Báo cáo khoa học: Kinetic mechanism for p38 MAP kinase a A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate potx

Kinetic mechanism for p38 MAP kinase a
A partial rapid-equilibrium random-order ternary-complex
mechanism for the phosphorylation of a protein substrate
Anna E. Szafranska
1
and Kevin N. Dalby
1,2
1 Division of Medicinal Chemistry, University of Texas at Austin, TX, USA
2 Graduate Programs in Biochemistry and Molecular Biology and the Center for Molecular and Cellular Toxicology, University of Texas at
Austin, TX, USA
Keywords
docking; inhibition; kinetic mechanism;
MAP kinase; p38 MAPK
Correspondence
K. N. Dalby, Division of Medicinal
Chemistry, College of Pharmacy, University
of Texas at Austin, TX 78712, USA
Fax: +1 512 232 2606
Tel: +1 512 471 9267
E-mail:
(Received 28 February 2005, revised
18 May 2005, accepted 20 June 2005)
doi:10.1111/j.1742-4658.2005.04827.x
p38 Mitogen-activated protein kinase alpha (p38 MAPKa) is a member of
the MAPK family. It is activated by cellular stresses and has a number
of cellular substrates whose coordinated regulation mediates inflammatory
responses. In addition, it is a useful anti-inflammatory drug target that has
a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a
number of transcription factors as well as protein kinases in its catalog
of known substrates. Fundamental to signal transduction research is the
understanding of the kinetic mechanisms of protein kinases and other pro-

tein modifying enzymes. To achieve this end, because peptides often make
only a subset of the full range of interactions made by proteins, protein
substrates must be utilized to fully elucidate kinetic mechanisms. We show
using an untagged highly active form of p38 MAPKa, expressed and puri-
fied from Escherichia coli [Szafranska AE, Luo X & Dalby KN (2005) Anal
Biochem 336, 1–10) that at pH 7.5, 10 mm Mg
2+
and 27 °C p38 MAPKa
phosphorylates ATF2D115 through a partial rapid-equilibrium random-
order ternary-complex mechanism. This mechanism is supported by a
combination of steady-state substrate and inhibition kinetics, as well as
microcalorimetry and published structural studies. The steady-state kinetic
experiments suggest that magnesium adenosine triphosphate (MgATP),
adenylyl (b,c-methylene) diphosphonic acid (MgAMP-PCP) and magnes-
ium adenosine diphosphate (MgADP) bind p38 MAPKa with dissociation
constants of K
A
¼ 360 lm, K
I
¼ 240 lm, and K
I
> 2000 lm, respectively.
Calorimetry experiments suggest that MgAMP-PCP and MgADP bind
the p38 MAPKa–ATF2D115 binary complex slightly more tightly than
they do the free enzyme, with a dissociation constant of K
d
 70 lm.
Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with
respect to ATF2D115, whereas MgADP exhibits an uncompetitive-like
pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP,

binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm aden-
ine or 2 mm MgAMP was detected, suggesting that the presence of a
b-phosphate is essential for significant binding of an ATP analog to the
Abbreviations
ATF2D115, glutathione S-transferase fusion protein of activating transcription factor 2 residues 1–115; ERK, extracellular signal-regulated
kinase; ITC, isothermal titration calorimetry; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MgADP, magnesium
adenosine diphosphate; MgAMP-PCP, adenylyl (beta,gamma-methylene) diphosphonic acid; MgATP, magnesium adenosine triphosphate;
MKK3, MAP kinase kinase 3; MKK6, MAP kinase kinase 6; MKP3, MAP kinase phosphatase; NADH, nicotinamide adenine dinucleotide;
p38 MAPKa, p38 mitogen-activated protein kinase alpha.
FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS 4631
All organisms, from bacteria and yeasts to mammalian
cells, respond to stimuli from the extracellular environ-
ment. Incoming signals are sent via a cascade of pro-
teins and enzymes from the surface of cells to their
interior, causing alterations in gene expression and
protein activity. These, in turn, generate cellular
responses, such as growth, differentiation, inflamma-
tion and apoptosis. In eukaryotic cells, the mitogen-
activated protein kinase (MAPK) module is a key
element in the propagation, amplification and trans-
port of extracellular signals to the nucleus [1]. The
MAPK superfamily includes the extracellular signal-
regulated kinases (ERKs), the Jun N-terminal kinases
(JNKs) and the p38 MAP kinases, among others.
These enzymes are terminal components of three-tiered
MAPK modules, each of which consists of a MAP
kinase (MAPK), a MAPK kinase (MAPKK) and a
MAPKK kinase (MAPKKK). MAPK modules oper-
ate in numerous biological settings where, through
largely unknown mechanisms, multiple components

impinge on a particular MAPKKK [1].
In recent years there has been substantial interest
in MAPKs due to their participation in numerous bio-
logical pathways and various human conditions and
diseases. One notable MAPK is p38 MAPKa whose
activity has been associated with diseases such as can-
cer [2] or those with inflammatory components [3–5].
p38 MAPKa is phosphorylated on Tyr180 and Thr182
by the upstream activators MAP kinase kinase 3
(MKK3) and MAP kinase kinase 6 (MKK6). Once
activated, p38 MAPKa exerts its effect by directly
phosphorylating transcription factors such as activa-
ting transcription factor 2 (ATF2) and MEF2, or indi-
rectly by activating downstream protein kinases such
as MAPKAP-K2 and MAPKAP-K3, which in turn
phosphorylate their own substrates [1].
Despite a wealth of biological information, there are
many unsolved issues concerning this and other
MAPK signaling cascades. Within the past decade,
four isoforms of p38 MAPK termed a, b, c and d have
been discovered, whose precise biological roles remain
to be defined [1]. Notably, the a and b isoforms are
inhibited by the classic family of pyridinyl inhibitors
related to SB 203580, whereas the c and d isoforms are
not. Thus, use of SB 203580, which has been the main
pharmacological tool employed to date, is transparent
to two of the p38 MAPK isoforms. Although a num-
ber of structural studies have been reported, showing
for example, inactive p38 MAPKa with and without
inhibitors bound at the ATP site [6–12], the structure

