From the Laboratoire de Cristallographie et de
Modélisation des Matériaux Minéraux et
Biologiques, Groupe Biocristallographie, UMR 7036, CNRS-Université Henri Poincaré, Faculté des Sciences,
54506 Vandoeuvre Cedex, France and § Maturation des ARN
et Enzymologie Moléculaire, UMR 7567, CNRS-UHP, Faculté des
Sciences, 54506 Vandoeuvre Cedex, France
Received for publication, October 29, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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The crystal structure of the phosphorylating
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus
stearothermophilus was solved in complex with its cofactor, NAD,
and its physiological substrate,
D-glyceraldehyde 3-phosphate
(D-G3P). To isolate a stable ternary complex, the
nucleophilic residue of the active site, Cys149, was
substituted with alanine or serine. The C149A and C149S GAPDH ternary
complexes were obtained by soaking the crystals of the corresponding
binary complexes (enzyme·NAD) in a solution containing G3P.
The structures of the two binary and the two ternary complexes are
presented. The D-G3P adopts the same conformation in the
two ternary complexes. It is bound in a non-covalent way, in the
free aldehyde form, its C-3 phosphate group being positioned in
the Ps site and not in the Pi site. Its C-1
carbonyl oxygen points toward the essential His176, which
supports the role proposed for this residue along the two steps of the
catalytic pathway. Arguments are provided that the structures reported
here are representative of a productive enzyme·NAD·D-G3P complex in the ground state (Michaelis complex).
Phosphorylating glyceraldehyde-3-phosphate dehydrogenases
(GAPDH)1 are tetrameric
enzymes that catalyze reversibly the oxidative phosphorylation of
D-glyceraldehyde 3-phosphate
(D-G3P) into 1,3-diphosphoglycerate (1,3-dPG) in the
presence of cofactor NAD(P) via a two-step chemical mechanism. This
mechanism has been well documented for the homotetrameric GAPDHs
involved in glycolysis (1). First, the acylation step leads to the
formation of a thioacylenzyme intermediate and NADH. This step
includes: 1) the binding of D-G3P to the binary
complex GAPDH·NAD; 2) the formation of a covalent thiohemiacetal
intermediate with D-G3P; and 3) the hydride
transfer from the thiohemiacetal intermediate toward the C-4 position
of the nicotinamide ring of NAD. Second, the phosphorylation step
consists of a nucleophilic attack of inorganic phosphate toward the
thioacylenzyme intermediate that leads to formation of 1,3-dPG. This
step, which is rate-limiting, includes the binding of inorganic
phosphate to the enzyme intermediate, possibly preceded by an
isomerization step consisting of a replacement of NADH by NAD (2).
Two amino acids are essential for the chemical mechanism,
Cys149 and His176. The high catalytic
efficiency of GAPDHs toward D-G3P implies prerequisites with respect to the chemical mechanism. First,
Cys149 should be in a thiolate form within the ternary
Michaelis complex to efficiently attack the C-1 aldehydic group of
D-G3P. This is ensured in part through the formation
of an ion pair with His176, which decreases
Cys149 pKapp from 8.0 to 5.9 (3).
Second, the hydride transfer should be efficient. This is assisted by
His176, which plays the role of a base catalyst. Moreover,
the different intermediates and transition states, including the
Michaelis complex, GAPDH·NAD·D-G3P, and the
thioacylenzyme intermediate, should be stabilized within the
active site. This is likely another role for His176
(Fig. 1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the postulated
catalytic mechanism of phosphorylating GAPDHs. The catalytic
mechanism can be divided into two steps. In the acylation step,
Cys149 and His176 form an ion pair in
holo-GAPDH (a). This decreases the
pKapp of Cys149, thus facilitating
the thiolate attack toward the C-1 of D-G3P.
