(Received for publication, May 10, 1995; and in revised form, June 28, 1995)
From the
The elongation factor 2 from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-2) binds
[H]GDP at 1:1 molar ratio. The bound
[
H]GDP is displaced by GTP or its nonhydrolyzable
analogue guanyl-5`-yl imidodiphosphate (Gpp(NH)p) but not by ATP, thus
indicating that only the two guanosine nucleotides compete for the same
binding site. The affinity of SsEF-2 for
[
H]GDP is higher than that for GTP and Gpp(NH)p.
On the contrary, in the presence of ribosomes the affinity of SsEF-2 for GDP is lower than that for Gpp(NH)p. SsEF-2 is endowed with an intrinsic hardly detectable GTPase
activity that is stimulated by ribosomes up to 2000-fold. The
ribosome-stimulated SsEF-2 GTPase (GTPase
) reaches
a maximum at pH 7.8 and is not affected by ATP but is competitively
inhibited by either GDP or Gpp(NH)p. Both K
for [
-
P]GTP and k
of GTPase
increase with increasing
temperature, and the highest catalytic efficiency is reached at 80
°C. The ADP-ribosylation of SsEF-2 does not significantly
affect either the binding of GDP and GTP or the kinetics of the
GTPase
. A hypothesis on the stimulation by ribosome of SsEF-2 GTPase is proposed.
In the course of the protein synthesis the translocation of the
peptidyl-tRNA from the A-site to the P-site of the ribosome is
catalyzed in eubacteria by the elongation factor G and in eukarya and
archaea by the elongation factor 2, both in the GTP-bound form.
Following the interaction of the elongation factor-GTP complex with the
ribosome, the GTP is hydrolyzed to GDP and P, and the
inactive GDP bound form of EF-2/EF-G (
)leaves the ribosome.
Therefore, the biological activity of EF-2/EF-G is regulated by the
alternate binding of GDP and GTP(1, 2, 3) .
In a previous study we have reported that the primary structure of the elongation factor 2 in the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-2, formerly aEF-2) possesses the consensus sequences typical of the GTP-binding proteins(4) . In the present report we show that SsEF-2 is able to bind guanosine nucleotides and to promote the hydrolysis of GTP in the presence of ribosomes as well. These findings support the hypothesis that the translocation step in the elongation of the polypeptide chain in archaeal systems involves similar mechanisms as those described for eukarya and eubacteria(1, 5, 6, 7) .
The following buffers were used: buffer A, 25
mM Tris/HCl, pH 7.8, 10 mM NHCl, 10
mM Mg(CH
COO)
; buffer B, 50 mM Tris/HCl, pH 7.8, 2 mM Mg(CH
COO)
,
50 mM KCl, 1 mM dithiothreitol; buffer C, 20 mM Tris/HCl, pH 7.8, 0.4 mM NH
Cl, 10 mM Mg(CH
COO)
, 0.05 mg/ml poly(U), 1 mM spermine, 1 mM ATP.
Ribosomes were prepared
according to the procedure described elsewhere (10) with an
additional ultracentrifugation step at 100,000 g at 20
°C in buffer A supplemented with 18% sucrose and 500 mM NH
Cl. The pellet was then suspended in buffer A
containing 50% glycerol and stored at -20 °C. One A
unit was taken to correspond to 25 pmol of
ribosomes.
Poly(U)-dependent poly(Phe) synthesis was performed as described previously(6) .
The amount of the SsEF-2[
H]GDP complex, formed upon
incubation of [
H]GDP with SsEF-2 in
buffer B, was estimated by counting the radioactivity sticking on
nitrocellulose filters(11) , using a Packard Tri-Carb 1500
liquid scintillation analyzer. The apparent dissociation equilibrium
constant, K`
, of the SsEF-2
GDP complex was determined by titrating SsEF-2 with [
H]GDP; the results were
plotted according to the Scatchard equation(12) , and both the K`
of the complex and the number of
binding sites for [
H]GDP on SsEF-2 were
derived. The values of K`
of the SsEF-2
GTP and SsEF-2
Gpp(NH)p complexes
were determined by competitive binding experiments between
[
H]GDP and GTP or Gpp(NH)p following the
procedure described previously for SsEF-1
(6) ;
other experimental details are given in the legend to Fig. 1.
Figure 1:
Binding of
[H]GDP, GTP, and Gpp(NH)p to SsEF-2. A, kinetics of the [
H]GDP binding to SsEF-2. 600 µl of buffer B containing 50 µM [
H]GDP (specific activity, 430 cpm/pmol),
and 0.38 µMSsEF-2 were incubated at 0 °C; at
the times indicated 90-µl aliquots were withdrawn, and the amount
of SsEF-2
[
H]GDP formed was
determined as described under ``Methods.'' B,
competitive binding on SsEF-2 between
[
H]GDP and ATP, GTP, or Gpp(NH)p. 160 µl
buffer B containing 6.5 µM [
H]GDP
(specific activity, 900 cpm/pmol) and 0.2 µMSsEF-2 were incubated in the presence of increasing
concentrations of GTP (
), Gpp(NH)p (
), or ATP (
).
After 30 min of incubation at 60 °C, a time chosen to be sure that
the equilibrium was attained, the amount of the SsEF-2
[
H]GDP complex formed was
determined in duplicate on 70-µl aliquots as described under
``Methods.'' Under these conditions the amount of GTP
hydrolyzed by SsEF-2 was negligible (from 0.04 to 1.7%). C, Scatchard plot for the dissociation of the SsEF-2
[
H]GDP complex. 300 µl
of buffer B containing 1 µMSsEF-2 and
0.5-40 µM [
H]GDP (specific
activity, 250-7000 cpm/pmol) were incubated for 30 min at 60 °C; at
each concentration of [
H]GDP the amount of SsEF-2
[
H]GDP formed was determined
in triplicate on 90-µl aliquots. The data were analyzed according
to the Scatchard equation; r, [
H]GDP
bound/SsEF-2 (by moles) at
equilibrium.