of an enzyme–substrate complex is notably lacking.
Although a number of mutagenesis studies have
mapped sites of protein–protein interaction, the basis
for and extent of the differences in specificity within
the p38 MAPK family are still poorly understood.
Thus, we have no clear picture of how p38 MAPKs
recognize protein substrates, or how this recognition is
regulated in vivo. Furthermore, we do not know how
cellular proteins such as scaffold proteins interact with
p38 MAPK isoforms, how these interactions are regu-
lated, how they interplay with catalysis, how they may
be exploited therapeutically or how they differ within
the family.
There is currently a lot of interest in understand-
ing the molecular recognition events associated with
MAPKs, because docking domains are thought to play
a major role in determining the specificity of sub-
strate–ligand and protein–ligand interactions [13–15].
A growing number of enzymes are thought to utilize
docking domains, which are substrate recognition ele-
ments lying outside the active site of the enzyme and
which govern the formation of an enzyme–substrate
complex [16–23]. Several years ago, we showed that
despite the presence of docking domains on p38
MAPKa, which could tether a protein substrate and
facilitate multiple phosphorylations in one collision,
p38 MAPKa phosphorylates ATF2D115 on Thr69 and
Thr71 in a nonprocessive manner [24]. Prior to this
study, LoGrasso et al. reported that p38 MAPKa
phosphorylates ATF2D115 via a compulsory-order

ternary-complex mechanism, in which the binding of
ATF2D115 must precede that of magnesium ATP
(MgATP) (Scheme 1B) [25]. This possibility is intrigu-
ing because: (a) the proposed mechanism would appear
to require novel communication between the enzyme
and substrates to ensure that p38 MAPKa exclusively
binds ATF2D115 before MgATP; and (b) such proper-
ties might be due to the employment of docking
domains in substrate recognition. However, the propo-
sal of LoGrasso et al. was challenged in a report that
enzyme. Surprisingly, we found that inhibition by the well-known
p38 MAPKa inhibitor SB 203580 does not follow classical linear inhibi-
tion kinetics at concentrations > 100 nm, as previously suggested, demon-
strating that caution must be used when interpreting kinetic experiments
using this inhibitor.
Kinetic mechanism for p38 MAP kinase a A. E. Szafranska and K. N. Dalby
4632 FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS
asserted that p38 MAPKa must bind MgATP before
it binds a peptide substrate (Scheme 1C) [26].
Recently, we established a new protocol for the pre-
paration of recombinant murine p38 MAPKa [27],
whose activity towards ATF2D 115 is some 10-fold
greater than previously reported [25]. Given the avail-
ability of a highly active untagged form of p38
MAPKa, the potential novelty of its docking domain-
dependent substrate recognition, the uncertainty of it
kinetic mechanism and the interest in the development
of protein–protein interaction inhibitors, we decided to
reinvestigate its kinetic mechanism using ATF2D115
as the substrate. We describe a steady-state kinetic

investigation of untagged p38 MAPKa and report
that rather than following a compulsory-order ternary-
complex mechanism, as previously reported [25],
p38 MAPKa phosphorylates ATF2D115 via a par-
tial rapid-equilibrium random-order ternary-complex
mechanism. We also show that nucleotides such as
MgATP and particularly magnesium ADP (MgADP)
bind preferentially to the binary p38 MAPKa–
ATF2D115 complex, whereas no binding of magnes-
ium AMP (MgAMP) or adenine was detected to
any enzyme form. This study provides the basis for
the design of further structure ⁄ function and tran-
sient kinetic studies aimed at defining the kinetic mech-
anism and physical properties of p38 MAPKa in
detail.
Results
Steady-state kinetics
Murine p38 MAPKa was expressed in Escherichia coli,
purified and fully activated by constitutively active
MKK6b according to the method of Szafranska and
Dalby [27] (Fig. 1). This preparation corresponds to
the highest reported activity against ATF2D115 for
this enzyme [26]. To examine the propensity of
p38 MAPKa to form a functional binary complex with
MgATP, the ATPase activity of the enzyme was
assessed. In line with a previous report, p38 MAPKa
displayed robust ATPase activity in the presence of
10 mm Mg
2+
at pH 7.6 (k

cat
¼ 0.3 s
)1
and K
m
¼
353 lm) [26]. The simplest mechanism accounting for
the ATP hydrolysis is shown in Scheme 2A. According
to this mechanism, MgATP reversibly binds p38
MAPKa in the active site to form the binary complex
EÆMgATP (k
a
). This binding renders it susceptible to
nucleophilic attack by hydroxyl nucleophiles, leading
to the nucleophilic addition of a water molecule to the
c-phosphoryl group of MgATP (k
p
), and the forma-
tion of MgADP and inorganic phosphate (P
i
). These
products then dissociate (k
diss
) from the active site.
Given the slow turnover (k
cat
¼ 0.3 s
)1
) for the hydro-
lysis reaction, and the relatively large Michaelis–

Menten constant for MgATP, we assume a rapid-equi-
librium mechanism where K
m
¼ k
–a
⁄ k
a
¼ 353 lm.A
conservative estimate for the second-order rate con-
stant of k
a
¼ 10
4
m
)1
Æs
)1
for the binding of MgATP to
p38 MAPKa gives a rate constant for the dissociation
of MgATP from the enzyme of k
-a
¼ 3.5 s
)1
, if the dis-
sociation constant K
A
¼ 350 lm is used. This value
exceeds k
cat
by one order of magnitude, supporting the

rapid-equilibrium assumption.
The ability of p38 MAPKa to bind MgATP and
facilitate the nucleophilic attack of a water molecule
with a turnover of 0.3 s
)1
, which is only fourfold lower
than the turnover of ATF2D115 (see below), supports
the notion that the EÆMgATP complex is not a dead-
end complex with respect to the binding and phos-
Scheme 1. (A) Random-order ternary-complex mechanism, (B)
compulsory-order ternary-complex mechanism (ATF2D115 binds
first, ATP second), (C) Compulsory-order ternary-complex mechan-
ism (ATP binds first, peptide second).
A. E. Szafranska and K. N. Dalby Kinetic mechanism for p38 MAP kinase a
FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS 4633
phorylation of ATF2D115. Given the binding mode
adopted by peptide substrates for a number of protein
kinases, it is reasonable to assume that a protein sub-
strate can bind productively to a preformed EÆMgATP
complex. Thus, as pointed out by Chen et al. [26], the
robust ATPase activity exhibited by p38 MAPKa
sheds some doubt on the compulsory-order ternary-
complex mechanism proposed by LoGrasso et al. [25].
We expressed and purified the glutathione S-trans-
ferase (GST) fusion protein of the N-terminal 115 resi-
dues of the transcription factor ATF2 (ATF2D115)
essentially as described previously [25], with some
minor modifications (Fig. 1) [27]. Having established
the kinetic competence of the EÆMgATP complex
(with respect to nucleophilic attack by water), we con-

