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Biophys J, November 2001, p. 2786-2794, Vol. 81, No. 5
and
*Computational Science Laboratory, Institute of Physical and
Chemical Research (RIKEN), Wako-shi, Saitama, 351-0198, Japan and
Laboratory of Physical Chemistry, Faculty of
Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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The ATP hydrolysis mechanism of myosin was studied using
quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the
energy-minimized structure of the myosin(S1dc)-ATP complex, which was
determined by molecular mechanics calculations. The result of QM
calculations suggested that the ATP hydrolysis mechanism of myosin
consists of a single elementary reaction in which a water molecule
nucleophilically attacked 
-phosphorus of ATP. In addition, we
performed molecular dynamics simulations of the initial and final
states of the ATP hydrolysis reaction, that is, the myosin-ATP and
myosin-ADP·Pi complexes. These calculations revealed roles of several
amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the
ATPase pocket. Lys185 maintains the conformation of 
- and
-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and
Ser237 are coordinated to a Mg2+ ion, which interacts with
the phosphates of ATP and therefore contributes to the stabilization of
the ATP structure. Arg238 and Glu459, which consisted of the gate of
the ATPase pocket, retain the water molecule acting on the hydrolysis
at the appropriate position for initiating the hydrolysis.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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Myosins are molecular motors that track along
actin filaments through the hydrolysis of ATP and play an important
role in diverse biological contractile events. Recently, the
three-dimensional structures of myosins have been determined by x-ray
crystallography (Rayment et al., 1993
; Fisher et al., 1995; Smith and
Rayment, 1996; Gulick et al., 1997). These structural data indicated
that nucleotide (ATP or ADP)- and actin-binding sites were located in
the globular head of myosin, called subfragment 1 or S1.
In Dictyostelium myosin II (S1dc), the nucleotide-binding
site (called the ATPase pocket) is surrounded by three loop structures; the P-loop (residues 179-186), the switch I loop (residues 233-240), and the switch II loop (residues 454-459). The ATPase pocket has a
gate for release of phosphate, a product of ATP hydrolysis. This gate
is closed or opened with the formation or disappearance of ionic bonds
between the side chains of Arg238 (in the switch I loop) and Glu459 (in
the switch II loop). Determination of the crystal structures of S1dc
with several nucleotides and its analogs (Fisher et al., 1995; Smith
and Rayment., 1996; Gulick et al., 1997) revealed that the switch I
loop undergoes a minor conformational change and that the switch II
loop undergoes a large conformational change. In the structures of S1dc
with MgADP/BeFx, MgAMPPNP, MgATPS, or MgADP, the switch II loop
moved away from the ATPase pocket, so that the gate was opened with the
disappearance of ionic bonds. In contrast, the structures of the S1dc
with MgADP/VO4 or MgADP/AlFx indicated that Arg238 formed ionic bonds
with Glu459 and, consequently, the gate was closed. From analysis of
these crystal structures, Rayment et al. (1993) suggested that the
conformation observed in the structure of S1dc with MgADP/VO4 (Fisher
et al., 1995), where the gate was closed, was necessary for the
hydrolysis of ATP. In addition, many experiments on site-directed
mutation of myosin have suggested that several amino acid residues of
the ATPase pocket play an important role in the hydrolysis of ATP (Sasaki and Sutoh, 1998; Li et al., 1998; Furch et al., 1999; Onishi et
al., 1998). From these studies, it was revealed that Lys185, Arg238,
and Glu459 were closely related with the hydrolysis of ATP. It is also
known that ionic bonds between Arg238 (in the switch I loop) and Glu459
(in the switch II loop) are required to support efficient ATP hydrolysis.
According to the suggestion by Bagshaw et al. (1974), the
mechanism of the binding and hydrolysis of ATP by myosin consists of
seven steps as shown in scheme 1, where M is myosin and Pi is a
phosphate. The asterisks refer to different conformational states as
detected by intrinsic protein fluorescence.
