Kinetic Analysis of the Hydrolysis of GTP by p21N

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Kinetic Analysis of the Hydrolysis of GTP by p21N
THEJOURNAL OF BIOLOGICAL
CHEMISTRY
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 263, No. 36,Issue of December 25, pp. 19718-19722,1988
Printed in U.S.A.
Kinetic Analysis of the Hydrolysis of GTP by p21N-"
THE BASAL GTPase MECHANISM*
(Received for publication, June 14,1988)
Susan E. Neal, John F. Eccleston, Alan Hall$,and Martin R. Webb4
From the Division of Physical Biochemistry, National Institutefor Medical Research, The Ridgeway, Mill Hill, London NW7
1AA and $The Institute of Cancer Research, Chester Beatty Laboratories, Fulhum Road, LondonS W3 6JB, United Kingdom
The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal
p21N-" (Gly-12) and an oncogenic mutant (Asp-12):
namely GTP binding, hydrolysis, phosphate release,
and GDP release. By extrapolation from data at lower
temperatures, the GTP association rate constant at
37 "cis 1.4 X 10' M" s-l for the normal protein and
4.8 X 10' M-' s-lfor the mutant. Other rate constants
were measured directly at 37 OC, and three processes
have similar slow values. GTP dissociation is at 1.0 X
s-l (normal) and 5.0 X
s-l (mutant). The
hydrolysis step is at 3.4 X
s-I.(normal) and 1.5 X
s-l (mutant). GDP dissociates at 4.2 X
s"
(normal) and 2.0 X
s" (mutant). GDP association
rate constants are similar to those for GTP, 0.5 X 10'
1"
s-l for normal and 0.7 X 10' M" s-' for mutant.
Both hydrolysis and GDP release therefore contribute
to rate limitation of the basal GTPase activity. There
are distinct differences (up to &fold)between rate
constants for the normal and mutant proteins at a
number of steps. The values are consistent with the
reduced GTPase activity for this mutant and suggest
little difference between normal and mutant proteins
in the relative steady-stateconcentrations of GTP and
GDP complexes that may represent active and inactive
states. The results are discussed in terms of the likely
role of p21" in transmembrane signalling.
understand the importance of various nucleotide complexes
and how they interconvert. Scheme 1 shows the minimal
number of steps for the GTPase activity, with p21" represented by R.
R
+ GTP + R.GTP R.GDP.Pi
1
2
4
3
P.GDP
e R + GDP
4
SCHEME
1
Each step is numbered so that the forward and reverse rate
constants for step i are
k+;and k-;, respectively. The hydrolysis
occurs with inversion of configuration and so is unlikely to be
via a phosphoenzyme': each process of nucleotide binding,
hydrolysis, and product release is shown as a single step. The
GTPase mechanism may be more complex than this scheme
because, forexample,noproteinconformationchanges
are included, although such changes have been found with
other transducing nucleoside triphosphatases. However, this
scheme is sufficient, based on current knowledge of the system. We describe here the measurement of rate constantsfor
nucleotide binding and release, and the hydrolysis step, for
both the normal (Gly-12) protein and also the transforming
single pointmutant (Asp-12) for p21N-"d,produced from
expression in Escherichia coli. A complete description of the
kinetic scheme of this basal GTPase is important for understanding differences between normal and mutant proteins
and as abasis for understanding which steps are modified by
interaction with receptors or effectors.
p21" is a 21,000 molecular weight protein with a single
polypeptide chain. When expressed in eukaryotic cells, it is
palmitoylated at a cysteine near the C terminus probably to
facilitate membrane association (Willumsen et al., 1984). Although p21 is expressed in many cells, its function is not
known, but itis likely to have a role in transmembrane signal
transduction associated with cell growth with a mechanism
analogous to G proteins (reviewedby Stryer and Bourne,
1986; Gilman, 1987). Certain single point mutants are found
in many cancer cells (reviewed by Barbacid, 1987), but the
molecular basis is not known by whichthese mutations effect
the presumed signal transduction, and so cell growth.
With G proteins (and hence by analogy, p21") the change
from inactive to active states with respect to signal transduction is induced by GTP binding. The GTP-bound state can
then activate an effector protein, although, at present, effectors in the p21 system have not been clearly identified. Because of this nucleotide-dependent activation, we wish to
We have defined a minimal mechanism for the hydrolysis
of GTP by p21N-" and measured most of the elementary rate
constants of this mechanism for both the normal (Gly-12)
protein and a transformingsingle point mutant (Asp-12).The
values of these rate constantsat 37 "C together with the GTP
and GDP association equilibrium constants calculated from
k+l/k-l and k-4/k+4,respectively, are shown in Table 1. The
missing rate constantsrelate to Pi release and rebinding which
are discussed below.