ducted initial rate studies at various concentrations
of ATF2D115 and MgATP. Reciprocal plots of initial
rate versus the concentration of ATF2D115 (Fig. 2A)
or ATP (Fig. 2B) revealed an intersecting pattern of
lines (> 1 ⁄ v ¼ 0), indicative of a sequential kinetic
mechanism, in which both substrates must bind to
form a ternary complex before catalysis occurs. Pre-
viously, we showed that ATF2D115 is phosphorylated
twice by p38 MAPKa on Thr69 and Thr71 in a non-
processive manner and that under initial rate con-
ditions, only the mono-phosphorylated forms of
ATF2D115 are produced at equal rates [24].
Our results differ in two significant aspects from
those previously reported for flag-tagged p38 MAPKa
[25]. First, in our case the double-reciprocal plots inter-
sect above the x-axis (compared with below the x-axis
for the flag-tagged enzyme). Second, the reported cata-
lytic constant towards ATF2 D115 is some 10-fold
higher. It is conceivable that these differences in activ-
ity reflect the presence of an N-terminal flag tag
and ⁄ or the method by which the enzymes were over-
expressed, activated and purified. In our case a sensi-
tive tryptic analysis indicates that the enzyme was fully
activated [27].
v
V
max
¼
AB
aK

A
K
B
þ aK
B
A þ aK
A
B þ AB
ð1Þ
The rapid equilibrium assumption is a powerful
approach used to simplify the analysis of enzyme
mechanisms and for a ternary-complex mechanism it
provides a good approximation to the reaction path-
way when ligand-binding events are fast compared
Fig. 1. Preparation of activated p38 MAPKa and ATF2D115. (A) 10% SDS ⁄ PAGE analysis showing activated, p38 MAPKa (lane 1) and its MS
analysis (M
r
41 731 Da observed; 41 726 Da calculated). (B) 12% SDS ⁄ PAGE showing ATF2D115 (lane 1) and its MS analysis (M
r
39 658 Da
observed; 39 650 Da calculated).
Scheme 2. (A) Mechanism of ATP hydrolysis by p38 MAPKa.(B)
Competitive inhibition of ATP hydrolysis with EÆI dead-end com-
plex.
Kinetic mechanism for p38 MAP kinase a A. E. Szafranska and K. N. Dalby
4634 FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS
with the interconversion of the central substrate and
product complexes. The lines in Fig. 2 represent the
best fit of the experimental data to Eqn (1), which
describes a rapid-equilibrium random-order ternary-

complex mechanism (Scheme 1A), according to the
parameters shown in Table 1. According to this fit,
p38 MAPKa binds both substrates in the mid micro-
molar range [K
B
¼ 39 lm (ATF2D115); K
A
¼ 360 lm
(MgATP)] to form the respective binary complexes.
We reasoned that with ligand binding to p38 MAPKa
occurring in the micromolar range and a relatively low
A
B
Fig. 2. Two-substrate dependence kinetic analysis of p38 MAPKa.
(A) Double-reciprocal plot of 1 ⁄ v versus 1 ⁄ ATF2D115 at five fixed
ATP concentrations (m,12l
M; n,25lM; r,50lM; d,100lM; .,
200 l
M). (B) Double reciprocal plot of 1 ⁄ v versus 1 ⁄ ATP at five
fixed ATF2D115 concentrations (.,2.5l
M; n,5lM; r,10lM; m,
20 l
M; d,40lM). Solid lines are the best fit through the experi-
mental data to Eqn (1).
Table 1. Kinetic constants for p38 MAPKa obtained from two-substrate steady-state kinetics, and ATPase activity and inhibition studies. C, competitive; UC, uncompetitive; M, mixed; ND,
not determined.
Activity Substrates
Substrate dependence constants (l
M)
Inhibitor

Varied
substrate
Inhibition
pattern
Inhibition constants
K
A
(lM) aK
A
(lM) K
B
(lM) aK
B
(lM) k
cat
,(s
)1
) K
I
(lM) bK
I
(lM)
Kinase ATF2D115 (B),
ATP (A)
360 ± 17 13.4 ± 15 38.6 ± 7 1.4 ± 0.1 1.1 ± 0.03 ADP ATF2D115
ATP
UC
C
>2000
a

9.7 ± 0.5
AMP-PCP ATF2D115
ATP
M
C
187.4 ± 37 8.6 ± 0.5
SB 203580 ATF2D115 ND – –
ATP C 0.021 ± 0.001
b
0.021 ± 0.001
b
ATPase ATP 353 ± 52 – – – 0.3 ± 0.01 AMP-PCP ATP C 241.5 ± 13 –
a
Lower estimate.
b
K
I
,andbK
I
set equal.
A. E. Szafranska and K. N. Dalby Kinetic mechanism for p38 MAP kinase a
FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS 4635
catalytic constant of k
cat
¼ 1.2 s
)1
for the phosphory-
lation of ATF2D115, the rapid equilibrium assumption
is likely to provide a reasonable description of the
reaction mechanism and could be used to distinguish

between several mechanistic possibilities. For example,
the rate-constant for the association of MgATP with
a protein kinase is typically of the order of
10
5
)10
6
m
)1
Æs
)1
, which, given a typical dissociation
constant of 10 lm for MgATP, indicates a rate con-
stant for MgATP dissociation of 1–10 s
)1
, which is at
least as fast as the observed k
cat
. Accordingly, we
noted that the pattern of intersecting lines in Fig. 2
excludes a rapid-equilibrium compulsory-order tern-
ary-complex mechanism where MgATP binds before
ATF2D115 (Scheme 1C), because this mechanism
requires that the lines in Fig. 2 intercept on the y-axis.
Thus, the observed ATPase activity, together with the
substrate-dependence kinetics, appears to rule out
possible compulsory-order ternary-complex mecha-
nisms and support instead a rapid-equilibrium ran-
dom-order ternary-complex mechanism. Interestingly,
the interaction coefficient of a ¼ 0.037 obtained from

the fit would indicate that both substrates are held
 27-fold more tightly in the ternary complex, com-
pared with their respective binary complexes, if the
mechanism was a full rapid-equilibrium mechanism.
More realistically however, the mechanism is likely to
be a partial rapid-equilibrium mechanism, where the
aK
A
represents a Michaelis–Menten constant and not
a dissociation constant. It should be noted that the
values of K
d
for MgATP obtained from both the single
and bisubstrate kinetics are essentially identical
(Table 1), which supports the mechanistic assignments.
Inhibitors
AMP-PCP
To examine the mechanism in more detail we exam-
ined the inhibition of p38 MAPKa by b,c-methylene
ATP (AMP-PCP), a nonhydrolyzable analog of ATP.
Lineweaver–Burk plots at different concentrations of
AMP-PCP show it to be a mixed inhibitor with respect
to ATF2D115 (Fig. 3A) and a competitive inhibitor
with respect to MgATP (Fig. 3B). Such patterns are
consistent with a partial rapid-equilibrium random-
order ternary-complex mechanism (Scheme 3) [28].
These lines represent the best fit of the experimental
data to Eqn (2) and correspond to values of K
I
¼