The role of His176 is also to stabilize the binding of the
substrate in the Michaelis complex GAPDH·NAD·D-G3P
(b), the thiohemiacetal intermediate (c), and the
thioacylenzyme intermediate (d). His176 also
plays a role as a base catalyst facilitating the hydride transfer from
the thiohemiacetal toward the nicotinamidium of NAD (c). In
the phosphorylating step, the binding of inorganic phosphate to the
thioacylenzyme is followed by its nucleophilic attack toward the
thioacyl intermediate (d), which leads via a
sp3-phosphorylated intermediate (e) to
the formation and release of 1,3-dPG (f). His176
is postulated to stabilize the tetrahedral intermediate (e)
and to facilitate, as an acid (d) or base
(e) catalyst, the 1,3-dPG formation. The exchange cofactor
step, which consists of NADH release prior to NAD and inorganic
phosphate binding, remains controversial (32-40). R'
represents the adenine-ribose-phosphate-phosphate-ribose part of the
cofactor, NAD. R represents the CH(OH)COPO3 part
of the substrate, D-G3P.
The chemical nature of the substrates, G3P and inorganic phosphate, and the product, 1,3-dPG, implies the presence of two anion recognition sites in GAPDHs. These two anion binding sites have been tentatively identified within the active site from the location of two sulfate ions coming from the crystallization medium (4). On the basis of model building of the lobster GAPDH thiohemiacetal intermediate, these two anion binding sites were postulated to correspond to those binding the C-3 phosphate group of D-G3P (Ps site) and the inorganic phosphate ion (Pi site) (4). In all bacterial and eukaryotic GAPDHs, the Ps site is composed of the side chains of residues Arg231 and Thr179 and the 2'-hydroxyl group of the nicotinamide ribose when NAD is bound; the Pi site includes the side chains of residues Ser148 and Thr208 and the main chain nitrogen of Gly209.
The Ps site has been found to occupy an identical location
in all the crystal structures of eukaryotic and bacterial GAPDHs solved
so far (4-14). But, the "true" location of the Pi site remains a matter of debate. Indeed, depending on the considered structure, the -strand-loop-
helix segment containing residues 206-210 (called loop 206-210) has been observed in two different conformations, giving rise to two alternative locations for the Pi site. The most common conformation is the one found in
the holoenzyme from Bacillus stearothermophilus (7), whereas
a second conformation is observed for instance in the holostructure from Leishmania mexicana and generates a Pi site
that is located closer to the catalytic Cys149 residue and
2.9 Å away from the former position (9).
The contribution of the Ps and Pi sites during the two steps of the catalytic mechanism also remains a matter of debate. Taking into account the revised orientation of the side chain of His176 in the refined holostructure of B. stearothermophilus GAPDH, Skarzynski et al. (7) noticed that it was no longer possible to build a reasonable model of the hemithioacetal with its C-3 phosphate bound in the Ps site. They proposed that although the Pi site must be the location of the inorganic phosphate in the phosphorylation step, the C-3 phosphate of the substrate would bind first to the Pi site in the acylation step and then flip from the Pi site to the Ps site in the phosphorylation step. This hypothesis was supported by: 1) the inspection of the crystal structure of a ternary covalent complex, GAPDH·NAD·glycidol 3-phosphate, from B. stearothermophilus, which localizes the C-3 phosphate of the inhibitor within the Pi site (7); and also 2) kinetic analyses showing that substitution of Arg231 of the Ps site with glycine or leucine does not significantly affect the first-order rate constants of the overall oxidoreduction step (15), whereas mutations in the Pi site decrease this constant 4-7-fold (mutants T150A and T208A, respectively; see Ref. 16). The recent structure of a binary complex between Escherichia coli apoGAPDH and D-G3P, in which the C-3 phosphate group of the substrate is bound in the so-called "new Pi site," also supports this hypothesis (17).