An essential feature of this work is that experiments were
performed using the apoprotein of p21N-" prepared by the
method of Feuerstein et al. (1987a) combined with the use of
substoichiometric amounts of nucleotide (ie.,single turnover
*This workwas supported by the Medical Research Council,
United Kingdom. The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
3 To whom correspondence should be addressed.
J. Feuerstein, R. S. Goody, M. R. Webb, unpublished results.
Portions of this paper including "Materials and Methods," "Results," Tables 2-4, and Figs. 1-5) are presented in miniprint at the
end of this paper. Miniprint is easily read with the aid of a standard
magnifying glass. Full size photocopies are included in the microfilm
edition of the Journal thatis available from Waverly Press.
EXPERIMENTALPROCEDURES
AND RESULTS*
DISCUSSION
19718
Kinetics of GTP Hydrolysis byp21""
TABLEI
Summary of rate constants ofp21 "N GTPase scheme, at 37 "C
Normal
k+, (M-' s")"
1.4 X
1.0 X
3.4 X
<0.17 X
4.2 X
0.51 X
14.0 X
1.2 X
Mutant
4.8 X 10'
10'
10-~
5.0 X
1.5 X 1 0 - ~
10-~
10" <0.08 X
10-4
2.0 X 10-4
0.70 X 10'
10'
9.6 X 10"
10"
10"
3.5 X 10"
(s-')
k+, (5-l)
k-2 ( s - ' ) ~
k+4 (s-')
k-l (16's-')O
GTP association constant (M-')
GDP association constant ("')
a These are obtained from values at 0 and 10 "Cby extrapolation
of an Arrhenius plot.
Limit from the measurement of the equilibrium constant for the
hydrolysis step 2 being completely (>95%) in favor of products.
k-1
19719
may correspond to theactive and inactive forms of the protein.
R.GTP
k+P
R.GDP
k+4
SCHEME2
Only these intermediates are present at high concentration,
because release of GDP will be immediately followed by the
rapid binding of GTP, andwe are assuming Pi release is rapid.
So [R.GTP]/[R.GDP] = k+4/k+2. Despite the 2-fold difference between k+4 and k+2 in the normal and mutant protein
(Table I), there is little effect on the ratio of these species:
1.2 for the normal protein, 1.3 for the mutant.
As mentioned in the Introduction, Scheme 1 only includes
intermediates with different nucleotide states. Currently,
there is no evidence for significant protein conformation
conditions). This additional purification procedure also lesschanges, although such changes are likely to occur, in order
ens problems due to protein inhomogeneity and removes to alter the p21 from active to inactive states in terms of
problems due to themixture of nucleotides (mainly GDP and signal transduction. An example of a nucleoside triphosphadGDP) bound to theE. coli-expressedp21. Wehave been able tase transducing system with well-recognized conformation
to measure the dissociation rate constant of GTP ( k - l ) , the changes is myosin (reviewed by Trentham et al., 1976; see
rate of GTP cleavage on the protein (k+2) and put an upper also Sleep and Hutton, 1980). With p21, the large free energy
limit on the value of k-2 using cold chase and single turnover change going from R. GTP to
R . GDP .Pi and R . GDP might
conditions. The values of the GDPdissociation rate constant well include a protein conformation change. We are probing
(k+4) have also been measured and agree with previously this using fluorescent-nucleotide analogues and oxygen-expublished results obtained mainly with p21Ha-" (Sweet et al., change studies.
1984; Feuerstein et al., 1987a, 198713). Values for the associaData from amino acid sequences and crystallographic studtionrateconstants
for GDP and GTP were obtained by ies indicate similarities between the nucleotide binding sites
from E. coli (Halmixing nucleotide with apoprotein. At 0 "C, values were ob- of p21 and elongation factor Tu (EF-Tu)~
liday, 1984; Jurnak, 1985; McCormick et al., 1985; de Vos et
tained over a range of nucleotide concentrationsandare
similar to those obtained by Feuerstein et al. (1987a) at a al., 1988). The two proteins probably have a similar common
single concentration. We have therefore shown these are true chemical mechanism for the hydrolysis: direct, in-line phospho transfer from GTP to water, with no phosphoenzyme
second-order rate constants.