187.4 lm and bK
I
¼ 8.6 lm (Table 1), where K
I
, but
not bK
I
is likely to be an equilibrium constant. Not
surprisingly, MgAMP-PCP and MgATP appear to dis-
play a similar degree of interaction with ATF2D115,
suggesting that the bridging b,c oxygen does not
contribute to MgATP binding. In addition to the
bisubstrate inhibition kinetics we also showed
that AMP-PCP inhibits the ATPase activity of
p38 MAPKa. Analysis of the inhibition data (not
shown), according to the mechanism in Scheme 2B,
suggests that AMP-PCP binds the free enzyme with a
dissociation constant of K
i
¼ 241 lm (Table 1), which
is in fairly good agreement with K
I
¼ 187.4 lm
obtained from the bisubstrate kinetics.
v
V
max
¼
A
aK

A
1 þ
K
B
B
þ
IK
B
K
I
B
þ
I
bK
I

þ A 1 þ
aK
B
B
ÀÁ
rearranged
v
V
max
¼
B
aK
B
1 þ

K
A
A
þ
IK
A
K
I
A

þ B 1 þ
aK
A
A
þ
aK
A
I
bK
I
A

ð2Þ
MgADP
We then examined the inhibitory effects of the product
MgADP. Lineweaver–Burk plots at different concen-
trations of MgADP and saturating MgATP (195 lm,
·7) suggest that MgADP is an uncompetitive-like
inhibitor with respect to ATF2D115 (Fig. 3C) and
a competitive inhibitor with respect to MgATP

(Fig. 3D). Such patterns are not normally expected for
a random-order ternary-complex mechanism, but can
arise if the inhibitor displays selectivity towards certain
enzyme forms. We believe the uncompetitive pattern
towards ATF2D115 (no slope effect) results because
MgADP does not bind the free form of the enzyme to
detectable levels under the conditions of the experi-
ment. The data in Fig. 3C do not rule out the possi-
bility of a slight slope effect, however (and weak
MgADP binding to the free enzyme), thus we conser-
vatively assign a lower limit of K
I
>2mm, the maxi-
mum concentration of MgADP used for the
dissociation constant, which is in line with other
reports [26].
MgAMP and adenine
We also tested whether adenine and MgAMP inhibit
p38 MAPKa. Surprisingly, neither compound inhibited
the activity of p38 MAPKa, suggesting that the pres-
ence of the b-phosphate is essential for ATP analogs
to bind.
SB 203580
The pyridinylimidazole inhibitor SB 203580 binds
within the ATP-binding pocket of both active and
Kinetic mechanism for p38 MAP kinase a A. E. Szafranska and K. N. Dalby
4636 FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS
inactive p38 MAPKa and has facilitated the dissection
of several signaling pathways involving p38 MAPKa
pathways [29,30]. In the course of their studies,

Lo-Grasso et al. reported that SB 203580 is an uncom-
petitive inhibitor of p38 MAPKa with respect to
ATF2D115 [25]. Such a mechanism seemed to contra-
dict the known predilection of the inhibitor for free
p38 MAPKa, thus we decided to re-examine the mech-
anism of inhibition. To do so, we first fixed the
concentration of ATF2D115 and varied SB 203580
over 0–80 nm. A competitive inhibition pattern was
obtained (not shown), as expected for an inhibitor that
binds in the ATP-binding site. The best fit to the kin-
etic data gave an approximate value for a competitive
inhibition constant that was in line with previous
reports [31]. Surprisingly, when we tried to extend our
study to higher concentrations of the inhibitor we were
not able to, because the mechanism of inhibition with
respect to ATF2D115 at concentrations > 200 nm did
not follow simple linear models of inhibition. We tried
two different preparations of SB 203580, a commercial
source and a sample provided to us by Kevan Shokat’s
laboratory. The kinetic results were identical. One
possible reason for the poor fit is that SB 203580,
which is fairly hydrophobic in character, aggregates at
higher concentrations [32,33]. The addition of 0.01%
(v ⁄ v) Triton X-100, whose use is suggested to identify
or reverse the action of aggregate-based inhibitors [34]
AB
CD
Fig. 3. Inhibition by MgAMP-PCP and MgADP. (Upper) (A) Double-reciprocal plot of 1 ⁄ v versus 1 ⁄ ATF2D115 at five fixed MgAMP-PCP con-
centrations (.,0m
M; n, 0.137 mM; r, 0.275 mM; ,0.55mM; d, 1.1 mM). The ATP concentration was fixed at 226 lM. (B) Double-recipro-

cal plot of 1 ⁄ v versus 1 ⁄ ATP at six fixed MgAMP-PCP concentrations (.,0l
M; n,32lM; r,64lM; ,128lM; d, 256 lM, m, 513 lM). The
ATF2D115 concentration was fixed at 58 l
M. (C) Double-reciprocal plot of 1 ⁄ v versus 1 ⁄ ATF2D115 at six fixed MgADP concentrations
(.,0m
M; d, 0.125 mM; r, 0.25 mM; m, 0.5 mM; , 1.0 mM; n, 2.0 mM). The ATP concentration was fixed at 195 lM. (D) Double-reciprocal
plot of 1 ⁄ v versus 1 ⁄ ATP at five fixed MgADP concentrations (d ,0m
M; .,0.25mM; n, 0.5 mM; r, 1.0 mM; m, 2.0 mM). The ATF2D115
concentration was fixed at 52 l
M. Points represent experimentally determined initial velocities. Solid lines are the best fit through the experi-
mental data according to Eqn (2).
A. E. Szafranska and K. N. Dalby Kinetic mechanism for p38 MAP kinase a
FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS 4637
proved inconclusive in our hands because the activity
of the enzyme was also affected by the presence of
Triton.
Isothermal titration calorimetry
To lend further support to our conclusions, we
examined the binding of ADP and AMP-PCP
to p38 MAPKa in the presence and absence of
ATF2D115 by isothermal titration calorimetry (ITC).
ITC is the most direct method for the determination of
macromolecular ligand dissociation constants (K
d
), if it
is feasible to conduct experiments in the appropriate
range of protein and ligand concentrations [35]. It is
useful because it can be used to determine the binding
stoichiometry, provided that the two interacting com-
ponents are titrated at concentrations higher than the