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(Scheme 1) |
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Based on the results of the aforementioned studies on x-ray crystallography and the site-directed mutation, we speculated that the ionic bonds between Arg238 and Glu459 were formed immediately before the hydrolysis of ATP (corresponding to the initial state of the third step in Scheme 1). The ATP hydrolysis in third step would be the core process in the sequence of Scheme 1 and has attracted the interest of many researchers. The detailed mechanism of ATP hydrolysis is, however, still unclear. In this study, we clarified the atomic-level reaction mechanism of the ATP hydrolysis by myosin using quantum chemical (QM) calculations and molecular dynamics (MD) simulations, and we examined the compatibility of our results with other experimental results.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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QM calculations
Construction of a model compound
For QM calculations of the ATP-hydrolysis mechanism, we used a model compound based on the energy-minimized structure of the myosin-ATP complex by molecular mechanics (MM) calculations (see MD simulation). Focusing on- and -phosphates, the model compound was constructed by extracting solid- and open-circle atoms from the
MM-minimized structure shown in Fig. 1
and replacing open-circle atoms with hydrogen atoms. The positions of
asterisked atoms were fixed during geometry optimization because these
constraints prevented the corruption of the basic structure of myosin.
This model compound consists of - and -phosphate groups of ATP, a
Mg2+ ion, a water molecule acting on the
hydrolysis,
CH3CH2NH3+
for Lys185,
CH3C(NH2)2+
for Arg238, CH3 COO
for
Glu459, two water molecules for Thr186 and Ser237 that interact with
the Mg2+ ion, and two water molecules
coordinating to the Mg2+ ion. A schematic
representation of the model compound is shown in Fig.
2.
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Computational details
The Schrödinger equation of the model compound was solved by the Hartree-Fock method using the 6-31G** basis set (Szabo and Ostlund, 1989). The model structures at the minimum points and transition states (TS) on the potential energy hypersurface were fully
optimized using the energy gradient method. Frequency analysis of the
structure of the TS indicated the presence of an imaginary frequency.
The lowest energy reaction path was determined by calculating the
steepest descent paths from each TS in both the forward and reverse
directions following the normal vibrational mode of the imaginary
frequency. The structures of a reactant and a product connected to the
TS resulted from this calculation. The intrinsic reaction coordinate in
Fig. 3 represents the lowest energy
reaction path expressed in mass-weighted coordinates. For checking the reliability of potential energy, we carried out calculations by the
density functional theory method using Becke's three-parameter hybrid
method (Becke, 1993) and using LYP correlation functions (B3LYP) (Lee
et al., 1988; Miehlich et al., 1989) on the structures of the stable
and TS which were determined at HF/6-31G**. The computational program
used was Gaussian 98 (Frisch et al., 1998).
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MD simulations
Construction of the initial structure
We carried out MD simulations of myosin-ATP and myosin-ADP·Pi complexes, which are the initial and final states of ATP hydrolysis. The initial structures of these complexes were constructed on the basis of the x-ray crystallographic structure of the myosin-ADP/VO4 complex (code in Protein Data Bank (Abola et al., 1987, 1996; Bernstein et al.,
1977): 1VOM (Smith and Rayment, 1996)). Because many amino acid
residues were not determined in the coordinate data of this x-ray
crystal structure, we repaired these amino acid residues
(Ala205-Ser208, Asn711, Ala716-Ser719, and Asp724-Leu730) by using a
homology module of Insight II (Insight II, version 95.0, Molecular
Simulations Inc., San Diego). The N-terminal amino acid residue, Met1,
of myosin (S1dc) was also not determined because of its flexibility,
and we therefore added it to the S1dc. Thus, the myosin (S1dc)
structure for MD simulations was constructed of 747 amino acid
residues. The structure of ATP was made by shortening the distance
between the vanadium atom of the vanadate VO4 and the oxygen atom of -phosphate to a normal P-O bond distance and replacing the vanadium atom with a phosphorus atom. A water molecule acting on the hydrolysis (lytic water molecule) was generated on the
basis of the position of the oxygen atom that had the longest V-O bond
among the four V-O bonds. The structures of ADP and phosphate Pi were
determined on the basis of the results of QM calculations (see
Results). We determined the parameters of ATP, ADP, and Pi by QM
calculations using the Hartree-Fock method/6-31G* basis set.
