the
assemblies
assemblies with tentoxin are
shown in Fig. 4. All of the assemblies,
with the exception of the
D20 mutant, were sensitive to
inhibition by tentoxin. There were, however, significant differences
among the mutant enzymes in the concentrations of tentoxin required to
reach maximum inhibition. The most obvious differences were with the
longer deletions. For example, a greater than 20-fold higher
concentration was required for 90% inhibition of the activities of the
D16 and
D18 mutants than that required to
inhibit the
WT to the same extent.
View larger version (14K):
[in a new window]
Fig. 4.
The effect of tentoxin on the Ca-ATPase
activities of the reconstituted, purified
assemblies.
Ca-ATPase activity was measured as described under "Experimental
Procedures." Each assembly was incubated with tentoxin to give the
indicated tentoxin/enzyme ratios in the ATPase assay medium for 10 min
at room temperature (~22 °C) then 2 min at 37 °C. The reactions
were started by addition of 5 mM CaCl2. The
ATPase activities in the absence of tentoxin were:
WT, 16.9 µmol·min
1·mg
1 (
);
D10, 35.1 µmol·min
1·mg
1 (
);
D12, 25.9 µmol·min
1·mg
1 (
);
D14, 33.9 µmol·min
1·mg
1 (
);
D16, 24.9 µmol·min
1·mg
1 (
);
D18, 15.0 µmol·min
1·mg
1 (
);
D20, 3.1 µmol·min
1·mg
1 (
).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit, more specifically the last 7-10 residues, is surrounded by a
sleeve of residues formed by part of the tightly packed
barrel
domains of the six
and
subunits. The sleeve residues, located
in the region marked A on the
subunit in Fig. 5, have an
overall hydrophobic character as do the nearby residues on the
subunit. A hydrophobic contact between
and the surrounding sleeve
could allow the
subunit to act as a spindle around which the
hexamer could rotate with minimum frictional resistance (2). The base
of the C-terminal helix of
is offset from the central axis of the
hexamer by ~7 Å, so that, provided it remained rigid, it would
sequentially and reversibly come into contact with regions of the
and
subunits during rotation to create the required asymmetry among
the nucleotide binding sites.
View larger version (38K):
[in a new window]
Fig. 5.
Cross-section through part of the beef heart
mitochondrial F1 structure indicating sites of interaction
between the ,
,
and
subunits. The
subunit contacts a
hydrophobic sleeve formed by the structures marked A on all
six of the
and
subunits. A second site of contact involves a
salt link between the
subunit and the structure marked B
on an adjacent
subunit (2).
The remarkably high amino acid sequence conservation among the and
subunits of F1 enzymes from different species, together with the fact that the structures of the
and
subunits of a thermophilic bacterium can be essentially superimposed upon those of
the mitochondrial F1 subunits (26), are cogent reasons for assuming that all of the F1 enzymes have a very similar
overall structure and utilize the same basic mechanism for ATP
synthesis. There is, however, some evidence suggesting that there may
be some minor structural differences among the F1 enzymes.
For example, site mapping studies of the chloroplast F1
using fluorescence resonance energy transfer (27) as well as chemical
cross-linking experiments (28) have indicated that cysteine 322, which
is the second last amino acid residue at the C terminus of the
CF1
subunit, is located near the base of the
hexamer, more than 60 Å away from its position in the mitochondrial
F1. The reason for this difference is not understood at
this time but is particularly intriguing given the significant amino
acid sequence homology which is apparent in the C-terminal domains of
subunits from different organisms (Ref. 4; also see Fig. 1).
Moreover, a different location for the
C terminus implies that the
idea that the C-terminal helix of
acts as a spindle for rotation is
probably not correct, at least not as originally envisioned based on
the mitochondrial F1 structure (2).
We have selectively deleted part of C terminus reasoning that if
this region of
was indeed acting as the tip of a spindle for
rotation, or if it was in any way critical for catalysis by CF1, the deletion should result in a complete loss of
catalytic activity. However, the enzyme containing mutant
subunits
missing up to 20 amino acids from the C terminus was still capable of significant catalytic activity, which, with the exception of the D20
mutant, was sensitive to specific allosteric inhibitors of CF1; a strong indication that the mutant enzymes followed
the usual cooperative catalytic pathway and that their activities were
not artifactual. It is noteworthy that a similar result was obtained
for E. coli F1 (29). In that case, membranes
containing the mutant enzyme retained a limited catalytic activity
(~10% of both ATP hydrolysis and synthesis) following deletion of 10 residues from the C terminus. Deletion more than 10 residues from the C
terminus resulted in a complete loss of activity in that case, although
the enzyme still apparently correctly assembled on the membrane. The
greater sensitivity of the E. coli enzyme to deletion of the
C terminus may reflect a different structural requirement for catalysis
by the F0-F1 complex than for the isolated F1. The activity of the E. coli F1
mutants following isolation from the membrane was not investigated in
that study.