K
d
. When binding occurs, it can be readily observed
from the change in shape of the binding isotherm.
The calorimetry experiments are significant for sev-
eral reasons. (The ITC experiments were designed so
that the c-value, the factor characterizing the shape of
titration curve, was not lower than 0.1. When c ¼ 0.1
the binding is very weak and yields a nearly horizontal
isotherm with a poorly defined binding constant, K
d
[39]. The c-value in our experiments was in the range
0.5–0.6, which corresponds to 65–77 lm p38 MAPKa
and represents a 13 000–15 400-fold increase in the
enzyme concentration in comparison with the kinetic
studies.) Notably, they show that the dissociation con-
stants for ADP and AMP-PCP from the binary com-
plex are fivefold higher than the values of bK
i
obtained
kinetically, suggesting that, as suspected, ligand bind-
ing is not completely at equilibrium during turnover
and that the mechanism is best described as a partial
rapid-equilibrium mechanism. For example, when
ADP (0–223 lm) was titrated into a mixture of
ATF2D115 (92 lm) and p38 MAPKa (68 lm), heat
was evolved indicative of favorable nucleotide binding
to the enzyme (Fig. 4B). The best fit according to the
two-component binding model provided a dissociation
constant of K

d
¼ 62 ± 7 lm, with n ¼ 0.52 ± 0.08
binding sites and the following thermodynamic para-
meters; DH ¼ )16 900 JÆmol
)1
, DS ¼ )37 ± 0.32 JÆ
mol
)1
ÆK
)1
. When MgAMP-PCP (0–183 lm) was titra-
ted into a mixture of ATF2D115 (97 lm) and the
enzyme (77 lm) (Fig. 5B), a similar amount of heat
was generated and the data analysis furnished the fol-
lowing values: K
d
¼ 69.6 ± 6 lm, n ¼ 0.52 ± 0.08,
DH ¼ )14 200 JÆmol
)1
, and DS ¼ )28.3 JÆmol
)1
ÆK
)1
.
The calorimetry analysis supports the notion of syn-
ergy between nucleotides and ATF2D115 upon binding
to p38 MAPKa. For example, the binding of AMP-
PCP to the binary complex appears to be at least
fivefold tighter than to the free enzyme. When
MgAMP-PCP (0–542 lm) was titrated into p38

MAPKa (194 lm), ~ 10-fold less heat was evolved
compared with when MgAMP-PCP was added to the
binary complex (Fig. 5A). The heat generated was not
sufficiently robust to enable an accurate titration, thus
the best fit to the binding model gave values of K
d
¼
300 ± 160 lm, n ¼ 1.4 ± 0.4, DH ¼ )1145 JÆmol
)1
,
and DS ¼ +12.3 JÆmol
)1
ÆK
)1
. This dissociation con-
stant is in line with the value of K ¼ 184 lm obtained
kinetically. Interestingly, when ADP (0–550 lm) was
titrated with p38 MAPKa (194 lm), no heat was
detected. This suggests like the inhibition data, that
binding is probably weak (> 300 lm) and beyond the
detection of the experiment. Interestingly, the calori-
metry experiments are consistent with only 0.5 binding
sites per binary p38 MAPKaÆATF2D115 complex,
suggesting that the enzyme either has only one func-
tional active site within the complex or that only 50%
of the preparation is functional.
Discussion
In recent years p38 MAPKa has emerged as a major
practicable drug target, associated with several severe
diseases of inflammation [3–5]. The identification in

1994 of the pyridinyl class of p38 MAPKa inhibitors
[29] fueled many studies aimed at exploiting the subtle
differences between the active sites of protein kinases.
Despite these efforts, to date, only a handful of ATP
competitive inhibitors have been developed that truly
Scheme 3. Random-order substrate binding with EÆIandEÆIÆSdead
end complexes.
Kinetic mechanism for p38 MAP kinase a A. E. Szafranska and K. N. Dalby
4638 FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS
exhibit sufficient specificity to warrant development
[36]. Thus, there is a keen need to exploit other sites
on protein kinases, such as cosubstrate or scaffold-
binding sites, which may offer alternative therapeutic
avenues. To this end, detailed kinetic and struc-
ture ⁄ function studies using protein substrates will help
us to understand the full compliment of molecular
interactions that govern the catalysis and regulation of
these enzymes.
Kinetic mechanism
Despite occupying an elevated position as a potentially
important target of signal transduction therapy, little
mechanistic work has been reported on p38 MAPKa
or related family members, and as such there remains
no clear model for their kinetic mechanisms. ERK2
was originally proposed to phosphorylate myelin basic
protein with rate-limiting (k
cat
¼ 10 s
)1
) phosphoryla-

tion [37]. However, with a physiologically relevant sub-
strate (Ets1), ERK2 was shown to be activated by
magnesium [38] and to follow a random-order ternary-
complex mechanism [39], with partially rate-limiting
phosphorylation (k
2
¼ 109 s
)1
) and product release
(k
3
¼ 56 s
)1
) [40].
In this study, we focus on the steady-state kinetic
mechanism of p38 MAPKa using the protein substrate
ATF2D115. Previous studies on p38 MAPKa have
been somewhat contradictory, suggesting that the
enzyme follows compulsory-order ternary-complex
mechanisms where the phosphoacceptor [25] or ATP
[26] must bind first. Compulsory-order mechanisms are
ruled out in this study through: (a) their inconsistency
with the substrate dependence and the dead end inhib-
itor kinetics (rules out the requirement that ATF2
must bind first); or (b) published structural studies and
binding studies, which show that a peptides derived
from a protein substrate can bind p38 MAPKa at
docking domains outside of the active site in the
absence of MgATP (rules out the requirement that
MgATP must bind first).