In the present study, the crystal structures of two mutant GAPDHs from
B. stearothermophilus in complex with the physiological substrate, D-G3P, have been determined at
2.0-2.2 Å resolution with the aim of evaluating the different
hypotheses on the contribution of both Ps and
Pi sites along the catalytic pathway. For this purpose, the
active site Cys149 residue has been substituted by Ala and
Ser. The C149A mutant GAPDH is inactive, whereas the C149S mutant GAPDH
possesses a low residual activity (18). New crystallization conditions
have been set up in the absence of any anions to avoid competitive binding with the C-3 phosphate of D-G3P. The
structures presented here constitute the first example of ternary
complexes mimicking the Michaelis complex, wild-type
GAPDH·NAD·D-G3P. The results strongly suggest that the
C-3 phosphate of D-G3P binds to the Ps site at least in the Michaelis ternary complex.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis, Production, and Purification of Mutant
B. stearothermophilus GAPDHs--
Overexpression and purification of
C149S mutant GAPDH were carried out by a procedure similar to that
described earlier (18). For C149A mutant GAPDH, overexpression was
carried out in E. coli HB101 strain
(supE44, hsdS20 (r,
m
), recA13, ara-14,
proA2, lacY1, galK2,
rpsL20, xyl-5, mtl-1) transformed with
a pBluescriptII-derived plasmid containing the gapA gene of
B. stearothermophilus under the control of the
lac promoter. Site-directed mutagenesis was performed using
the QuikChange site-directed mutagenesis kit (Stratagene). Purification
of C149A mutant GAPDH was performed as described previously for other
B. stearothermophilus mutant GAPDHs (3), except that a
supplementary step was added to separate the B. stearothermophilus mutant GAPDH totally from the wild-type
E. coli GAPDH produced by the HB101 cells. For this, enzyme
solution was applied onto a phenyl-Superose HR column (Amersham
Biosciences) equilibrated with Tris-HCl 50 mM, EDTA
2 mM, (NH4)2SO4 1.7 M, pH 8 buffer, followed by a linear gradient from 1.7 to
0.7 M (NH4)2SO4. Under
these conditions, C149A mutant B. stearothermophilus GAPDH
was eluted at 1070 mM (NH4)2SO4, and E. coli
GAPDH at 850 mM.
Both mutant GAPDHs were isolated as apoforms, as judged by the A280/A260 ratio of 2. The purity of the enzymes was checked by SDS-PAGE and mass spectrometry.
Activity Assay of GAPDH--
Initial rate measurements were
carried out at 25 °C on a Kontron Uvikon 933 spectrophotometer by
following the absorbance of NADH at 340 nm. Thermostatted sample
holders using a circulating water bath for all of the measurements
maintained the temperature of the solutions at 25 °C. The
experimental conditions were 1 mM, D,L-G3P and
1 mM NAD in 40 mM triethanolamine, 2 mM EDTA, 50 mM K2HPO4
buffer, pH 8.9. Protein concentrations were calculated using a molar
extinction coefficient at 280 nm of 1.17.105
M1 cm
1. Activities were
expressed per monomer in s
1.
Crystallization and Data Collection-- Crystals of the binary complexes I and II (C149A GAPDH·NAD and C149S GAPDH·NAD) were obtained at 293 K using the hanging-drop vapor diffusion technique (19). Crystals of the C149A mutant protein were grown from a solution composed of 8% (w/v) polyethylene glycol (PEG) 4000, 0.1 M sodium acetate buffer, pH 4.6, 2 mM EDTA, 2 mM dithiothreitol, and 1.5 mM NAD, whereas the C149S mutant protein crystallized in quite similar conditions but in Tris-HCl buffer, pH 7.5, and with 10% (w/v) PEG 4000 as the precipitating agent.
Ternary complexes I and II (C149A GAPDH·NAD·D-G3P and C149S GAPDH·NAD·D-G3P) were obtained by soaking the crystals for 10 min in the crystallization solution containing 1.9 mM D,L-G3P. The crystals were then flash-frozen in liquid nitrogen by using 25% (v/v) 2-methyl-2,4-pentadiol as a cryoprotectant.
The four data sets were collected at 110 K on an area detector
(DIP2030) with a Phi goniometer using CuK radiation from a rotating
anode generator (Model FR591, Nonius B. V.). The data sets were
processed and scaled with the HKL suite (20). Binary complex I
crystallized in space group P3121 with one
homotetramer/asymmetric unit. The crystal of the monoclinic binary
complex II belongs to space group C2 and contains two
independent dimers in the asymmetric unit (Fig. 2). The crystals of
binary complex I and of ternary complex I diffracted to 2.2 Å resolution, whereas the crystals of binary complex II and ternary
complex II diffracted to 2.1 and 2.0 Å, respectively. The statistics
of the data sets are summarized in Table I.