Calculations
MM potential energy minimizations and MD simulations were carried out using the program package AMBER (version 5.0, University of California) (Pearlman et al., 1995). Calculations were performed using
an all-atom force field of Cornell et al., (1995). The system was
solvated in a rectangular box of ~108 Å × 85 Å × 76 Å in size, and 20,240 TIP3P water molecules (Jorgensen et al., 1983) were generated in the box. The periodic boundary condition was applied and
the pressure was kept constant in the system. The temperature was kept
constant according to Berendsen' s algorithm with a coupling time of
0.2 ps (Berendsen et al., 1984). To simplify this calculation, the
SHAKE method was used (Ryckaert et al., 1977). The nonbonded interactions were calculated by the dual cutoff method. The distance of
the first cutoff was 12 Å and that of the second cutoff was 15 Å. The
integration time step of the MD simulations was 1 fs.
The strategy of our simulations was as follows. First, potential energy
minimizations were performed on the initial systems. In the potential
energy minimization, the steepest descent method was used for the early
cycles and then the conjugate gradient method was used later. The
minimized structure of the myosin-ATP complex was used for the
construction of the model compound for QM calculations, as described
before. Next, MD simulations were performed on the energy-minimized
systems, taking careful consideration of the temperature setting. After
10-ps MD simulations at 300 K only for the solvent water molecules with
the enzyme, nucleotide, and crystal water molecules fixed, the
temperature of all systems was gradually increased by heating to 300 K
for the first 60 ps, and then it was kept at 300 K for the next 500 ps.
The trajectories at this temperature (300 K for 500 ps) were considered
to be the most probable structure under physiologic conditions and were analyzed in detail.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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QM calculation: analysis of potential energy
As seen in the potential energy curve (Fig. 3), QM calculations at
HF/6-31G** revealed that a single elementary reaction comprised the
mechanism of ATP hydrolysis. We determined the structure of the TS from
the initial model compound. Fig. 4 shows
the unique imaginary frequency of the structure of this TS. The normal
vibrational mode of the imaginary frequency indicated that the oxygen
and hydrogen atoms of the water molecule combined with the phosphorus and oxygen atoms of the -phosphate, respectively. The potential energy drop along the steepest descendent path in the forward direction
of the hydrolysis reaction transformed the TS into the final state, S2,
the product of the hydrolysis. In contrast, the potential energy drop
in the opposite direction led to the initial state of the hydrolysis
reaction, S1 (Fig. 3). The hydrolysis reaction required an activation
energy of 58.63 kcal/mol, and the potential energy of S2 was 2.17 kcal/mol lower than that of S1. For more exact analysis of energy, we
calculated the potential energies of S1, TS, and S2 by the density
functional theory method. The results are shown in Table
1. QM calculations at B3LYP/6-31G** revealed that its activation energy was 41.97 kcal/mol, which was lower
than that of HF/6-31G** by ~17 kcal/mol. The potential energy of S2
was more stable than that of S1 by 6.19 kcal/mol.
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QM calculations: analysis of structures
The structures of the stable states (S1, S2) and the TS that appeared in the hydrolysis reaction are shown in Fig. 5.
