(Received for publication, July 10, 1995)
From the
Glu-185 of the Escherichia coli H
-ATPase (ATP synthase)
subunit was
replaced by 19 different amino acid residues. The rates of multisite
(steady state) catalysis of all the mutant membrane ATPases except
Asp-
185 were less than 0.2% of the wild type one; the Asp-
185
enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase
activity. The purified inactive Cys-
185 F
-ATPase
recovered substantial activity after treatment with iodoacetate in the
presence of MgCl
; maximal activity was obtained upon the
introduction of about 3 mol of carboxymethyl residues/mol of
F
. The divalent cation dependences of the S-carboxymethyl-
185 and Asp-
185 ATPase activities
were altered from that of the wild type. The Asp-
185,
Cys-
185, S-carboxymethyl-
185, and Gln-
185
enzymes showed about 130, 60, 20, and 50% of the wild type unisite
catalysis rates, respectively. The S-carboxymethyl-
185
and Asp-
185 enzymes showed altered divalent cation sensitivities,
and the S-carboxymethyl-
185 enzyme showed no
Mg
inhibition. Unlike the wild type, the two mutant
enzymes showed low sensitivities to azide, which stabilizes the enzyme
Mg
ADP complex. These results suggest that Glu-
185 may form a
Mg
binding site, and its carboxyl moiety is essential
for catalytic cooperativity. Consistent with this model, the bovine
glutamate residue corresponding to Glu-
185 is located close to the
catalytic site in the higher order structure (Abrahams, J. P., Leslie,
A. G. W., Lutter, R., and Walker, J. E.(1994) Nature 370,
621-628).
The H-ATPase (ATP synthase) of Escherichia
coli synthesizes ATP similar to those of mitochondria or
chloroplasts (see (1, 2, 3, 4) for
reviews). The catalytic site of the enzyme is in the
subunit of
the membrane extrinsic F
sector. Studies on mutant enzymes
indicated that Lys-
155 and Thr-
156 in the
subunit
phosphate loop or conserved glycine-rich sequence
(Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149-156; conserved
residues underlined) and Glu-
181 and Arg-
182 in the conserved
Gly-Glu-Arg sequence (residues 180-182) are essential catalytic
residues(5, 6, 7) . Affinity labeling with
ATP analogues indicated that Lys-
155 bound the
and
phosphate moiety of ATP(8) . The crystal structure (9) of the bovine F
sector reported recently is
essentially consistent with these results.
The purified
F (
or
F
-ATPase) hydrolyzes ATP through unisite (single site) or
multisite (steady state) catalysis. The multisite rate is
10
-10
-fold faster than the unisite one
due to the cooperativity of the multiple catalytic
sites(10, 11) . Conformational transmission for
cooperativity may be initiated from a specific region(s) or residue(s)
in the single catalytic site of the
subunit. Mutations near
catalytic site residues often dramatically lower the multisite rate
without changing unisite
catalysis(6, 11, 12) , possibly due to the
defective conformational transmission between catalytic sites essential
for the catalytic cooperativity. However, the role of a specific
residue or region for the cooperativity has been questionable because
all mutations so far introduced at a certain position did not always
have the same effects on multisite catalysis. A typical example is the
result of mutations at Gly-
149 of the phosphate loop; the
Ala-
149 or Ser-
149 enzyme exhibited similar ATPase activity
to the wild type, whereas the Cys-
149 enzyme had only 8% of the
wild type ATPase activity(13) , indicating that Gly-
149 is
not an essential residue for conformational transmission.
In this
study, we were interested in conserved Glu-185, which is near
essential catalytic residues (Glu-
181 and Arg-
182) described
above and substituted it with 19 different residues. Surprisingly, all
the mutants except Asp-
185 exhibited no multisite catalysis (less
than 0.2% of the wild type activity); Asp-
185 had about 16% of the
wild type membrane ATPase activity. Purified F
-ATPases with
Asp-
185, Gln-
185, and Cys-
185 residues showed unisite
catalysis with rates of a similar order of magnitude to that of the
wild type. The Cys-
185 enzyme showed substantial multisite
catalysis upon chemical modification with sodium iodoacetate (IAA). (
)These results clearly indicate that Glu-
185 is the
first residue identified as being absolutely essential for multisite
catalysis. The roles of Glu-
185 are discussed on the basis of the
properties of the mutant enzymes.
