Product-assisted catalysis as the basis of the reaction specificity of threonine synthase

Article abstract of DOI:10.1074/jbc.M110.186205

Threonine synthase (TS), which is a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the γ-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at Cβ to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k_cat and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, α-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the Cβ of the PLP-α-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the Cβ of the analog. These results indicated that the phosphate ion released from OPHS by γ-elimination acts as the base catalyst for the addition of water at Cβ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/ deprotonation at Cγ.

Full text of DOI:10.1074/jbc.M110.186205

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 4, pp. 2774–2784, January 28, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Product-assisted Catalysis as the Basis of the Reaction
□
S
Specificity of Threonine Synthase*
Received for publication, September 17, 2010, and in revised form, October 27, 2010 Published, JBC Papers in Press, November 17, 2010, DOI 10.1074/jbc.M110.186205
Takeshi Murakawa, Yasuhiro Machida, and Hideyuki Hayashi1
From the Department of Biochemistry, Faculty of Medicine, Osaka Medical College, Takatsuki 569-8686, Japan
Threonine synthase (TS), which is a pyridoxal 5ꢀ-phosphate
(PLP)-dependent enzyme, catalyzes the elimination of the
␥-phosphate group from O-phospho-L-homoserine (OPHS)
and the subsequent addition of water at C␤ to form L-threo-
nine. The catalytic course of TS is the most complex among
the PLP enzymes, and it is an intriguing problem how the ele-
mentary steps are controlled in TS to carry out selective reac-
tions. When L-vinylglycine was added to Thermus thermophi-
lus HB8 TS in the presence of phosphate, L-threonine was
formed with kcat and reaction specificity comparable with
those when OPHS was used as the substrate. However, in the
absence of phosphate or when sulfate was used in place of
phosphate, only the side reaction product, ␣-ketobutyrate, was
formed. Global analysis of the spectral changes in the reaction
of TS with L-threonine showed that compared with the more
acidic sulfate ion, the phosphate ion decreased the energy lev-
els of the transition states of the addition of water at the C␤ of
the PLP-␣-aminocrotonate aldimine (AC) and the transaldi-
mination to form L-threonine. The x-ray crystallographic anal-
ysis of TS complexed with an analog for AC gave a distinct
electron density assigned to the phosphate ion derived from
the solvent near the C␤ of the analog. These results indicated
that the phosphate ion released from OPHS by ␥-elimination
acts as the base catalyst for the addition of water at C␤ of AC,
thereby providing the basis of the reaction specificity. The
phosphate ion is also considered to accelerate the protonation/
deprotonation at C␥.
hydratase, tryptophan synthase, and cysteine synthase, consti-
tutes the ␤-family of PLP enzymes (2, 3). Structurally, these
enzymes form fold type II PLP enzymes (4). Because TS is
found only in bacteria (5), yeasts (6), and plants (4, 7), it can
be a target for developing antibacterial drugs (5). For this pur-
pose, elucidation of the mechanism of action of TS is crucial.
The reaction mechanism of TS is considered to be the most
complicated one catalyzed by the PLP enzymes, proceeding
through all the types of intermediates formed during the ca-
talysis of the PLP enzymes (summarized in Scheme 1 (1, 3, 8,
9)). In this mechanism, OPHS reacts with the PLP-Lys aldi-
mine (internal aldimine 1) to form the external aldimine (2)
and liberates the side chain of the Lys residue (transaldimina-
tion). The intermediate 2 is then converted to the ketimine
(3) via a 1,3-prototropic shift. The electron-withdrawing im-
ino group promotes deprotonation at C␤, and after the for-
mation of the enamine (4), the ␥-phosphate group is elimi-
nated, yielding ␤,␥-unsaturated ketimine (5) and a phosphate
ion. In 5, deprotonation at C4Ј of the cofactor and protona-
tion at C␥ of the substrate moiety occur to form the PLP-␣-
aminocrotonate aldimine, 6). The stereospecific addition of a
water molecule from the Re face (C␤ being taken as the
prochiral center) of 6 to the C␣-C␤ double bond yields the
PLP-L-threonine aldimine (8) via the quinonoid intermediate
(7). Finally, attack of the ⑀-amino group of Lys on 8 releases
the product L-threonine and regenerates the internal aldimine
(1).
The remarkable feature of this catalytic reaction is that TS
can carry out many regiospecific (C4Ј, C␣, C␤, and C␥) and
stereospecific proton transfers at the coenzyme-substrate
complexes. Therefore, it is an intriguing problem how such
specific proton transfers are enabled to optimize the reaction
specificity of TS.