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The initial state of hydrolysis, S1, was the myosin-ATP complex (see
Fig. 5, S1). In the structure of S1, a lytic water molecule was between
the gate of the ATPase pocket (Arg238 and Glu459) and the -phosphate
of ATP. The distance between the water molecule and -phosphate was
3.41 Å; thus, there is an effective interaction to initiate the
hydrolysis reaction. This water molecule formed a hydrogen bond with
Arg238 rather than with Glu459. At this time, the
Mg2+ ion was surrounded by six ligands in an
octahedral arrangement. The Mg2+ ion interacted
with oxygen atoms O1B and O1G of - and -phosphate in ATP and
therefore contributed to maintenance of the appropriate conformation of
phosphates in the ATPase pocket. The OG atom of Thr186, the OG atom of
Ser237, and two water molecules were also coordinated to the
Mg2+ ion. Lys185 formed two hydrogen bonds with
the O3G and O2B atoms of - and -phosphates (O3G(ATP)-NZ(Lys185):
3.11 Å, O2B (ATP)-NZ(Lys185): 2.84 Å), so that the conformation of
-phosphate was maintained in the ATPase pocket.
The TS structure appeared when the oxygen atom of the lytic water
molecule approached the phosphorus atom PG of the -phosphate (see
Fig. 5, PG(-phosphate)-O(water): 2.17 Å). At this time, the
hydrogen atom was just in the process of transferring to the oxygen
atom O3G of the -phosphate. The movement of the hydrogen atom
increased the distance between the O3G atom of -phosphate and the NZ
atom of Lys185.
The final state of the hydrolysis, S2, was the myosin-ADP·Pi complex
(see Fig. 5, S2). In this structure, the OH group from the lytic water
molecule was linked to the PG atom of the -phosphate, and the
hydrogen atom initially attached to the water molecule transferred
completely to the O3G atom of -phosphate. These movements resulted
in dissociation of phosphate Pi from the ATP molecule. At this time,
the distance between -phosphate and Pi was 3.29 Å. This
dissociation caused the disappearance of the hydrogen bond between the
O3G atom of Pi and the NZ atom of Lys185 and the formation of a
stronger hydrogen bond (low-barrier hydrogen bond) between the O2B atom
of -phosphate and the NZ atom of Lys185. The Pi was moved from the
nucleotide (ADP) and approached the gate of the ATPase pocket. The
conformation of ligands interacting with the Mg2+
ion was different from that of the S1 structure. The structure of
-phosphate was changed by the dissociation of Pi from ATP, so that
the interaction between O3B and Mg2+ atoms became
stronger, whereas the interaction between O1B and Mg2+ atoms became a little weaker. The
interaction between O1G and Mg2+ atoms also
became weaker.
Fig. 6 shows an interesting feature of
the structural change in this ATP hydrolysis mechanism. The dihedral
angle O1G-O2G-O3G-PG of -phosphate was 28.37° in S1, 9.94° in
TS, and 
33.04° in S2. Information on the dihedral angles indicated
that inversion of the -phosphate occurs. As shown in Fig. 5, a lytic
water molecule approached the PG atom on a PG-O3B bond line. This fact
revealed that a Walden inversion reaction occurs in ATP hydrolysis.
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MD simulations of myosin-ATP and myosin-ADP·Pi complexes
MD simulations were carried out for both myosin-ATP and myosin-ADP·Pi complexes. Figs. 7 and 8 show the average structures of these two complexes.