Phasing and Refinement-- The structure of the binary complexes were solved by molecular replacement (21) using the wild-type holostructure (Protein Data Bank code 1gd1) as the starting model and data from the 10 to 5 Å resolution range. Cycles of refinement (crystallography NMR software (CNS) (22)) alternated with manual rebuilding (TURBO-FRODO (23)) and inclusion of higher resolution data were carried out to improve the models.
Finally, the ordered water molecules were added. Each peak contoured at
3 in the Fo
Fc maps
was identified as a water molecule, provided that hydrogen bonds would
be allowed between this site and the protein. The models were then
adjusted and refined up to a final convergence, using all of the
reflections (sigma cut-off = 0.0) with R and
R-free values of 17.7 and 21.0%, respectively, for the
C149A binary complex and 19.8 and 24.9% for the C149S binary complex
(Table I).
The ternary complexes were refined from the final models of the binary complexes using an identical procedure. The R and R-free values were 18.0 and 21.4%, respectively, for the C149A ternary complex and 19.3 and 23.5% for the C149S ternary complex (Table I).
Structure Analyses and Final Structure Parameters--
The
geometry of the models was checked with PROCHECK (24). Statistics
concerning the geometry of the final models are given in Table I. All
non-glycine residues are located in favorable regions of the
Ramachandran plot (25), except Asp186 and
Val237, which belong to external loops and have side chains
that are very well defined in the (2Fo Fc) density maps. The location of these residues
outside the allowed regions has already been noticed in the crystal
structures of GAPDHs isolated from other sources (11). The coordinates
and the structure factors of the binary and ternary complexes have been
deposited to the Protein Data Bank at Research Collaboratory for
Structural Bioinformatics (ID codes 1NPT, 1NQ5, 1NQA, and 1NQO). Figs.
2-5 were drawn with MOLMOL (26) or TURBO-FRODO (23).
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RESULTS |
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Biochemical Properties of Mutant GAPDHs--
The B. stearothermophilus C149A mutant GAPDH was overproduced in
E. coli cells, purified, and separated from the E. coli GAPDH by taking advantage of its lower hydrophobicity. This
separation was accompanied by a drastic diminution of the activity from
5.104 s
1 to less than 1.10
6
s
1. This result clearly shows that the C149A mutant GAPDH
displays no significant dehydrogenase activity in contrast to the
E. coli C149A mutant GAPDH, which was shown to display a
significant non-phosphorylating activity (27). This finding suggests
subtle structural differences between the active sites of E. coli and B. stearothermophilus GAPDHs (see below).
On the other hand, the C149S mutant GAPDH displayed, as already
published (18), a low but significant phosphorylating dehydrogenase
activity with a kcat of 8.10
3
s
1.
Overall Structures--
All GAPDH structures solved thus far share
a common conserved fold composed of a NAD(P) binding domain and a
catalytic domain. The enzyme is active as a homotetramer in which four
subunits, designated O, P, Q, and R, are related by three perpendicular 2-fold axes, P, Q, and R (for nomenclature, see
Ref. 28), with the choice of the first monomer for denomination "O"
being arbitrary. Because the crystals of the wild-type holoenzyme from
B. stearothermophilus (7) had been obtained in the presence
of ammonium sulfate, which could have competed with the substrate for
the binding to the anion recognition sites, new crystallization
conditions had to be set up for the present study. Two new crystal
forms have been obtained using PEG 4000 as the precipitant. The
crystals of the binary complex C149A GAPDH·NAD (binary complex I) are
trigonal and contain one typical homotetramer per asymmetric unit. The asymmetric unit of the monoclinic crystals obtained from the binary complex C149S GAPDH·NAD (binary complex II) is composed of two independent dimers (Fig. 2). Although
having a different orientation, these two dimers are equivalent and
were thus called OQ and O'Q'. Two biological tetramers (called A and B)
were generated from these dimers OQ and O'Q' by using the
crystallographic 2-fold axis. In tetramer A, the crystallographic axis
is along the 2-fold axis P, whereas in tetramer B, it
corresponds to the R axis. A striking feature of this
monoclinic form is the high compactness of the tetramers in the unit
cell (Matthews coefficient, (VM = 2.19 Å3/Da)) when compared with the trigonal form
(VM = 2.92 Å3/Da).