Threonine synthase (TS)2 catalyzes the last step of the L-
threonine biosynthesis, conversion of O-phospho-L-homo-
serine (OPHS) into L-threonine and inorganic phosphate (1).
TS is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme and,
together with the L- and D-serine dehydratases, threonine de-
To provide a structural basis for resolving this issue, we
previously determined the x-ray crystallographic structures of
a thermostable TS from Thermus thermophilus HB8 in the
unliganded form and a complex form with the substrate ana-
log, 2-amino-5-phosphonopentanoic acid (AP5) (8). The
structure of the complex with AP5 showed an sp2 hybridiza-
tion configuration at C␣ and C␤ and an sp3 hybridization
configuration at C4Ј, indicating that it is an enamine analo-
gous to 4. In this structure, the ⑀-amino N of Lys-61 is 3.5, 3.3,
and 3.6 Å away from C4Ј, C␣, and C␤, respectively. Consider-
ing the fact that the PLP binding lysine residue is the general
base catalyst for the 1,3-prototropic shift in aminotransferases
(10), we can expect that the ⑀-amino group of Lys-61 is the
general base catalyst for the protonation/deprotonation at
these atoms, which is involved in steps 2–3, 3–4, and 7–8.
* This work was supported in part by Grants-in-aid for Scientific Research
21570120 (to H. H.) and 91445990 (to T. M.) from the Japan Society for
the Promotion of Science.
□S
The on-line version of this article (available at http://www.jbc.org) con-
tains supplemental Figs. S2–S3.
The atomic coordinates and structure factors (codes 3AEY and 3AEX) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural Bioin-
formatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
1 To whom correspondence should be addressed: Dept. of Biochemistry,
Faculty of Medicine, Osaka Medical College, 2-7 Daigakumachi, Takatsuki
569-8686, Japan. Fax: 81-72-684-6516; E-mail: hayashi@art.osaka-med.
ac.jp.
2 The abbreviations used are: TS, threonine synthase; OPHS, O-phospho-L-
homoserine; AP5, 2-amino-5-phosphonopentanoic acid; 10, (E)-4-(3-hy-
droxy-2-methyl-5-(phosphonooxymethyl)pyridin-4-yl)-2-oxobut-3-enoic
acid.
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Product-assisted Catalysis of Threonine Synthase
In contrast to these steps, however, the catalytic steps in-
volving protonation/deprotonation at C␥ are difficult to un-
derstand. The ⑀-amino N of Lys-61 is too far (4.7 Å) from C␥
to directly catalyze the protonation/deprotonation at C␥, and
no appropriate protein side chains that can donate/accept a
proton are found in the proximity of C␥. Based on the crystal
structure of the T. thermophilus HB8 TS complexed with
AP5, we pointed out the possibility that the phosphate re-
leased during 4 3 5 may reside at the active site and act as a
base catalyst for the protonation/deprotonation at C␥ (8).
However, the involvement of the phosphate ion in the proton
transfer steps and the reaction specificity of TS have not been
experimentally studied.
of reagent (1 g of semicarbazide HCl plus 0.9 g of sodium ace-
tate in 100 ml of H2O) was added to terminate the reaction.
After incubation for 15 min at 298 K, 3 ml of H2O was added,
and the absorbance due to the semicarbazone was read at 254
nm. Steady-state kinetic parameters were determined by
systematically changing the concentration of L-threonine,
phosphate, or sulfate. The kinetic data were fitted to the
Michaelis-Menten equation by nonlinear regression using
KaleidaGraph (Version 3.0, Synergy Software).
Spectrophotometric Measurement—The absorption spectra
were measured at 298 K using a Hitachi (Japan) U-3300 spec-
trophotometer. Stopped-flow spectrophotometry was per-
formed using an Applied Photophysics SX.18MV spectropho-
In this study we performed kinetic and spectroscopic analy- tometer. Typically, equal volumes (75 ␮l each) of solution A
ses of the reactions of the T. thermophilus HB8 TS with
OPHS, L-vinylglycine, L-threonine in the presence or absence
of phosphate or sulfate and showed that the phosphate ion
functions as the catalyst for the addition of water at the C␤ of
the PLP-␣-aminocrotonate aldimine, thus enabling TS to un-
dergo the ␤-synthase reaction rather than the ␥-lyase reac-
tion. Additionally, the phosphate was also suggested as the
catalyst for the protonation/deprotonation at C␥.