We show that, like the c-isoform [41], p38 MAPKa
displays robust ATPase activity with a catalytic
constant of k
cat
¼ 0.3 s
)1
, which is very similar to
Fig. 4. Isothermal titration calorimetry measurements of MgADP binding. (A) (upper) Titration of MgADP (1.47 mM,21· 8 lL) with activated
p38 MAPKa (68 l
M)at27°C; (lower) integrated enthalpy change for each injection. (B) (upper) Titration of MgADP (1.47 mM,21· 8 lL) with
a mixture of activated p38 MAPKa (68 l
M) and ATF2D115 (91.8 lM); (lower) integrated enthalpy change for each injection.
A. E. Szafranska and K. N. Dalby Kinetic mechanism for p38 MAP kinase a
FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS 4639
the catalytic constant for the phosphorylation of
ATF2D115 that has a value of k
cat
¼ 1.2 s
)1
. This and
the work of Chen et al. [26] are consistent with the
notion that MgATP can bind the enzyme to form a
functionally active complex.
There is substantial evidence to support the notion
that p38 MAPKa binds protein substrates at its
C-terminus in the absence of MgATP [42]. Specific-
ally, studies from the laboratories of both Goldsmith
[43] and Ahn [44] showed that the inactive form of
p38 MAPKa can bind to a peptide derived from the
p38 MAPKa substrate MEF2A. This peptide con-

tains a consensus motif for docking of R ⁄ K-X
4
-F
A
-
X-F
B
(where X represents any amino acid and F
represents a hydrophobic residue: Leu, Ile, or Val)
and binds in a groove in the C-terminus of
p38 MAPKa between the helices a
D
and a
E
and the
reverse turn between strands b
7
and b
8
[43]. As this
consensus sequence is also present in ATF2, it is
probable that p38 MAPKa binds ATF2 in the same
groove as MEF2. Given that the unphosphorylated
form of p38 MAPKa, whose active site is not prop-
erly molded, still binds these pepides, it is extremely
unlikely that the binding of MgATP must precede
ATF2.
Thus, taken together, these observations and our
data support a partial rapid-equilibrium random-order
ternary-complex mechanism (Scheme 1A), where

both substrates (MgATP and ATF2D115) bind to
p38 MAPKa with moderate affinities (MgATP, K
A
¼
360 lm; ATF2D115, K
B
¼ 39 lm). (It is well known
that steady-state kinetic studies do not identify the
extent to which binary complexes are actually func-
tionally productive and that transient kinetic studies
are required to determine this unequivocally. However,
protein kinases are considered to have extremely
flexible active sites that can accommodate both
substrates before they adopt a more closed conforma-
tion that facilitates catalysis [45]. Therefore, it seems
reasonable to assume that, for p38 MAPKa, which
utilizes a docking domain, both binary complexes lie
on the reaction pathway.) Jointly with calorimetry
experiments, a fivefold synergy in substrate binding
is indicated.
Fig. 5. Isothermal titration calorimetry measurements of MgAMP-PCP binding. (A) (upper) Titration of AMP-PCP (1.58 mM,19· 8 lL) into
activated p38 MAPKa (75 l
M)at27°C; (lower) integrated enthalpy change for each injection. (B) Titration of AMP-PCP into a mixture of
activated p38 MAPKa (75 l
M) and ATF2D115 (97 lM); (lower) integrated enthalpy change for each injection.
Kinetic mechanism for p38 MAP kinase a A. E. Szafranska and K. N. Dalby
4640 FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS
Our conclusions and results differ on a number
of counts to a previous report by LoGrasso et al.
who proposed a compulsory-order mechanism with

ATF2D115 binding before MgATP [25]. The major dif-
ferences we found are as follows. Firstly, MgAMP-
PCP exhibits a mixed inhibition pattern and not
an uncompetitive pattern (Fig. 3A) with respect to
ATF2D115, which is consistent with a partial rapid-
equilibrium random-order ternary-complex mechanism
(Scheme 1A). LoGrasso et al. reported an uncompeti-
tive pattern [25].
Secondly, as with the previous study we found that
MgADP exhibits an uncompetitive-like inhibition pat-
tern with respect to ATF2D115 (Fig. 3C). We interpret
this as being due to its inability to bind free
p38 MAPKa and not because MgATP, the substrate,
binds free p38 MAPKa weakly. That is, the observed
pattern of inhibition reflects the selectivity of the prod-
uct inhibitor ADP for the different enzyme forms and
not the substrate ATP. To corroborate this interpret-
ation we showed that the binding of MgADP to
p38 MAPKa could not be detected using microcalori-
metry (Fig. 4A), whereas robust binding of MgADP
to the EÆATF2D115 binary complex (Fig. 4B) was
detected.
Thirdly, LoGrasso et al. reported that SB 203580
displays an uncompetitive pattern with respect to
ATF2D115 [25]. This observation was curious because
SB 203580 is known to bind p38 MAPKa very tightly
[31]. Therefore, the ability of SB 203580 to bind the
same enzyme form as ATF2D115 predicts that there
should be a significant ‘slope effect’ in a Lineweaver–
Burk plot of 1 ⁄ v against 1 ⁄ ATF2D115. Driven by this

curiosity, we examined the mechanism of inhibition by
SB 203580 and found that it is not well described by
any classical linear inhibition models, possibly because
it aggregates in solution at concentrations above
200 nm, leading to nonspecific inhibition effects as seen
for other protein kinase inhibitors [32–34].
It is difficult to fully explain the differences between
this study and LoGrasso’s. While it is possible that the
N-terminal FLAG tag utilized by LoGrasso et al. for
purification purposes [25] imparts an influence on sub-
strate and nucleotide binding that could influence the
inhibition patterns, other differences between our
results cannot be explained by the tag. It must be sta-
ted that the mechanism proposed by LoGrasso et al.is
unique for a protein kinase and neither followed by
ERK2 [39] nor supported by the data presented here.
We propose, therefore, that the kinetic mechanism for
p38 MAPKa should be reassigned as a partial rapid-
equilibrium random-order ternary-complex mechanism
for the phosphorylation of ATF2D115.
In conclusion, activated p38 MAPKa has been
prepared without a purification tag to the highest
specific activity reported. The enzyme phosphorylates
ATF2D115 by a partial rapid-equilibrium random-
order ternary-complex mechanism and not through a
compulsory-order mechanism as previously suggested
[25]. Interestingly, neither MgAMP nor adenine inhib-
ited p38 MAPKa suggesting that the b-phosphate is
necessary for MgATP binding. It will be interesting to
understand the basis for this selectivity.