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In the average structure of the myosin-ATP complex (Fig. 7), a lytic
water molecule existed between the gate of the ATPase pocket (Arg238
and Glu459) and -phosphate of ATP, as in the S1 structure determined
by QM calculations. The distance between the lytic water molecule and
-phosphate was 3.30 Å, similar to the value obtained by QM
calculations (3.41 Å). However, the distance between the lytic water
molecule and Arg238 was 3.79 Å, which is longer by 0.9 Å than the
value of the S1 structure. The reason for this difference would be that
the model compound for the QM calculations does not take Gly457 into
consideration. In the MD simulation, the main chain NH group of Gly457
formed a hydrogen bond with this lytic water molecule. By this hydrogen
bond, the position of the lytic water molecule was closer to ATP than
that of the S1 structure. In the same manner as that of the of S1
structure, the NZ atom of Lys185 formed hydrogen bonds with O3G and O2B
atoms and helped to maintain the conformation of the - and
-phosphates in the ATPase pocket. The conformation involving the
Mg2+ ion is similar to the crystal structures and
the results of QM calculations. The Mg2+ ion
interacts with six ligands (Thr186, Ser237, - and -phosphates, and two water molecules). The side chain of Asp454 faced the ATPase pocket and formed hydrogen bonds with Thr186 and the water molecule, but Asp454 did not directly interact with the
Mg2+ ion. In these aspects, the results of MD
simulation of the myosin-ATP complex almost accorded with the results
of QM calculations.
The results of MD simulation of the myosin-ADP·Pi complex are as
follows. In the MD simulation (Fig. 8), the dissociating Pi formed
hydrogen bonds with the NZ atom of Lys185, N and O atoms of Ser237, and
the N atom of Gly457, and it was therefore maintained stably in the
ATPase pocket. The hydrogen bond between the NZ atom of Lys185 and the
O2B atom of -phosphate was maintained during the simulation, in the
same way as that of the S2 structure. The conformation of the ligands
involving the Mg2+ ion was similar to the results
of QM calculations; namely, the Mg2+ ion
interacted with the oxygen atom (O3B atom) of -phosphate in addition
to the six ligands observed in MD simulation of the myosin-ATP complex.
MD simulation of myosin-ATP complex in the mutation of G457A or D454A
In our QM calculations, Gly457 and Asp454 were not included in the
model compounds. Some experimental study with mutation technique
suggested that these amino residues, Gly457 and Asp454, had a relation
to the activity of ATP hydrolysis as well as Lys185, Arg238, and Glu459
(Sasaki and Sutoh, 1998). Accordingly, MD simulations were also
performed for two mutants, G457A and D454A, to examine the involvement
of these residues in ATPase activity. The MD computational method was
the same as the previous section.
Table 2 shows the average interatomic distances during the 500-ps MD simulations at 300 K for three types of myosin-ATP complexes (wild, G457A, and D454A). No prominent conformational differences are seen among the three. In the simulation of G457A, the distance between the NH1 atom of Arg238 and lytic water increased by 0.7 Å compared with the wild-type. This is attributed to the absence of the hydrogen bond between the O3G atom of ATP and the N atom of Ala457. The G457A mutation caused the steric hindrance of the side chain of alanine and the N atom of Gly457 could make a hydrogen bond only with the lytic water. In the wild-type, the N atom of Gly457 has two hydrogen bonds with the lytic water and the O3G atom of ATP. Hence, the position of the lytic water was shifted toward ATP. No other obvious difference in interatomic distances between wild-type and G457A is seen in Table 2.
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In the simulation of D454A, most interatomic distances are consistent between wild-type and D454A mutant with the exception of the OE1 atom of Glu459, the O atom of lytic water. Because Glu459 is located at the opposite position to Asp454, the D454A mutation seems not to be a direct reason for the change of the OE1 atom of Glu459, the O atom of lytic water distance.
Despite an experimental suggestion of low activity of ATP hydrolysis in
the G457A and D454A mutants, the present MD simulation did not
demonstrate any significant difference between wild-type myosin and
those mutants. In the section QM calculations: analysis of structures,
the QM calculation could give a reaction path of ATP hydrolysis without
those amino residues. Consequently, it would be concluded that Gly457
and Asp454 had a minor role for the ATP 
ADP conversion reaction. We
speculate that a low ATPase activity in G457A and D454A mutant is
attributable to other reasons, such as problems in incorporating ATP or
water at the catalytic site.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
|
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Our QM calculations revealed that the mechanism of ATP hydrolysis
by myosin was a single elementary reaction accompanied by Walden
inversion of -phosphate. This result explained the outline of the
mechanism. In addition to the information obtained by QM calculations,
MD simulations clarified the effects of many amino acid residues in the
ATPase pocket. We show the roles of the amino acid residues in the
ATPase pocket by analyzing our computational results.