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No significant structural differences are observed between the four
monomers within each asymmetric unit. In the binary I and binary II
complexes, the mean root-mean-square values obtained from the different
pairwise superimpositions of the C atoms are 0.26 and
0.30 Å, respectively. For the ternary I and ternary II complexes,
these values are 0.26 and 0.31 Å, respectively. In the four monomers
of the binary or ternary complexes, the NAD molecule is well defined in
the (2Fo
Fc) electron
density maps (Fig. 3) and possesses
average temperature factors similar to those of the protein (Table
I).
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Structure of the Binary Complexes: Comparison with the Wild-type
Holoenzyme--
Because the structure of the wild-type holoenzyme
revealed a quasi-perfect 222 symmetry (7), the least-squares
superimpositions involving the 334 C atoms of subunit O
of the wild-type holoenzyme were realized with each subunit of the two
binary complexes. The resulting root-mean-square differences are less
than 0.45 Å, showing that the overall structures of the binary I and
binary II complexes are very close to that of the wild-type holoenzyme.
The largest differences mainly concern either solvent-exposed loops or
regions involved in different contacts with symmetry-related molecules. In addition, differences can be noted for loop 206-210. This loop is
located in the vicinity of the Pi site, which is
"empty" in both the binary I and binary II complexes, whereas the
side chain of Thr208 and the main chain nitrogen of
Gly209 are directly involved in the binding of a sulfate
anion in the structure of the wild-type holoenzyme (7). The
displacement of residues 206-210 range from 0.2 to 1.1Å depending on
the subunit. Furthermore, the average temperature factors calculated
for all the atoms of the loop is 5 Å2 higher than the
average B-factor calculated for all the atoms of the subunit. These
differences in B-values are not observed in the wild-type
holostructure, in which the presence of the sulfate anion bound at the
Pi site could stabilize the conformation of this loop.
Nevertheless, the amplitude of the displacement remains small and
insignificant when compared with that observed for the E. coli GAPDH·G3P binary complex (17). In the present work, the
main conformation of loop 206-210 is rather similar to that observed
in the structure of B. stearothermophilus wild-type GAPDH (7), as confirmed by omit maps (not shown) calculated in this region.
Therefore, the Pi site must still be structurally competent to bind sulfate (or phosphate) even if the Pi site does not
contain any sulfate anion in the mutant structures, because of the
crystallization conditions used in this study. In both binary
complexes, a broad electron density peak, identified as a sulfate ion,
is observed within the Ps site. Because the crystallization
trials have been conducted in the absence of sulfate, this anion arises
most probably from the ammonium fractionation step of the purification
and has not been eliminated despite the extensive dialyses carried out prior to the crystallization experiments.
Structures of the Ternary Complexes of Mutant GAPDHs--
The
comparisons of each ternary complex with its corresponding binary
complex give root-mean-square differences of less than 0.12 Å showing
that no significant conformational changes have been induced upon
D-G3P binding. The analysis of the
(Fo Fc) maps of the
two ternary complexes clearly revealed a peak of electronic density
corresponding to the substrate D-G3P (Fig.
4).
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The substrate adopts the same conformation in both structures and shares almost the same interactions with the enzyme (Table II). It is bound in a non-covalent way, its C-3 phosphate being positioned within the Ps site (Fig. 5), whereas the Pi site is devoid of density. The oxygen atoms of the C-3 phosphate group form hydrogen bonds with the conserved side chains of the residues Arg231 and Thr179 of the Ps site and with the 2'-hydroxyl group of the ribose adjacent to the nicotinamide of NAD (Table II, Fig. 5). In the holostructure of the wild-type enzyme (7), the nonconserved Arg195 residue of B. stearothermophilus GAPDH was also shown to interact with the sulfate anion at Ps site via a water molecule. This water molecule is present in the ternary complex I (WAT-440, see Table II) but is not found in the ternary complex II, although Arg195 occupies the same position as in the wild-type structure and in ternary complex I. Nevertheless, in both ternary complexes, the direct interactions shared between the phosphate group of the substrate and the protein are exactly the same as those observed for the sulfate ion occupying the Ps site in the holostructure of the wild-type enzyme (7).