Experimental procedures
Buffers, reagents and plasmids
Trizma base was purchased from EM Industries (Gibbs-
town, NJ, USA), Hepes from Sigma (St. Louis, MO, USA)
and ammonium carbonate from Fisher (Fair Lawn, NJ,
USA). Glutathione (GSH) agarose and glutathione
(reduced form) for GST-fusion protein purification were
obtained from Sigma. NiSO
4
and Ni-NTA agarose for
His
6
-p38 MAPKa purification were provided by Qiagen
Inc. (Santa Clarita, CA, USA) and Sigma, respectively. The
His
6
tag was removed from p38 MAPKa using thrombin
from Novagen (Madison, WI, USA). MgCl
2
for
p38 MAPKa activation and kinetic studies, as well as ATP
analogs used in inhibition studies (adenine, AMP, AMP-
PCP, ADP) and SB 203580 were purchased from Sigma.
Kinase assays were conducted with Roche (Indianapolis,
IN, USA) special quality ATP and [
32
P]ATP[cP] from ICN
(Costa Mesa, CA, USA). Reagents for coupled enzyme
assay, such as lactate dehydrogenase, pyruvate kinase,
NADH and phosphoenol pyruvate, were supplied by

Sigma. All other buffer components and chemicals were
obtained from Sigma. The plasmid used to express His
6
-
p38 MAPKa [45] and GST-ATF2 (1–115) [27] have been
reported previously. The plasmid used to express GST-
MKK6b (S207E T211E) was a gift from R. Copeland
(DuPont Merck, Delaware, USA).
Proteins
Expression and purification of tagless p38 MAPKa
DNA sequences encoding p38 MAPKa cloned into
pET14B (Novagen) was used to express p38 MAPK a as an
N-terminal, hexa-histidine fusion protein in E. coli BL21
(DE3) pLysS. The enzyme was expressed and purified
according to the method of Szafranska et al. [27]. Follow-
ing removal of the hexa-histidine tag the enzyme prepar-
ation was dialyzed overnight at 4 °C into storage buffer S1
[25 mm Hepes, 2 mm dithiothreitol, 50 mm KCl, 5% (v ⁄ v)
glycerol, pH 7.5], and stored at )80 °C at a concentration
of approximately 2 mgÆmL
)1
until further use. The concen-
tration of p38 MAPKa was determined using the molar
A. E. Szafranska and K. N. Dalby Kinetic mechanism for p38 MAP kinase a
FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS 4641
extinction coefficient (A
280
) of 52501 cm
)1
Æm

)1
, following
the method of Gill and von Hippel [46]. The homogeneity
of the protein was routinely verified by 10% SDS ⁄ PAGE
and by MS.
Expression and purification of GST-ATF2 (1–115)
Residues 1–115 of the transcription factor ATF2 was
expressed as a GST fusion protein from a pGEX-5T vector
in E. coli BL21 (DE3) pLysS and purified according to the
previously reported method with two anion-exchange chro-
matography steps. The protein substrate was stored at
)80 °C in buffer S1 lacking glycerol. The homogeneity of
the protein was routinely verified by 12% SDS ⁄ PAGE and
by MS. The concentration of ATF2D115 was determined
using the extinction coefficient (A
280
) of 48 490 cm
)1
Æm
)1
,
following the method of Gill and von Hippel [46].
Expression and purification of GST-MKK6b
(S207E T211E)
MKK6b (S207E T211E) was expressed as a GST fusion
protein in E. coli BL21 (DE3) pLysS. The protein was puri-
fied by GSH-affinity chromatography according to stand-
ard protocols (Sigma Product Information for G4510).
Pooled protein fractions were dialyzed at 4 °C into buffer
S1, concentrated and stored at )80 °C [27].

Activation of p38 MAPKa by GST-MKK6b
(S207E T211E)
p38 MAPKa (10.7 lm, 14 mg) and GST-MKK6b (0.26 lm,
1.6 mg) were incubated for 5 min at 27 °C prior to the
addition of ATP (4 mm) in 50 mL of activation buffer P1
(25 mm Hepes, 2 m m dithiothrietol, 20 mm MgCl
2
, 0.5 mm
EDTA, pH 7.5) and repurified, essentially according to the
method of Szafranska et al. [27]. The activated, tagless
p38 MAPKa was stored in buffer S1 at )80 °C until fur-
ther use. The homogeneity of the protein was routinely
verified by 10% SDS ⁄ PAGE and by MS. The concentra-
tion was assessed using calculated extinction coefficient
(A
280
) of 52501 cm
)1
m
)1
, according to the method of Gill
and von Hippel [46].
Electrospray mass spectrometry of proteins
Protein samples were rid of excess salt by injection on a RP-
HPLC C
18
column (Grace Vydac, 218TP54; Columbia, MD,
USA) and elution over a standard gradient of 0–100%, (v ⁄ v)
acetonitrile over 80 min at a flow rate of 0.6 mLÆmin
)1

.
Collected peaks were lyophilized, the residue re-suspended
in 30 lL water: acetonitrile mixture (1 : 1) containing 0.1%
(v ⁄ v) trifluoroacetic acid and analyzed by LC-MS with
a Finnigan-MAT LCQ (Finnigan ⁄ Thermoquest, San Jose,
CA) electrospray, ion trap mass spectrometer coupled with a
Magic 2002 Microbore HPLC (Microchrom BioResource,
Auburn, CA). The mass spectrometer was scanned over a
300–2000 m ⁄ z mass range using a 1 : 1 mixture of mobile
phase A (acetonitrile ⁄ water ⁄ acetic acid ⁄ trifluoroacetic acid;
2 : 98 : 0.1 : 0.02; v ⁄ v ⁄ v ⁄ v) and B (acetonitrile ⁄ water ⁄ acetic
acid ⁄ trifluoroacetic acid; 10 : 90 : 0.009 : 0.02; v ⁄ v ⁄ v ⁄ v) at
a flow rate of 10 lLÆmin
)1
.
Steady-state kinetics
p38 MAPKa assays were conducted at 27 °C in assay buf-
fer A1 (20 mm Hepes, 2 mm dithiothreitol, 100 mm KCl,
0.1 mm EDTA, 0.1 mm EGTA, 10 lgÆmL
)1
bovine serum
albumin, pH 7.6) containing 5–10 nm activated p38
MAPKa and 10 mm MgCl
2
in a final volume of 100 lL.
At 10 mm magnesium the activity of the kinase is optimum.
The concentrations of substrates ranged as follows:
ATF2D115 (2.5–52 lm), ATP (12.5–200 l m, 100–1000
c.p.m.Æpmol
)1