The initial activation of enzyme turnover upon deletion of up to 14 residues from the C terminus of occurred for both the calcium- and
the magnesium-dependent ATPase activities. One likely explanation for this effect is that the deletions resulted in a partial
loosening of the structure of the enzyme to a point where it weakened
binding of the cation-ADP reaction product at the catalytic site(s) in
the interfacial region between
and
subunits. Since the off-rate
of the cation-ADP limits the overall reaction rate, the end result is
to increase the Kcat of the enzyme. This would
also explain why the sulfite-stimulated activity is inhibited rather
than stimulated by the deletions. Assuming that the presence of high
concentrations of sulfite maximally reduce the off-rate of cation-ADP
so that the on-rate of the cation-ATP now becomes rate-limiting, a
further reduction in nucleotide affinity at the catalytic site caused
by the
deletions would result in a reduction in the on-rate for the
cation-ATP substrate and thus a reduction in the
Kcat. This could also explain the reduced
apparent affinities for the
subunit and for tentoxin, which
resulted from the C-terminal deletions. Both inhibitors are known to
block cooperative release of bound nucleotides, probably by stabilizing a rigid inhibited conformation of CF1 (1, 12). Thus a
loosening of the F1 structure might favor the activated
conformation over the inhibited conformation. The structural
change resulting in altered inhibitor affinity does not
necessarily have to be large, since small perturbations of
structure, such as reduction of the
disulfide bond or single-site
cleavage of
by trypsin, are known to markedly decrease the
affinity of CF1 for the
subunit (30).
The chloroplast subunit has a glycine residue at position 310, 14 residues in from the C-terminal end (Fig. 1). Most secondary structural
prediction algorithms predict a break in the C-terminal helix of
at
Gly-310. If the CF1
were to turn back on itself at this
point, the cysteine at position 322 would face toward the bottom part
of CF1 (i.e. toward the membrane in
CF1-F0) and come close to the position of this
residue determined by fluorescence distance mapping (27). This would
create a three-helix bundle in the central cavity of the enzyme rather
than the two-helix bundle identified in the mitochondrial enzyme (2).
If this is the case, the important binding interactions between
and the
subunits identified in the mitochondrial enzyme, and which are likely to be primarily responsible for creating asymmetry among the
different catalytic sites, might also be preserved in CF1.
Deleting the 14 C-terminal residues from CF1
would
remove the third helix from the central bundle, possibly decreasing the number of contacts between
and the
subunits. This could
feasibly have the effect of loosening the structure, thereby weakening the nucleotide affinity. The sharp decrease in catalytic activity, which occurred upon deleting residues beyond the first 14, may have
resulted from an interference with the important
-
interactions. For example, the arginines at positions 254 and 256 in
MF1 are surrounded by a ring of 9 charged residues located
on six loop segments of the
and
subunits marked
"B" on one of the
subunits in Fig. 5. In the
MF1 structure, Arg-254 and Gln-255 form hydrogen bonds with
adjacent residues in the
subunit loop to form one of the few sites
of direct contact between
and the
and
subunits (2).
Assuming that CF1 has the same arrangement in this region of
, deleting residues in the near vicinity of the site of contact would be expected to significantly compromise catalytic function as was observed.
Interestingly, an earlier study of the E. coli enzyme (31)
showed that mutations near the C terminus of the subunit were able
to restore catalytic function to functionally impaired enzymes which
contained mutations near the N terminus. The original interpretation of
these results was that the two mutations are in close proximity to each
other. If this is true it would place the C terminus of the E. coli
in a location very close to that of the chloroplast
as determined by the fluorescence mapping experiments, assuming of
course that the position of the N terminus of the E. coli
is similar to that of the mitochondrial enzyme.
In conclusion, the results of this study have demonstrated that the
extreme C-terminal 20 residues of the subunit of CF1 are not essential for normal cooperative catalytic turnover by the
isolated enzyme. The results eliminate the possibility of a catalytic
mechanism that is universal to all F1 enzymes in which the
tip of the C terminus of the
subunit must act as a spindle for
rotational catalysis. The lack of functional importance of this part of
the
subunit for rapid turnover by CF1 is consistent with earlier work indicating that the conformation of the C-terminal portion of the
subunit of the chloroplast ATP synthase may differ from that of the mitochondrial enzyme. The results further suggest that
the contacts between the
,
, and
subunits of the enzyme, which are essential for rotational catalysis must be provided by
regions of the
subunit other than the extreme C terminus.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant OSR-9255223 and United States Department of Agriculture Grant 93-37306-9633.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.
Current address: Massachusetts Eye and Ear Infirmary, Harvard
Medical School, Cambridge, MA 02114.
§ Current address: Dept. of Physiology, University of California at Los Angeles, Los Angeles, CA 90095-1662.
¶ To whom correspondence should be addressed. Tel.: 785-864-3334; Fax: 785-864-5321; E-mail: markl{at}kuhub.cc.ukans.edu.
2 M. Sokolov, L. Lu, W. Tucker, F. Gao, P. A. Gegenheimer, and M. L. Richter, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CF1, chloroplast coupling factor 1;
CF1(), CF1 deficient in the
and
subunits;
PCR, polymerase
chain reation;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
WT, wild
type.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|