It should be noted that thevalues of GDP dissociation rate intermediate (Eccleston and Webb, 1982).' However, the kiand GTP cleavage rate for both normal and mutant protein netic data reported here for p2lN." show thatthere are
are such that p21".GTP cannot be prepared by incubating significant differences between the interaction of the two
~21". GDP with excess GTP since appreciable hydrolysis will proteins with guanosine nucleotides. Both GTP and GDP
bind to p21N"- at similar rates which at 37 "C would be close
occur during the exchange process. This feature has implicato those expected for diffusion controlled reactions (Guttions for experiments which require a GTP complex to test
freund, 1987), whereas the association rate constants of GTP
for the active state ofp21" and to define structural differ- and GDP to EF-Tu at0 "C are 1 X lo4 M" s" and 2.6 X lo6
ences from the p21".GDP complex.
M" s-l, respectively (Fasano et al., 1978). This difference
The results in this paper do not address step 3,which
between the rate constants of GTP and GDP binding to EFinvolves Pi release. We are measuring the rate of Pi release Tu has been confirmed at physiological concentrations of
using phosphate-water oxygen exchange measurements reactants using chromophoric analogs of GTP and GDP with
(Webb et al., 1986), and these indicate that Pi release may be rapid-reaction techniques (Eccleston, 1981). The results were
fast andso would not contributeto ratelimitation of the basal interpreted as showing a two-step binding mechanism, that
GTPase activity? This is assumed in the discussion below.
is, binding itself followed by a conformation change of the
A comparison of rate constants for the normal and mutant complex. Distinctive differences in solution structure between
proteins shows differences at most steps. Thus, there is a 3- EF-Tu .GTP and EF-Tu. GDP have been reported (Kaziro,
5-fold increase in both GTP binding and release steps, but 19781, but no such differences have yet been documented for
this only leads to a 1.5-fold difference in the equilibrium p21. The rate constant for the cleavage step of EF-Tu is 5 X
s" at 24 "C (Kalbitzer et al., 1984) although when the
association constant. The two steps which contribute most to
the steady-state rate of GTP hydrolysis, k+z and k+4,are both EF-Tu. GTPcomplex with aminoacyl-tRNA binds to the A
reduced by a factor of 2 in the mutant. Under conditions site of programmed ribosomes, this rate is accelerated to 50
prevailing in the cell when [GTP] >> [GDP], thesteady-state s" (Eccleston et al., 1985). The rateof GDP release from EFGTPase rateis given byk+2.k+J(k+Z k+4),and so our results Tu in uiuo is also accelerated by interaction with another
predict a decrease in this rateby 50% for the mutantprotein. protein factor, EF-Ts (Chau et al., 1981). It remains to be
Our steady-state measurements indicate a reduction by 70% seen whether other cellular components can effect the rate of
(data not shown), although calculation of steady-state rates the cleavage step of p21 or of the rateof release of nucleotide.
Recently, there has been reported a widespread, cytosolic
as kcat values are critically dependent on the accurate measprotein GAP ("GTPase activating protein"), which interacts
urement of active p21" concentrations.
with normal p21", increasing GTPase activity, but does not
Also under these conditions, we can consider a simplified seem to affect transformingmutants(Traheyand
Mcscheme (Scheme 2) to relate the major intermediates that
+
J. Hunter and M. R. Webb, unpublished results.
The abbreviations used are: EF-Tu, elongation factor Tu; HPLC,
high performance liquid chromatography.
Kinetics
19720
of GTP
Cormick, 1987; Cales et al., 1988; Adari et al., 1988). There is
evidence in this work that GAP is an effector
for the ras
signal transducing
system. It will be of great interest to see
how interaction
with GAP affects individual
rate constants
of the ~21” GTPase mechanism. For example, if the GTPase
stimulation
is preferentially
in step 2, this may effect the
quasiequilibrium
of Scheme 2 and cause deactivation
of ~21.
Conversely, interaction
with activated receptor, by accelerating step 4, would induce the postulated
active GTP-bound
conformation
via nucleotide exchange.