The ribosome-stimulated GTPase activity of SsEF-2 was
measured in buffer C; ATP was maintained throughout even though it was
essential only when the GTPase was tested in the presence of partially
purified ribosomes. The [-
P]GTP hydrolyzed
was estimated from the amount of
P
released,
measured by the charcoal method(13) ; the reaction was followed
kinetically at 60 °C. Blanks run in the absence of SsEF-2
were subtracted. The rate of [
-
P]GTP
breakdown was calculated from the slope of the linear kinetics of the
hydrolytic reaction. The values of K
and K`
for
[
-
P]GTP, k
and K`
of GDP and Gpp(NH)p for GTPase
were determined as reported previously (13) .
The
ADP-ribosylation of SsEF-2 was performed as previously
described (8) using 12 µMSsEF-2, 100
µM NAD, and 1 µM diphtheria
toxin. The ADP-R-F2 was freed of diphtheria toxin, nonmodified SsEF-2, and NAD
excess by chromatography on a
Mono Q column equilibrated with 20 mM Tris/HCl, pH 7.8,
connected to a fast protein liquid chromatography system (Pharmacia
Biotech Inc.) and eluted by a linear 0-150 mM KCl
gradient in the same buffer. ADP-R-F2 was collected at about 60 mM KCl, whereas the residual SsEF-2 was eluted at about 100
mM KCl.
Protein concentration was determined by the method of Bradford (14) using bovine serum albumin as standard.
Figure 2:
Hydrolysis of
[-
P]GTP promoted by SsEF-2 in the
presence of ribosomes. 250 µl of buffer C containing 100 µM [
-
P]GTP (specific activity, 180
cpm/pmol) were incubated at 60 °C, and the amount of
P
released was determined on 40-µl
aliquots as described under ``Methods.'' A, time
course of the GTPase activity in the presence of 0.1 µMSsEF-2 (
), 0.5 µM ribosomes (
), or
0.1 µMSsEF-2 plus 0.5 µM ribosomes
(
). B, GTPase activity of ribosomes at increasing
concentrations in the absence (
) or in the presence (
) of
0.1 µMSsEF-2. Blanks were
subtracted.
Figure 3:
Effect of pH on SsEF-2
GTPase. The reaction mixture contained 0.5 µM ribosomes, 100 µM [
-
P]GTP
(specific activity, 80 cpm/pmol), and 0.1 µMSsEF-2 in 50 µl of buffer C in which 20 mM Tris/HCl was substituted for 50 mM of each of the
following buffers: sodium citrate (
), glycine/NaOH (
),
imidazole/acetate (
), Tris/HCl (
). The reaction was carried
out for 3 min at 60 °C, and the
P
released
was determined; blanks were subtracted.
In order to evaluate the effect of temperature on
the rate and on the affinity of GTPase for
[
-
P]GTP, K
and k
were determined by
Lineweaver-Burk plots that in the temperature range 50-91 °C
were all linear. The data reported in Table 1show that at
increasing temperatures both k
and K
increased; the catalytic efficiency,
expressed by the ratio k
/K
, reached a
maximum at 80 °C. The possibility that the increase with
temperature of K
of SsEF-2
GTPase
for [
-
P]GTP could also
be due to a competitive inhibition by the GDP produced upon hydrolysis
of [
-
P]GTP was ruled out since such an
amount was negligible compared to that required to get a significant
increase of K
(see below). A linear
Arrhenius plot gave a value for the energy of activation equal to 85
kJ/mol; at 87 °C the calculated values for
H*,
S*, and
G* were 82
kJ
mol
, -3
J
mol
K
, and 83
kJ
mol
, respectively. These energetic data were
quite close to those reported for Escherichia coli EF-G
GTPase
at 30 °C(19) , thus indicating that the
two enzymatic systems have similar energetic requirements.
It is
remarkable that at the growth temperature of the respective source
organisms, the affinity for GTP of GTPase is very similar
between S. solfataricus SsEF-2 (K
= 32 µM) and E. coli EF-G (K
= 41 µM) (20) .
Figure 4:
Competitive inhibition of GTPase by GDP and Gpp(NH)p. 200 µl of buffer C contained 0.07
µMSsEF-2, 2-200 µM [
-
P]GTP (specific activity,
84-7600 cpm/pmol), in the absence (
) or in the presence of
30 µM GDP (
) or 32 µM Gpp(NH)p
(
); the amount of [
-
P]GTP hydrolyzed
was followed kinetically at 60 °C on 40-µl aliquots. The
reaction rate (v) was expressed as picomoles of
[
-
P]GTP hydrolyzed per
min.
To our knowledge, this is the first report on the characterization
of an archaeal hyperthermophilic EF-2 regarding either its affinity for
GDP and GTP or its ribosome-stimulated GTPase activity. Our recent
report that the elongation factor SsEF-1 binds GDP and
GTP (6) and elicits an intrinsic GTPase (13) and the
data reported in the present work prove that the S. solfataricus elongation factors are functionally similar to the corresponding
bacterial and eukaryal counterparts (1, 3, 5, 7) despite that the
temperature for optimum growth of S. solfataricus is about 50
°C higher.