(28 ␮M enzyme and 100 mM phosphate or sulfate) and solu-
tion B (various concentrations of L-threonine) were mixed.
The mixing dead time was 2.3 ms under an N2 gas pressure of
500 kPa. The time-resolved spectra were collected at 298 K
using SX.18MV equipped with a photodiode array accessory
and XScan (Version 1.0, Applied Photophysics). The spectral
data were analyzed by ProKineticist II (Version 1.9, Applied
Photophysics) to obtain the spectra of the reaction intermedi-
ates and the kinetic parameters. The buffer solution for the
spectrophotometric measurements contained 50 mM PIPES,
100 mM KCl, 0.1 mM EDTA, pH 8.0. TS was equilibrated with
this buffer by gel filtration using a PD-10 column before the
measurements.
EXPERIMENTAL PROCEDURES
Materials—The recombinant TS was isolated and purified
as previously described (8). All other chemicals were of the
highest grade commercially available. (E)-4-(3-hydroxy-2-
methyl-5-(phosphonooxymethyl)pyridin-4-yl)-2-oxobut-3-
enoic acid (10) was synthesized according to the method of
Schnackerz et al. (11). Removal of the cofactor from TS was
Crystallization, Data Collection, and Refinement—The
apoenzyme of TS was crystallized by the microdialysis
method. The enzyme solution (8 mg/ml) was placed in a
performed by precipitation with ammonium sulfate at low pH 50-␮l dialysis button and dialyzed at 293 K against 1.45 M am-
according to the method of Toney and Kirsch (10) except that monium sulfate in 0.1 M MES, pH 6.5. Single crystals with the
a 100-fold molar excess of hydroxylamine was added to the
enzyme to remove PLP from the Lys residue before the addi-
tion of ammonium sulfate. The apoenzyme preparation was
passed through a PD-10 (GE Healthcare) column twice to
remove ammonium sulfate. The concentration of sulfate in
approximate dimensions of 0.1 ϫ 0.1 ϫ 0.1 mm grew in about
2 weeks, and then the dialysis buttons were transferred to a
new reservoir solution supplemented with 40% (v/v) glycerol
as a cryoprotectant and kept at 293 K for 5 h. The complex of
TS with 10 was crystallized by the hanging drop vapor diffu-
the final preparation, measured after heat denaturation (3 min sion method. The drop was composed of 5 ␮l of protein solu-
in boiling water) of the protein, was below the detection limit
(1 ␮M) of the photometric method using bromthymol blue
(12).
tion (8 mg/ml apoenzyme in 5 mM Tris-HCl, pH 6.2), 5 ␮l of
reservoir solution (10% (v/v) PEG3350 in 0.1 M Na2HPO4 and
KH2PO4, pH 6.2), and 1 ␮l of 10 mM 10. Single crystals with
the approximate dimensions of 0.2 ϫ 0.1 ϫ 0.1 mm were ob-
tained by equilibration of the mixture solution against the
reservoir solution at 293 K for about 1 week. The crystal was
Enzymatic Formation of L-Threonine—TS was incubated
with various concentrations of OPHS in a final volume of 100
␮l of 50 mM PIPES, 100 mM KCl, 0.1 mM EDTA, pH 8.0. After
incubation at 298 K for various periods, 100 ␮l of 100 mM per- soaked for 10 s in a new reservoir solution supplemented with
chloric acid was added to terminate the reaction. A 100-␮l
aliquot of 100 mM phenyl isothiocyanate and 100 ␮l of 1 M
30% (v/v) glycerol as a cryoprotectant. The crystals were
mounted on thin nylon loops (ø, 0.2–0.3 mm) and frozen by
triethylamine were then added. After incubation for 1 h at 298 flash-cooling to 100 K in a cold N2 gas stream. The x-ray dif-
K, 400 ␮l of hexane was added and mixed, and the lower layer fraction data were collected at 100 K by synchrotron X-radia-
was loaded onto a Wakosil PTC column (Wako, Japan) using
a Beckman System Gold analyzer. The phenylthiocarbamyl
L-threonine was detected by monitoring the absorbance at
254 nm.
tion (␭ ϭ 0.9 Å) using a DIP6040 detector (Bruker AXS, Mad-
ison, WI) in the station BL44XU at SPring-8 (Hyogo, Japan).