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Adari, H., Lowy, D. R., Willumsen,
B. M., Der, C. J., and McCormick,
F. (1988) Science 240,518~521
Barbacid,
M. (1987) Annu. Reu. Biochem.
66, 779-827
Calis, C., Hancock,
J. F., Marshall,
C. J., and Hall, A. (1988) Nature
332,548~551
Chau, V., Romero,
G., and Biltonen,
R. L. (1981) J. Biol. Chem. 266,
5591-5596
de Vos, A. M., Tong, L., Milburn,
M. V., Matias,
P. M., Jancarik,
J.,
Noguchi,
S., Nishimura,
S., Miura,
K., Ohtsuka,
E., and Kim,
S.-H. (1988) Science 239,888-893
Eccleston,
J. F. (1981) Biochemistry
20, 6265-6272
Eccleston,
J. F., and Webb, M. R. (1982) J. Biol. Chem. 257, 50465049
Eccleston,
J. F., Dix, D. B., and Thompson,
R. C. (1985)
J. Biol.
Chem. 260,16237-16241
Edsall,
J. T., and Gutfreund,
H. (1983)
Biothermodynamics:
The
Study of Biochemical
Processes
at Equilibrium,
John Wiley & Sons,
New York
Fasano,
O., Bruns,
W., Crecket,
J., Sander,
G., and Parmeggiani,
A.
Hydrolysis by ~21”
(1978) Eur. J. Biochem.
89,557-565
Ferguson,
K. M., Higashijima,
T., Smigel, M. D., and Gilman,
A. G.
(1986) J. Biol. Chem. 261, 7393-7399.
Feuerstein,
J., Goody,
R. S., and Wittinghofer,
A. (1987a)
J. Biol.
Chem. 262,8455-8458
Feuerstein,
J., Kalbitzer,
H. R., John, J., Goody, R. S., and Wittinghofer, A. (1987b) Eur. J. Biochem.
162,49-55
Gilman,
A. G. (1987) Annu. Reu. Biochem.
66,615-649
Gutfreund,
H. (1987) Biophys.
Chem. 26, 117-121
Hall, A., and Self, A. J. (1986) J. Biol. Chem. 261, 10963-10965
Halliday,
K. R. (1984) J. Cyclic Nucleotide
Res. 9,435-448
Jurnak,
F. (1985) Science 230,32-36
Kalbitzer,
H. R., Goody,
R. S., and Wittinghofer,
A. (1984) Eur. J.
Biochem.
141,591-597
Kaziro,
Y. (1978) Biochim.
Biophys.
Acta 506,95-127
McCormick,
F., Clark,
B. F. C., La Cour, T. F. M., Kjeldgaard,
M.,
Norskov-Lauritsen,
L., and Nyborg,
J. (1985) Science 230, 78-82
Sedmark,
J. J., and Grossberg,
S. E. (1977) Ad.
Biochem.
79,544-
552
Sleep, J. A., and Hutton,
R. L. (1980) Biochemistry
19, 1276-1283
Sweet, R. W., Yokoyama,
S., Kamata,
T., Feramisco,
J. R., Rosenberg,
M., and Gross, M. (1984) Nature
311, 273-275
Stryer,
L., and Bourne,
H. R. (1986) Annu. Reu. Cell Bid. 2,391-419
Taylor,
R. S., and Weeds, A. G. (1977) FEBS I&t. 76.55-60
Trahey,
M., and McCormick,
F. (1987) Science 238,542-545
Trentham,
D. R., Eccleston,
J. F., and Bagshaw,
C. R. (1976) Q. Rev.
Biophys.
9,217-281
Webb, M. R., and Eccleston,
J. F. (1981) J. Biol. Chem. 266, 7734-
7737
Webb, M. R., Hibberd,
M. G., Goldman,
Y. E., and Trentham,
D. R.
(1986) J. Biol. Chem. 261,15557-15564
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B. M., Norris,
K., Papageorge,
A. G., Hubbert,
N. L., and
Lowy, D. R. (1984) EMBO
J. 3,2581-2585
Kinetics of GTP Hydrolysis byp21"
neasvreaent
19721
GTp hVdrolVSis
For the single-turnover and cold-chase experiments. samples (125 "1) from
the reaction m x t u r e were taken at tined intervals after addition Of nucleotide
and added to 150 u l ice-cold 1% perchloric acid. The SOlutlOn was immediately
adjusted to pH 4 by the addition of 60 ill 0.2811 NaOH/0.12 LIR sodium acetate to
stabilize the nucleotides. 10" 01 GMP, 20" 01 GDP and 4Onmal GTP were added as
carrier to each sample.
The ['HIGTP
and ['HIGDP
in each aliquot were analyzed
fro
by anion exchange HPLC, on a Partisil-lo SAX coiunn (250 x 4.611
.in-'
Whatman). eluting with 0.5M ( N H 4 1 2 H m adjusted to pH4.0 with ncl at 2
(Webb and Eccleston. 1981).
The elulion was monitored by absorption aif 254nm.