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Whereas D,L-G3P was used for the soaking experiments, the C-2 carbon adopts an R configuration, which accounts for the selective binding of the D-G3P, as expected because D-G3P is a better substrate than L-G3P (29). The C-2 hydroxyl group of D-G3P points toward the NH main chain of residue 149, with a distance of 3.6 Å between D-G3P-O-2 and Ser (or Ala)149 NH (Table II). The C-2 hydroxyl group also interacts with a water molecule (WAT-621) in the four monomers, which is stabilized through hydrogen bonding to the 2'O of the ribose adjacent to the nicotinamide ring (Fig. 5).
Although the equilibrium between the aldehyde and the gem-diol forms is
strongly shifted in favor of the hydrated form in solution, analysis of
the electron density allows us to conclude unambiguously that the
substrate is bound under its reactive form (free aldehyde) within the
active site, even for the C149A mutant GAPDH. This result correlates
with the lack of activity of the B. stearothermophilus C149A
mutant GAPDH but is nevertheless quite surprising, because the C149A
mutant GAPDH from E. coli was shown to use the hydrated form
as a substrate (27). Therefore, one could have expected to observe the
gem-diol form bound into the active site in the case of the ternary
complex I even at the pH of 4.6 of the crystallization medium. Some
subtle differences in the active site between the E. coli
and B. stearothermophilus C149A mutant GAPDHs probably
account for this difference in behavior, which remains however
unexplained because the residues composing the active site and their
geometry are conserved in the two wild-type proteins (11). In the case
of the ternary complex II of C149S mutant GAPDH, the lack of a covalent
bond between D-G3P and Ser149 is
consistent with the kinetic properties of the mutant protein. Indeed,
the rate of the acylation step, of which the nucleophilic attack of
Ser149 was shown to be rate-limiting, is decreased
105-fold compared with that of the
kobs of the wild type enzyme (18). In the
crystallization medium, which contains PEG 4000, the acylation rate is
probably decreased even more than in solution, which likely explains
why, under the experimental x-ray conditions that include a
flash-freezing after 10 min of soaking, the non-covalent ternary complex remains chemically stable within the crystal.
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DISCUSSION |
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With the aim of better understanding the catalytic mechanism of GAPDH and of evaluating the hypothetical models deduced from kinetic and structural studies, we solved the crystal structure of the B. stearothermophilus GAPDH in complex with its physiological substrate, D-G3P. For this purpose, the active site nucleophilic residue, Cys149, was substituted by alanine or serine. The mutant GAPDHs used herein are either inactive (Ala149) or possess a low residual activity (Ser149). New crystallization conditions have been set up in the absence of any anions in order to avoid competitive binding with the C-3 phosphate group of the substrate. The ternary complex structures have been solved and constitute the first example of a ternary complex, GAPDH·NAD·D-G3P. The substrate presents the same conformation in both structures. It is bound in a non-covalent way, under its aldehydic form, with its C-3 phosphate located in the Ps site. The binding of the C-3 phosphate group in the Ps site rather than in the Pi site is fully consistent with the observation that in the binary complexes, the Ps site was occupied with a sulfate ion coming from the purification step, whereas the Pi site was empty. This suggests a higher affinity of Ps site versus Pi site for phosphate and has been observed already in the structure of B. stearothermophilus wild-type enzyme. In this structure, the Ps site was fully occupied with sulfate coming from the crystallization medium, whereas the occupancy of the Pi site was only partial. These observations also support the hypothesis shared by Chakrabarti (30) and Copley and Barton (31) that the presence of an arginine residue within an anion binding site enhances its affinity for anions. In this regard, examination of the residues lining the Ps and Pi sites shows that Arg231 is involved in the formation of the Ps site, although no arginine residue contributes directly to the Pi site. Electrostatic potential calculations, carried out on the monomer structure, confirmed that the local positive electrostatic potential is higher for Ps than for Pi sites (results not shown) and could explain the affinity differences between these two anion recognition sites.