From QM and MD calculations of the myosin-ATP complex (see Figs. 5 (S1)
and 7), Lys185 prevents the deformation of the - and -phosphates
of ATP by forming hydrogen bonds with O3G and O2B atoms, and it plays a
role in maintaining the appropriate structure of ATP for generation of
hydrolysis. In MD simulation of the myosin-ADP·Pi complex (Fig. 8),
Lys185 formed hydrogen bonds with the O3G atom of Pi and the O2B atom
of -phosphate in ADP and contributed to the stabilization of
myosin-ADP·Pi complex structure, which seemed to be in a semistable
state. This lysine commonly exists in kinesin and G-protein, each of
which has the activity of ATP or GTP hydrolysis. Studies on their
crystal structures indicated that lysine played a common role in these
proteins because the similar hydrogen bonds involving lysine were
formed. In our QM calculations, because the distances between the NZ
atom of Lys185 and the O3G atom of -phosphate in S1, TS, and S2
structures are 3.11, 3.49, and 4.19 Å, respectively, the hydrogen bond
must have been lost with the progress of the reaction. In S1, the NZ atom of Lys185 formed a hydrogen bond with the O3G atom, and then the
O3G atom acted as a proton acceptor for a lytic water molecule, and, as
a consequence, the hydrogen bond was lost. In contrast, this hydrogen
bond was maintained in the MD simulation of the myosin-ADP·Pi complex
because the O3G atom of Pi was maintained by the formation of many
hydrogen bonds with amino acid residues in the ATPase pocket. These
results suggest that the reason why K185Q mutant myosin did not exhibit
ATPase activity (Li et al., 1998), in which Gln185 was not able to form
hydrogen bonds with - and -phosphate, and thus the structure of
- and -phosphates was deformed and the lytic water molecule could
not attack the -phosphate. From this point, it is thought that
Lys185 plays a role in maintaining the appropriate structure of
phosphate groups in the nucleotide.
Thr186 and Ser237 are coordinated to the Mg2+
ion, which interacts strongly with the - and -phosphates of the
nucleotide in QM and MD calculations. These two amino acid residues
maintain the conformation of these phosphates of the nucleotide through the Mg2+ ion. According to the results of an
experiment on site-directed mutation (Sasaki and Sutoh., 1998), S237A
mutant myosin exhibited a low ATPase activity. We speculate that the
disappearance of the hydroxy group because of the replacement of
alanine causes a change in the conformation involving the
Mg2+ ion, thus preventing the occurrence of ATP
hydrolysis. In the myosin-ATP complex, six ligands were coordinated to
the Mg2+ ion in good balance, and the structure
of -phosphate became suitable for ATP hydrolysis. Thus, Thr186 and
Ser237 are important amino acid residues.