Fractions (1m11 were collected and radioactivity was Counted in 20nl Beckman
"Readysafe" scintillation Cocktail. A white precipltate forms due to the
mi
counting aver a period of 24 h.
Kinetic data
exponential using
-3
in this experiment and others were fltted to a single
"on-linear least squares procedure (Edsall and Gutfreund,
20
1983).
0'
a & GDp
kinetics
measurement Or
50
100
200
150
250
k+l & k-41
The second-order rate Constants for the b'nding Of GT and CDP to p21N-=
were msaaured were by incubating p21 with I'HlGTP
or I'HICDP
and analysing
samplas of the
reaction mixture for protein-bound nucleotide at time intervals.
For ease of analysis.
using the filter binding assay (Hall and Self, 1986).
such experiments are usua11y done usinq pseudo-first-order conditions in which
there is an excess Of nucleotide over protein. For GTP binding, the obsarved
k
k+l[GTPI + k-l.
A plot of k against [GTP]
rate Of complex formation
therefore gives a straiqdt I h e Of slope ktl and intercept .
:
k
A similar
equation applies forGDP.
-
Normal (Nl and nutant (g) p 2 l N - W apaprotein ( 1 OM] in buffer A Yere
incubated at 37OC with 0.8
H [ HIGTP. Aliquots were removed at various time6
and analyzed for ('HIGTP and ['KIGDP.
The solid liner are the bast fit of the
data to a sinale exoonential.
Data points were also obtained at 360 min,
which gave 0%-GTP.
In the case of nucleotide binding to p21, this approach is limited by the
fact that at high concentrations Of nucleotide or at high temperatures, the
rate Of binding becomes too fast to measure by the manual sampling pchniques
a ailable. However, we have been able to measure this process for [ HIGDP and
[%]GTP binding to p21 over a 1D-fold range Of nucleotide concentrations at
ooc
.
Pig. 2A shows the binding of I1 nll [3H)GDP to normal a r d mutant p21 at
80th p ~ o c e 8 s e scan be fitt d to a single exponential. ?he experiment was
repeated at 2.2 nn and 22 nn [%]CDP and the effect of concentration on the
observed rate of binding is shown in Fig. 28. War this l m i t e d concentration
0%.
TEMP ("C)
to the dieaociation rate
conatant k- o r k+4. These rate constants are measured
more precisely below. MearvramantE &r=
Is0 made at 0% although these could
be at only the lowest concentratmn of ['HIGDP
and ['HICTP
since the reaction
Was too fast to measure at higher concentrations.
Valuer of the second-order
rate ConPtants are given in Tabie 2, and show little difference between GDP and
GTP. but in all cases, the nucleotide binds approximately 5 t m e s faster to
mutant than to the normal proteln.
3000
,
1
I
S
0. I
Normal
Noma1
+ 12 nn Pi
RATE CONSTANT'
22
0.042
0.71
37
3.36
0
X
10-4
x
x 10-4(3)
0
22
37
TIME <*I
ris, 2
(GOPI ("MI
Tinecourse ef L ' m W
to normal and mufanr
mN-k3S
['HIGDP
was added to a solution Of 0 . 4 4 nH normal IN) and
Spmplea were taken at intervals and analyzed for
mutant "
'
:
2
p
'
d
(
at 0%.
protein-bound nucleotide (R.[ HIGDP) by the filter assay.
The solid line
represents the best fit to a single exponential.
8.
Effect Of GDP Concentration on the Observed arsociation rate
constant (ka)
lyl
GDP
GTP
GDP
GTP
x
x
x
x
IO6
5.31
x
7.21
11.7
23.3
x
106
106
IO6
1.27
0
0
1.01
3.79
4.09
10
10
106
106
lo6
(b) Mutant
GDP
GTP
GDP
GTP
0
0
10
iD
x
x
lo6
s l o es of the
Values Of rate Constants at
Doc were Obtained from
concentration dependences Of k
as shown in Fig. 28. Values at 10% were from
one concentration and aseune k;"
k+l[GTP] or k_4[GDPI.
Sinsle-turnover
(Measurement
a b2& h-21
The rate of CDP formation at different temperatures following binding Of
GTP to
the ammrotein Yere measured for the noma1 and mutant proteins.
Substoichiametric amounts o f ['HIGTP (0.8 moles GTP/mole PrOteln) Were bound to
the apoprateln and the timecourse of COP formation was followed using high
performance llquid chromatography to analyze nucleotides. Fig. 3
shows the
results Obtained with the normal and mutant protein at 37OC.