Unisite catalysis was assayed using 0.25 µM
[-
P]ATP and 0.5 or 10 mM MgSO
at 25 °C (20, 21, 22, 23) . The ATP binding
rate (k
) was measured as the decrease of ATP
in the medium using hexokinase and glucose(5) . The rate of ATP
synthesis was assayed at 25 °C by the published
method(16) . Protein concentration measurement (24) using bovine serum albumin as a standard and
polyacrylamide gel electrophoresis (25) were described
previously.
Asp-185 exhibited about 16% of
the wild type membrane ATPase activity, whereas other mutants exhibited
no membrane ATPase activity (less than 0.2% of wild type multisite
catalysis). Membrane ATPase of the Cys-
185 mutant became
detectable after incubation with IAA but not with the same
concentration of iodoacetoamide; activity of about 0.05 units/mg
protein became detectable after incubation of Cys-
185 membranes
with 100 µM IAA in 50 mM Tris-HCl, pH 8.0, at
room temperature for 10 min (ATPase activity of Cys-
185
F
-ATPase without IAA treatment, about 0.01-0.02
units/mg). This result suggests that the
S-carboxymethylated enzyme has activity. Detailed studies
of the IAA effects were then carried out below using purified
Cys-
185 F
-ATPase. Consistent with the low membrane
ATPase activity and negative growth by oxidative phosphorylation,
mutant membranes (Gln-
185 or Cys-
185) did not show
significant ATP synthesis (Table 2, right column). Membranes
treated with IAA also did not show ATP synthesis, because the mutant
membranes lost respiration-driven proton transport after IAA treatment
(data not shown). A similar IAA effect was observed for wild type
membranes.
The mutant enzymes showed unisite catalysis
with initial rates of about 50 (Gln-185), 60 (Cys-
185), and
130% (Asp-
185) of that of the wild type, and Asp-
185
F
-ATPase exhibited a k
value
(rate of ATP binding) of a similar order of magnitude as that of the
wild type (Table 2). The k
values for
Gln-
185 and Cys-
185 were slightly lower than that of the wild
type. The wild type and all the mutant enzymes except for Cys-
185
F
-ATPase showed cold chase in unisite catalysis, consistent
with the partial release of the
subunit from F
during
purification(23) . These results clearly indicate that the
major defect of the mutant enzymes is not in the catalytic reaction
itself but in the catalytic cooperativity required for multisite
catalysis.
Figure 1:
Activation of the
Cys-185 enzyme with iodoacetate. a, effects of varying
concentrations of IAA on the Cys-
185 enzyme. Mutant
F
-ATPase (0.4 mg/ml) was mixed with varying concentrations
of IAA in the presence (open circles) or the absence (closed circles) of 20 mM MgCl
. After 2 h
at 30 °C, the mixtures were diluted 200-fold with 2 mM Tris-HCl, pH 8.0, containing 2 µg/ml bovine serum albumin and
2 mM dithiothreitol, and then ATPase activity was immediately
assayed with 4 mM ATP and 10 mM MgCl
. b, effects of varying concentrations of MgCl
or
CaCl
on the IAA-dependent activation of Cys-
185
F
-ATPase. The mutant enzyme (0.4 mg/ml) was mixed with 100
µM IAA and varying concentrations of MgCl
(open circles) or CaCl
(open
diamonds). After 2 h at 30 °C, the mixtures were diluted
200-fold with the above buffer, and then ATPase activity was
immediately assayed as shown above.
Figure 2:
Binding of the
[C]carboxymethyl moiety to the Cys-
185
enzyme. F
-ATPase (1.5 mg/ml) was mixed with 100 µM sodium [2-
C]iodoacetate in 50 mM HEPES, pH 8.0, with (+) or without(-) 20 mM MgCl
. After 5 or 30 min at 30 °C, the mixture was
denatured and then subjected to polyacrylamide gel electrophoresis. The
gel was dried, and radioactivity was scanned with an image scanner,
BAS1000. The positions of F
subunits are indicated. The dye
front is indicated by an open
arrow.
