Kinetic mechanism for p38 MAP kinase aA partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate Anna E.. We show using an untagged
Trang 1Kinetic mechanism for p38 MAP kinase a
A partial rapid-equilibrium random-order ternary-complex
mechanism for the phosphorylation of a protein substrate
Anna E Szafranska1and Kevin N Dalby1,2
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: Dalby@mail.utexas.edu
(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 Mg2+ and 27C 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 KA¼ 360 lm, KI¼ 240 lm, and KI> 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 Kd 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.
Trang 2All 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 concentrainhibi-tions > 100 nm, as previously suggested, demon-strating that caution must be used when interpreting kinetic experiments using this inhibitor
Trang 3asserted 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 ATF2D115 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 Mg2+ at pH 7.6 (kcat¼ 0.3 s)1 and Km¼
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 (ka) 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 (kp), and the forma-tion of MgADP and inorganic phosphate (Pi) These products then dissociate (kdiss) from the active site Given the slow turnover (kcat¼ 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 Km¼ k–a⁄ ka¼ 353 lm A conservative estimate for the second-order rate con-stant of ka¼ 104m)1Æs)1for the binding of MgATP to p38 MAPKa gives a rate constant for the dissociation
of MgATP from the enzyme of k-a¼ 3.5 s)1, if the dis-sociation constant KA¼ 350 lm is used This value exceeds kcatby 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).
Trang 4phorylation 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 ATF2D115 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
Vmax
aKAKBþ aKBAþ aKABþ 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
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.
Trang 5with 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 [KB¼ 39 lm (ATF2D115); KA¼ 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.
experi-mental data to Eqn (1).
Varied substrate Inhibition pattern
KA
KB
kcat
KI
KI
Trang 6catalytic constant of kcat¼ 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
105)106m)1Æs)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 kcat 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 a¼ 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
aKA represents a Michaelis–Menten constant and not
a dissociation constant It should be noted that the
values of Kdfor 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 KI¼
187.4 lm and bKI¼ 8.6 lm (Table 1), where KI, but
not bKI 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 Ki¼ 241 lm (Table 1), which
is in fairly good agreement with KI¼ 187.4 lm obtained from the bisubstrate kinetics
v
Vmax
aKA 1þK B
B þIK B
KIBþ I
bKI
þ A 1 þaKB
B
rearranged
v
Vmax
aKB 1þK A
A þIK A
K I A
þ B 1 þaK A
A þaK A I
bKIA
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 KI> 2 mm, 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
Trang 7inactive 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]
experi-mental data according to Eqn (2).
Trang 8proved 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
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 (Kd), 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
Kd 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 c¼ 0.1
the binding is very weak and yields a nearly horizontal
isotherm with a poorly defined binding constant, Kd
[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 bKiobtained
kinetically, suggesting that, as suspected, ligand bind-ing is not completely at equilibrium durbind-ing 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 Kd¼ 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)1ÆK)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: Kd¼ 69.6 ± 6 lm, n ¼ 0.52 ± 0.08,
DH ¼)14 200 JÆmol)1, andDS ¼)28.3 JÆmol)1ÆK)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 Kd¼
300 ± 160 lm, n¼ 1.4 ± 0.4, DH ¼)1145 JÆmol)1, and DS ¼ +12.3 JÆmol)1ÆK)1 This dissociation con-stant is in line with the value of K¼ 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 EÆI and EÆIÆS dead
end complexes.
Trang 9exhibit 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 (kcat¼ 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 (k2¼ 109 s)1) and product release (k3¼ 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 kcat¼ 0.3 s)1, which is very similar to
Trang 10the catalytic constant for the phosphorylation of
ATF2D115 that has a value of kcat¼ 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-X4-FA
-X-FB (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 aD and aE and the
reverse turn between strands b7 and b8 [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, KA¼
360 lm; ATF2D115, KB¼ 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