The formation Of
GDP Can be fitted to a single exponential with an end-point at 0% GTP. There
is no indication of an initial rapid phase of GDP
formation which would
indicate that there is a rapid equilibrium of the cleavage etep: such a rapid
phase is BEmn with Other systems, for example with myosin eubfrayent 1 (Taylor
and Weeds. 19771.
Similar experiments were done at 22% at
D C
but in the
last case although the reaction was too slow to follow to comp1at;on. the data
could be Well fitted t o a single exponential. The end point was D% GTP at 37°C
The rate constants
and 22%.
and this was also assumed to be the case at 0%.
obtained are summarized in Table 3.
A comparison of the rate constants
obtainad for the normal and mutant pr teins (Table 3) BhoYe that the single
turnover rate Constants for normal"'12p
were 2-3 times greater than the
mutant protein.
Duplicate measurements Of the rate constant Of GTP hydrolysis under these
single turnover conditions were made at 0%. 22% and 37%.
An Arrhenius plot
of the data qave e straight line (R > 0.99) which gave values for the
activation energy af - 8 ktcal.mol- for bath normal and mutant prateina.
Tha rate EOnStantS for the dissociation of GTP from the p2l-.GTP
complexes Y re determined by displacement with GDP.
A subatoichiometric
aubatoichionetric
Was added to nucleotide-free p 2 1 u at DOC.
amount of [%]GTP was
After 5 min
off L
a aliquot by g e l filtration showed that all the ['HIGTP
analysis o
was bo&
to the protein and none remained free in solution.
A large excess
e x c o ~ s of "onradioactive GDP
t 3 7 °OCc. .
GDP was
Was then added and the
the solution
eolut'on was incubated
inc bated a
at
sampl
Samples were taken at intervals and analyasd for ['HlGDP
by
HPLC.
and ['HIGTP
In thin experiment, the bound [3H]GTP can either dissociate from the
protein or be hydrolysed to ['HIGDP.
The presence of the large excess or
unlabeled GDP prevents any y.ssociation Of [3H]GTP after it has been released.
The rrte of formation Of [ HIGDP as in Scheme 1 is a first-order process with
an Observed first order rate constant (kc) equal to
+ k-2 + k-l).
This
simplifies to
beCaU e k
.
is V e r y small.
At the end Of the
r action when
tkl)bound ['y]CTb
h y been Converted either to released
[%]GTP or to [ HIGDP, the ratio [ H]GDP/[ HIGTP is equal to k+2/k-1.
Hence,
values for both rate constants can be obtained.
$i20;
Kinetics of GTP Hydrolysis by p21 O
's
19722
The rate constant for GDP dissociation vas measured by displacing ['HIGDP
from its co plex vith p21 using a large eXOesh Of GTP.
A substoishiometric
amaunt Of ['HIGDP
was added to the apoproteln at 0%.
After 5 nln a large
excess Of u labeled GTP was added. the solution vas incubated at:'73
and the
amount or [ % p o p remaining bovnd to the protein wsa measured at various times
using the filter assay. The results of one experiment are Shown in Fig. 5 dnd
the rate constants are shovn in Table 1.
It vas found that GDP release was
sensitive to ammonium sulfate concentration. 80 that at
-100 nW ammonium
sulfate
was -5-fold larger.
This type Of dependence has been found vith
Ye also found that GTP t"ele).Be Vaa
G pratiin?(Ferguson
pf &,
1986).
sensitive to ammonium sulfate, so all measurements were done under conditions
when ammonium sulfate vas reduced to less than 8 nW.
Table 1 shows that the
dlssociatmn of GDP from mutant is about 2-fold slover than from the normal
protern.
40
20
Beoroducibility
'0
50
100 200 150
250
nf
Figs. 2-5 show that 4006 fits to single exponentials are obtained in a11
experiments.
Calculated rate constants or each individual experiment using
the same or different preparations Of ~ 2 1 ' ~
differed by 18.6 than SI.
TIME h l n )
0.064 UM c3H]GTP vas added to a solution of 0.08
I4 p2lNat O°C.
After
5 nin. the solution was made 1 ml4 in unlabeled GDP and rn "bated at
I C
Samples were taken at
intervals and analyzed for total ['HIGDP
and
[';];TP
by HPIC. Data are for the noma1 (N) or mutant ( M ) protein. The solid
lmes represent the best flt t o a ~ m g l eexponential.
100
y
a
0
u
=
eo
60
40
S
20
&
0
IO

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