Whereas a racemic mixture of G3P was used for the soaking experiments, only the D-enantiomer is bound. The molecular factors explaining this stereoselectivity of GAPDHs have been the subject of controversy in the literature. Modeling the hemithioacetal intermediate within the active site of the lobster enzyme led Moras et al. (4) to propose that the preference for the D-isomer could be explained by an interaction of the C-2 hydroxyl group of the substrate with the side chain of Ser148. This hypothesis had been then refuted by Skarzynski et al. (7) who observed in the refined holostructure of B. stearothermophilus enzyme the presence of a hydrogen bond between the OH group of Ser148 and the main chain NH group of Thr151, thus excluding the involvement of Ser148 in the stereoselectivity. More recently, Duée et al. (11) modeled the hemithioacetal intermediate in the crystal structure of E. coli GAPDH and proposed that an interaction between the O-2 atom (Fig. 4) and the NH group of Cys149 would explain this stereoselectivity. In our complexes, the side chain of Ser148 forms a hydrogen bond with the amide group of Thr151, excluding any contribution of Ser148 to the stereoselectivity of substrate. The distance between the C-2 hydroxyl group of D-G3P and the NH group of residue 149 is consistent with the interpretation of Duée et al. (11). Furthermore, the interaction between the C-2-OH and a water molecule, stabilized through hydrogen bonding to the 2'O of the ribose adjacent to the nicotinamide ring (Fig. 5), might also contribute to the stabilization of the C-2 hydroxyl group.
In both ternary structures, the relative position of residue 149 with
respect to the C-1 atom of the substrate suggests that, assuming a
similar positioning of Cys149, the nucleophilic attack of
Cys149 in the wild-type will occur on the Re face of
the D-G3P. The O-1 oxygen of
D-G3P is orientated for both mutant GAPDHs in
such a way that it can accept a hydrogen bond from the
N2 atom of the catalytic His176
residue and also from the side chain of Ser149 for the
ternary complex II (Table II). This is consistent with the role
postulated for His176 in the stabilization of the substrate
(3) (Fig. 1). Altogether, our results suggest that the structures of
the ternary complexes presented here are representative of the
productive enzyme·NAD· substrate complex in the ground state
(Michaelis complex). Indeed, the use of a C149S mutant GAPDH displaying
a Km value for D-G3P similar
to that of the wild-type and the ideal orientation of the carbonyl O-1
oxygen with respect to the essential His176 residue let us
suppose that the substrate is bound in a catalytically competent
manner, analogous to the one it adopts in the Michaelis complex in the
case of the wild-type enzyme.
These results, suggesting that the Ps site constitutes the binding site for the C-3 phosphate group of the substrate, are however a bit surprising because former structural studies carried out with the aim of obtaining a structure that mimics the hemithioacetal intermediate pointed to the involvement of the Pi site. Indeed, the structure of B. stearothermophilus holoenzyme in complex with a substrate analogue, glycidol 3-phosphate (7), as well as the structure of E. coli apoGAPDH in complex with D-G3P (17) both revealed that the C-3 phosphate group was located in the Pi site. From this point of view, the structures of the ternary complexes presented here are in apparent contradiction with the structural studies and the biochemical results mentioned in the Introduction. Several reasons, which are not mutually exclusive, can be put forward to explain this apparent discrepancy.
First, the structure of the E. coli enzyme in complex with D-G3P was carried out on the apoform (without NAD). It is, however, well established that NAD contributes to the formation of the Ps site (7), and therefore the lower affinity of a nonfunctional Ps site might explain why the C-3 phosphate was found in the Pi site in this structure. Furthermore, for this latter structure, Yun et al. (17) noticed that the C-1 hydroxyl group of D-G3P and the nicotinamide base would be close to each other and proposed that the positive charge of the nicotinamidium contributes to the stabilization of the tetrahedral transition intermediate. It was also observed that His176 was involved in the binding of the phosphate group of D-G3P and thus would be unable to play a role in stabilizing the tetrahedral intermediate and as a base catalyst for facilitating hydride transfer, which does not correlate with the conclusions drawn from former site-directed mutagenesis experiments (3).