We describe below the roles of Arg238 and Glu459, which comprise the
gate of the ATPase pocket. The results of QM and MD calculations showed
that Arg238 and Glu459 formed ionic bonds with one another during the
process of ATP hydrolysis. In the S1 structure (Fig. 5), Arg238 formed
a hydrogen bond with a lytic water molecule (water molecule-Arg238:
2.89 Å). MD simulation of the myosin-ATP complex revealed that the
distance between a lytic water molecule and Arg238 was long enough for
initiation of the hydrolysis, although the effect of the other amino
acid residues containing Gly457 increased the distance to 3.79 Å. It
is interesting that the oxygen of the lytic water molecule faces toward
the opposite direction of the nucleophilic attack on -phosphorus of
ATP. This is why Arg238 acts as a proton donor. According to the
experimental data of R238E/E459R double mutant myosin, the ATPase
activity of this mutant myosin was equal to that of the wild-type
myosin (Furch et al., 1999). This suggests that the ATPase hydrolysis
does not depend on the orientation of the water molecule. Arg238 plays a role in maintaining the appropriate position of the water molecule for ATP hydrolysis. However, it is thought that Glu459 did not directly
contribute to the dissociation reaction of ATP (Figs. 5, 7, and 8),
though it formed ionic bonds with Arg238. The results of our
calculations revealed that the distance between a lytic water molecule
and Glu459 was ~5 Å, and there was therefore not a sufficient
interaction. In addition, Glu459 formed ionic bonds with Arg238 during
ATP hydrolysis. Therefore, it is unlikely that Glu459 acts as a base in
the hydrolysis. However, we believe that Glu459 plays a role in the
opening and closing of the ATPase pocket and in the capture of a lytic
water molecule acting on the hydrolysis. Experiments on site-directed
mutation of Arg238 and Glu459 (Sasaki and Sutoh, 1998; Li et al., 1998;
Furch et al., 1999; Onishi et al., 1998) showed that substitution of
Ile, Ala, or Glu for Arg causes a decrease in ATPase activity, and that
the substitution of Arg or Ala for Glu also causes a decrease in ATPase
activity. However, ATPase activity of R238K mutant myosin was very
similar to that of the wild-type myosin (Li et al., 1998). Furthermore, the double substitutions of Glu and Arg for Arg and Glu exhibited the
same ATPase activity as that of wild-type myosin (Furch et al., 1999;
Onishi et al., 1998). These experimental findings suggest that the
formation of ionic bonds between Arg238 and Glu459 is essential for the
initiation of ATP hydrolysis. We speculated the roles of Arg238 and
Glu459 as follows: when ATP and myosin combine, a water molecule is
trapped near the -phosphate of the ATPase pocket, and this triggers
the formation of ionic bonds between Arg238 and Glu459 by electrostatic
interactions. As a consequence, the ATP hydrolysis reaction starts
without any influence of bulk water molecules. Therefore, Arg238 and
Glu459 play roles in the capture of a lytic water molecule from
numerous solvent water molecules and in the supply of a lytic water
molecule into the ATPase pocket by closing the gate of the ATPase pocket.
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CONCLUSION |
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|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
|
|---|
Our QM calculation revealed that the mechanism of ATP hydrolysis
by myosin is a single-elementary reaction with Walden inversion of
-phosphate, and QM and MD calculations suggested that there is an
effect of amino acid residues in the ATPase pocket. Lys185 plays a role
in maintaining the appropriate structure of phosphate groups in the
nucleotide. Thr186 and Ser237 maintain the conformation of the
phosphates of the nucleotide through the Mg2+
ion. Arg238 and Glu459 play roles in keeping a lytic water molecule at
the appropriate position for initiating the hydrolysis.
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ACKNOWLEDGMENTS |
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This work was supported in part by the super computer Vpp700e in Institute of Physical and Chemical Research (RIKEN). The authors thank the Research Center for Computational Science, Okazaki. The computations were also carried out by the DRIA System at the Faculty of Pharmaceutical Sciences, Chiba University.
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FOOTNOTES |
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Received for publication 29 September 2000 and in final form 31 July 2001.
Address reprint requests to Dr. Noriaki Okiomoto, Institute of Physical and Chemical Research (RIKEN), Computational Science Laboratory, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. Tel.: 81-48-467-9417; Fax: 81-48-467-4078; E-mail: okimoto{at}atlas.riken.go.jp.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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S, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain.
Biochemistry.
36:11619-11628-phosphate binding site of myosin on its motor function.
J. Biol. Chem.
273:27404-27411
Biophys J, November 2001, p. 2786-2794, Vol. 81, No. 5
© 2001 by the Biophysical Society 0006-3495/01/11/2786/09 $2.00
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