The Asp-185 and S-carboxymethyl-
185
enzymes showed altered requirements for MgCl
(Fig. 3). The wild type enzyme showed the highest activity
with 2 mM MgCl
and 37% maximal activity with 10
mM MgCl
when assayed in the presence of 4 mM ATP, confirming previous results(28, 29) . On the
other hand, the S-carboxymethyl-
185 enzyme showed maximal
activity with 10 mM and only 6% activity with 2 mM MgCl
. It was of interest that S-carboxymethyl-
185 enzyme did not show Mg
inhibition, although the wild type enzyme was inhibited with a
higher Mg
concentration. On the other hand, unisite
catalysis of the S-carboxymethyl-
185 enzyme showed
similar Mg
dependence to that of the wild type
(slightly lower rate in 10 mM MgSO
; data not
shown), suggesting that Mg
had different effects on
the catalytic cooperativities of the wild type and S-carboxymethyl-
185 enzymes. The Asp-
185 enzyme
showed maximal activity with 6 mM MgCl
and was
slightly inhibited with a higher concentration. It is noteworthy that
the S-carboxymethyl-
185 and Asp-
185 enzymes had very
low ATPase activities, which were dependent on Ca
;
the Ca
-dependent activity of the mutant enzymes was
only 6-9% of the Mg
-dependent activity when
10-20 mM of the divalent cations were used. These
results suggested that Glu-
185 or its vicinity is closely related
to the Mg
binding required for multisite catalysis
and that the length of the side chains for the carboxyl moiety affected
the divalent cation dependence of the catalysis.
Figure 3:
Effects of varying concentrations of
MgCl on the mutant and wild type F
-ATPases. The
Asp-
185 (squares), S-carboxymethyl-
185 (triangles), and wild type (circles)
F
-ATPases were assayed with 4 mM ATP in the
presence of varying concentrations of MgCl
(open
symbols) and CaCl
(closed symbols). The S-carboxymethyl-
185 enzyme was obtained as described in
the legend to Fig. 1after removal of excess IAA on a centrifuge
column.
Figure 4:
Sodium azide sensitivities of the S-carboxymethyl-185 and Asp-
185 enzymes. The mutant (S-carboxymethyl-
185 (squares) and Asp-
185 (triangles)) and wild type (circles)
F
-ATPases were assayed with varying concentrations of
NaN
in the presence of 4 mM ATP and 10 mM
MgCl
. The results are expressed as relative rates of
percentage of control (without azide). The control values for the
mutant and wild type enzymes were: S-carboxymethyl-
185,
12.6; Asp-
185, 26.4; and wild type, 32.4
µmol/mg
min.
The S-carboxymethyl-185 and Asp-
185 enzyme became highly
sensitive to salts such as LiCl, NaCl, KCl, or
Na
SO
. About 70 and 90% of the activities of S-carboxymethyl-
185 and Asp-
185 enzymes were
inhibited, respectively, by 150 mM LiCl (Fig. 5),
whereas less than 10% of the wild type enzyme was inhibited. Similar
results were obtained with other salts. Therefore, the inhibition of
the mutant ATPase activities with high NaN
might be due to
the effect of the high salt concentrations.
Figure 5:
Effects of LiCl on the S-carboxymethyl-185 and Asp-
185 enzymes. The mutant (S-carboxymethyl-
185 (triangles) and
Asp-
185 (squares)) and wild type (circles)
F
-ATPases were assayed with varying concentrations of LiCl
in the presence of 4 mM ATP and 10 mM MgCl
. The results are expressed as relative rates of
percentage of control (without LiCl). The control rates were given in
the legend to Fig. 4.
Extensive mutagenesis studies on F-ATPase showed
that the Lys-
155 and Thr-
156 residues of the phosphate loop (5) and Glu-
181 (6, 7) and Arg-
182 (6) of the conserved Gly-Glu-Arg (positions 180-182)
sequence are essential residues for uni- and multisite catalysis. Thus,
the roles of other residues near the phosphate loop and the Gly-Glu-Arg
sequence are of interest. We were interested in the Glu-
185
residue, which is conserved in all the
subunits so far sequenced
(57 different species; SWISS PROT Release 30). It was surprising to
find that all the mutants except Asp-
185 were unable to grow by
oxidative phosphorylation and exhibited no functional multisite
catalysis. The purified Gln-
185 and Cys-
185
F
-ATPases also exhibited no multisite catalysis.
Cross
and co-workers (27) showed recently that E. coli F-ATPase, similar to chloroplast or mitochondrial
F
(30) , is inhibited by the catalytic site-bound
Mg
ADP. They proposed that the effect of Mg
ADP should be
considered before kinetic results are interpreted. However, we think
that the possibility of highly increased Mg
ADP inhibition of
mutant enzymes is low because phosphoenolpyruvate and pyruvate kinase
(treatment to release Mg
ADP) did not increase the activities of
the Gln-
185 and Cys-
185 F
-ATPases. Furthermore,
the S-carboxymethyl-
185 and Asp-
185 enzymes were not
inhibited by Mg
, as discussed below.