). The reaction mixture was incubated for
5 min before the addition of ATP. Aliquots (10 lL) were
taken at set time points and applied to a 2 · 2 cm P81
cellulose papers, which were allowed to air-dry, washed
with 50 mm phosphoric acid (5 · 10 min), then in acetone
(1 · 10 min) and dried. The incorporation of radioactivity
was determined by counting in 1.5 mL CytoScint on a
Packard 1500 scintillation counter at a r-value of 2. Each
assay was performed at least twice. Protein concentrations
were determined at 280 nm using the following molar
extinction coefficients: e ¼ 52 501 m
)1
Æcm
)1
(p38 MAPKa),
48 490 m
)1
Æcm
)1
(ATF2D115). ATP concentration was
determined at 259 nm using e ¼ 15 400 m
)1
Æcm
)1
. The ini-
tial velocities were fitted using scientist for Windows v 2.0
(MicroMathÒ).
Enzyme inhibition studies
In general, enzyme inhibition studies were performed as des-
cribed for two-substrate kinetics. When ATF2D115 was the

varied substrate (2.5–55 lm), ATP was fixed at 190–220 lm
(100–1000 c.p.m.Æpmol
)1
). When ATP was the varied
substrate (20–320 lm), ATF2D115 was fixed at 40–55 lm.
The concentrations of inhibitors ranged as follows: ADP
(0.2–2 mm), SB 203580 (0.025–1.0 lm), AMP-PCP (0.18–
1.4 mm), AMP (0.5–2 mm) and adenine (0.21–1.7 mm).
Where possible, the extinction coefficients were used to
determine the accurate concentrations of inhibitors (e ¼
15 400 m
)1
Æcm
)1
for ADP, AMP-PCP and AMP, and
13 300 m
)1
Æcm
)1
for adenine). Protein concentrations were
determined using extinction coefficients as described above
(Steady-state kinetics section). In each case inhibition reac-
tions were performed twice. The initial velocities data from
the inhibition studies were fitted to the general velocity equa-
tions using scientist for Windows v 2.0 (MicroMathÒ).
Kinetic mechanism for p38 MAP kinase a A. E. Szafranska and K. N. Dalby
4642 FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS
Isothermal titration calorimetry
Isothermal titration calorimetry was performed using an
MCS isothermal titration calorimeter (Microcal Inc., Nor-

thampton, MA) connected to a water bath to maintain the
constant temperature of 27 °C. Ligand solutions were pre-
pared in calorimetry buffer C1 (20 mm Hepes, pH 7.5–7.6,
adjusted with 1 m KOH, containing 100 mm KCl, 2 mm
2-mercaptoethanol, 0.1 mm EDTA, 0.1 mm EGTA and
10 mm MgCl
2
). Protein solutions were dialyzed overnight
at 4 °C into the same buffer. All solutions were centrifuged
at 13 200 g for 5 min and degassed under vacuum for at
least 30 min prior to use. After a stable baseline was
achieved, ligand solutions were titrated into the stirred
(410 r.p.m.) sample cell (1.325 mL) containing equilibrated
(30 min) samples of the proteins at 27 °C. The injection
sequence consisted of an initial 2 lL injection (not used in
data fitting) followed by 26 injections of 8–10 lL, each at
180 s intervals until saturation was reached. To correct for
the heat of dilution and mixing, blank titrations of ligand
into buffer were subtracted from the experimental titra-
tions. The reference offset was set at 20%. Data evaluation,
integration, analysis, and determination of binding parame-
ters were performed using origin 5.0 software (Microcal,
Inc.). Final protein and ligand concentrations were as
follows: 65–77 lm activated p38 MAPKa, 91–97 lm
ATF2D115, 223 lm ADP and 183 lm AMP-PCP. To facili-
tate detection of binding between the enzyme and ATP
analogs, additional ITC experiment was carried out where
their concentrations were approximately doubled (194 lm
p38 MAPKa, 549 lm ADP, 542 lm AMP-PCP).
ATPase activity assay

The ATPase activity of activated p38 MAPKa was charac-
terized using the coupled assay method. The decrease in
NADH was monitored at 340 nm using a Cary 50 UV
spectrophotometer. The temperature was maintained at
27 °C using a VWR Refrigerated Circulator (Suwanee, GA,
USA). The molar absorption coefficient (e) for NADH of
6.22 · 10
)3
m
)1
Æcm
)1
at 340 nm was used for calculations.
The coupled assay enzymes were purchased from Sigma as
3.2 m (pyruvate kinase) and 2.4 m (liver alcohol dehydro-
genase) suspensions in (NH
4
)
2
SO
4.
Prior to the experiment
they were buffer-exchanged with the assay buffer
(4 · 400 lL for every 10 lL of enzyme suspension) using
0.5 mL Microcon centrifugal filter devices (13 200 g,4°C)
to rid of excess ammonium sulfate. The resultant enzyme
solutions were concentrated down to the approximate con-
centrations of 1.4 units (pyruvate kinase) and 3 units (liver
alcohol dehydrogenase) per 10 lL. Both NADH and phos-
phoenolpyruvate solutions were prepared in 25 mm Hepes,

pH 7.6 immediately prior to use. The final assay volume of
200 lL contained: activated p38 MAPKa (0.4–0.45 lm),
ATP (0.085–1.36 mm), PEP (1 m m), NADH (0.18–
0.220 mm), pyruvate kinase (1.4 U) and liver alcohol dehy-
drogenase (3 U) in assay buffer 1. Where applicable,
AMP-PCP (0.25–2.2 mm) was added to the assay. All assay
components were preincubated at 27 °C for 5 min before
addition of ATP and the data was acquired for the follow-
ing 5 min under the same conditions.
Acknowledgements
This research was supported in part by the Welch
Foundation (F-1390) and the NIH (GM59802). We
are indebted to Dr Melanie Cobb (UT South-Western
Medical Center) and R. Copeland (DuPont Merck) for
generously providing us with DNA encoding his
6
-p38
MAPK and GST-MKK6b (S207E T211E), respect-
ively. Mass spectra were acquired by Dr Herng-Hsiang
Lo in the CRED Analytical Instrumentation Facility
Core supported by NIEHS center grant ES07784.
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