Second, when compared now with the B. stearothermophilus
GAPDH·NAD·glycidol 3-phosphate structure, the use of the true
substrate of the enzyme in our ternary complexes shows that an H-bond
is formed between the C-1 carbonyl and
His176-N2, which could explain
the different positioning of glycidol 3-phosphate. Indeed, the lack of
oxygen atom at the C-1 position in the latter molecule prevents the
formation of such an H-bond.
A third difference concerns the absence of a covalent link between the C-1 carbonyl atom and the catalytic residue Cys149 in our complex when compared with the two structures mentioned above.
In previous kinetic studies (15, 16), changing Ps site residues was shown to strongly decrease the kcat, whereas changing Pi site residues led to moderate kcat effects. In all mutant GAPDHs, the limiting step was shown to take place after the hydride transfer, and the Km values of D-G3P were not drastically modified. The fact that changing Pi site residues decreased the first-order overall oxidoreduction rate, whereas changing Ps site residues did not, was interpreted as evidence in favor of an initial binding of the C-3 phosphate at the Pi site. Although it seems probable from the current structures that the Ps site in the wild-type enzyme constitutes the binding site for the C-3 phosphate group of the substrate in the non-covalent complex, GAPDH·NAD·D-G3P, our results do not rule out the possibility that the formation of the covalent bond between Cys149 and D-G3P promotes the repositioning of the C-3 phosphate in the Pi site before the hydride transfer. Modeling the hemithioacetal intermediate in the B. stearothermophilus wild-type enzyme (not shown) with the C-3 phosphate group positioned either in the Ps or in the Pi site leads to models equivalent to those already reported by Duée et al. (11) or Kim et al. (9). It is furthermore obvious from these modeling experiments that changing the position of the C-3 phosphate group from Ps site to Pi site requires only a rotation around the C-1-C-2 bond. This rotation can be achieved without significant changes in the position of the C-1 atom and, whatever the location of the C-3 phosphate group in either the Ps or Pi site, leads to a complex in which the relative position of His176 with respect to the C-1 hydroxyl group is consistent with the role of base catalyst proposed for His176. Therefore, it is not possible to use these models to discriminate between the two alternative positioning for the C-3 phosphate group at the hemithioacetal intermediate level, except that binding in the Ps site seems to be more favorable from an energetic point of view, as already discussed by Kim et al. (9).
In conclusion, the structures of the ternary complexes
presented here are probably representative of that of the wild-type enzyme·NAD·D-G3P ternary complex in the ground state.
But it remains to be known whether the substrate will keep the same
position, in particular with regard to its C-3 phosphate group, when
the covalent bond within the ternary complex is formed between
Cys149 and D-G3P. The fact that the
thioacylenzyme intermediate can accumulate in the absence of phosphate
could allow the determination of its crystal structure and thus the
localization of the C-3 phosphate within one of the different covalent
complexes formed along the catalytic mechanism.
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr. S. Azza for the production and purification of enzymes and to Drs. A. Van Dorsselaer and S. Sanglier-Cianferani for mass spectrometry analyses. We thank the Service Commun de Diffraction X sur Monocristaux (Université Henri Poincaré-Nancy 1) for providing access to crystallographic experimental facilities.
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FOOTNOTES |
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* This work was supported by the Centre National de la Recherche Scientifique, the French Ministère de la Recherche et de l'Enseignement Supérieur, and local funds from the Région Lorraine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1NPT, 1NQ5, 1NQA, and 1NQO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence may be addressed. Tel.: 33-3-83-68-43-04; Fax: 33-3-83-68-43-07; E-mail: guy.branlant@maem.uhp-nancy.fr.
To whom correspondence may be addressed. Tel.:
33-3-83-68-48-69; Fax: 33-3-83-40-64-92; E-mail:
aubry@lcm3b.uhp-nancy.fr.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M211040200
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ABBREVIATIONS |
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The abbreviations used are: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G3P, glyceraldehyde 3-phosphate; 1, 3-dPG, 1,3-diphosphoglycerate; PEG, polyethylene glycol.
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