Despite the
absence of multisite catalysis, the purified mutant
F-ATPases (Gln-
185 and Cys-
185) retained
substantial unisite catalysis. Furthermore, multisite catalysis of the
Cys-
185 enzyme was recovered on the introduction of a
carboxymethyl group after treatment with IAA, whereas the same
treatment did not increase the unisite catalysis of the enzyme. Taken
together with the observation of Asp-
185 mutant, these results
indicate that the carboxyl moiety at position 185 is required for
catalytic cooperativity. It is noteworthy that Glu-
185 is the
first residue found to be essential for multisite catalysis. Similar
residues were not identified previously because multisite catalysis was
lost to varying degrees depending on the residues
substituted(16, 31) .
MgCl had a
dramatic effect on the activation of Cys-
185 F
-ATPase
with IAA; ATPase activity obtained with MgCl
was about
5-fold higher than that on incubation without it. About 3 and 2 mol of S-carboxymethyl residues were incorporated into the mutant
enzyme, respectively, on incubation with and without MgCl
,
respectively. Thus, all three Cys-
185 residues bound carboxymethyl
moieties in the presence of Mg
and became fully
active, consistent with the requirement of three active
subunits
for multisite activity(11) .
The S-carboxymethyl-185 and Asp-
185 enzymes had
interesting properties. Their ATPase activities showed divalent cation
dependences different from those of the wild type: the two mutant
enzymes required more MgCl
for maximal multisite catalysis
than the wild type and exhibited very low CaCl
-dependent
activity. Interestingly, S-carboxymethyl-
185 enzyme
activity is accelerated by excess MgCl
(4 mM ATP
and 10 mM MgCl
), suggesting the importance of free
Mg
ion. On the other hand, the divalent cation
requirements of the mutant enzymes for unisite catalysis were similar
to those of the wild type (data not shown). Thus, a change in the side
chain length of the carboxyl moiety (Asp, Glu, and S-carboxymethyl) at position 185 affected the divalent cation
requirement for multisite catalysis. These results suggest that the
carboxyl group of the Glu-
185 residue may be close to
Mg
at the catalytic site or forming the
Mg
binding site. The bovine glutamate (position 192)
residue corresponding to E. coli Glu-
185 is actually
located in the catalytic site close to the Mg
ion in
the x-ray structure of bovine F
-ATPase (9) . Thus,
we propose that the Glu-
185 residue contributes to the catalytic
cooperativity through Mg
binding. In this regard,
Weber and co-workers reported that the cooperativity for ATP binding is
dependent on Mg
(32) .
In contrast to the
strong inhibition of the wild type enzyme by MgCl at higher
than 3 mM (about 60% inhibition with 5 mM
MgCl
), excess MgCl
did not inhibit the
multisite catalysis of S-carboxymethyl-
185 and only
slightly inhibited the Asp-
185 enzyme (about 10% inhibition with
10 mM MgCl
). The Mg
inhibition
of the ATPase activity of the wild type enzyme was shown to be due to
the Mg
ADP binding to the catalytic site(27) . Similar to
wild type enzyme, S-carboxymethyl-
185 and Asp-
185
enzyme retained about five bound nucleotides detected after passing
through a centrifuge column (data not shown). Thus, the low
Mg
inhibition of the mutant enzymes suggests that the
affinity of the Mg
ion to the catalytic site bound
ADP was lower in the mutant than in the wild type. In addition, the
azide sensitivities of the S-carboxymethyl-
185 and
Asp-
185 enzymes were decreased by more than 2 orders of magnitude.
Azide inhibits F
-ATPase by stabilizing the
enzyme-Mg
ADP complex(27, 30) , suggesting that
the low azide sensitivity of Asp-
185 or S-carboxymethyl-
185 is because the mutant
enzyme-Mg
ADP complex is not stabilized by azide. Previously, we
reported that azide did not inhibit unisite catalysis(22) .
Thus, azide may change the environment around the Mg
ion binding site including the Glu-
185 residue, resulting in
strong inhibition of the catalytic cooperativity through stabilization
of the enzyme-Mg-ADP complex.