The diffraction data collected for these crystals were pro-
cessed and scaled using HKL2000 (14). Both crystals belong to
Enzymatic Formation of ␣-Ketobutyrate—The method used the space group P3221. The details and statistics of the data
was a modification of that described by Mo¨ckel et al. (13).
Briefly, TS was incubated with each concentration of sub-
strate in a final volume of 1 ml of 50 mM PIPES, 100 mM KCl,
0.1 mM EDTA, pH 8.0. After incubation at 298 K for 1 h, 1 ml
collection are summarized in Table 4. The programs used for
the refinements, calculation of the electron-density maps, and
assignment of the solvent molecules were CCP4 program
suite Version 6.12 (15) and CNS Version 1.2 (16). Manual re-
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Product-assisted Catalysis of Threonine Synthase
SCHEME 1. The reaction mechanism of TS. There is an ambiguity as to the protonation state of the pyridine N1 of the PLP moiety. In this scheme, all the
structures were assumed to be protonated at N1. The intermediate 7 is generally called the quinonoid intermediate for its other resonance structure.
building was performed using the Xfit module of the Xtal-
View software package (17). The structure of TS in the ho-
loenzyme form (PDB code 1UIN, Ref. 8) and the structure of
TS complexed with AP5 (PDB code 1V7C, Ref. 8) were used
as the initial model for the apoenzyme of TS and the TS com-
plexed with 10, respectively. After being subjected to the rig-
id-body refinement, the initial structures of the apoenzyme
and the complex with 10 were further refined through simu-
lated annealing from 3000 K and several cycles of B-factor and
positional refinements. The model of 10 was built using MOE
Version 2007.09 (Chemical Computing Group), and then the
Fc, Fo Ϫ Fc, and omit maps. These models were refined
through several additional cycles of the B-factor and posi-
tional refinements after introducing solvent molecules. The
details and statistics of the crystallographic refinement are
also summarized in Table 4. The coordinates for the apoen-
zyme and TS complexed with 10 have been deposited in the
PDB with accession codes 3AEY and 3AEX, respectively.
RESULTS
Catalytic Reaction of TS with OPHS and L-Vinylglycine—
TS catalyzes the formation of L-threonine from OPHS as a
topology and parameter files for refinement in CNS were gen- physiological reaction (1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3
erated with XPLO2D in the X-UTIL package (18). Water mol- 1 in Scheme 1). In addition, TS catalyzes the formation of
ecules, phosphate ion derived from the reservoir solution, and ␣-ketobutyrate from OPHS as a side reaction (1 3 2 3 3 3
residues in the active-site region were examined using 2Fo Ϫ
4 3 5 3 6 3 1). The kcat and kcat/Km values for the L-threo-
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Product-assisted Catalysis of Threonine Synthase
TABLE 1
L-Threonine and ␣-ketobutyrate formation by TS from OPHS and L-vinylglycine
TS was incubated with various compounds, and the solutions were analyzed for L-threonine and ␣-ketobutyrate (see “Experimental Procedures”). Values in parentheses
are s.d.
L-Threonine
formation
Reaction
␣-Ketobutyrate formation
specificityb
Phosphate or
sulfatea
Substrate
OPHS
kcat
s
k
M
cat/Km
kcat
s
k
M
cat/Km
kcat
k
cat/Km
Ϫ1s
Ϫ1s
%
Ϫ1
Ϫ1
Ϫ1
Ϫ1
0.80
5200
(700)
—
0.0069
28
99
99
(0.03)
(0.0012)
(6)
c
L-Vinylglycine
LL--VViinnyyllggllyycciinnee
—
0.015 (0.0002)
0.013 (0.0006)
0.0034
0.37 (0.004)
0.13 (0.001)
0.037 (0.010)
ϳ0
98
ϳ0
98
Phosphate
Sulfate
0.61 (0.12)
—
5.9 (0.5)
—
ϳ0
ϳ0
(0.0023)
a Concentrations were 50 mM.
b Reaction specificity was calculated as (value for the L-threonine formation)/(sum of the values for the L-threonine formation and the ␣-ketobutyrate formation).
c Not detected.
nine formation were about 120- and 190-fold higher, respec-
tively, than those for the ␣-ketobutyrate formation, yielding a
high reaction specificity of 99% in both the kcat and kcat/Km
values for OPHS indicate the importance of the phosphate group
of OPHS for binding to TS. In contrast to the reaction of L-vinyl-
glycine with TS in the presence of phosphate, the reaction in the
terms (Table 1). That the ratio of the kcat values for the L-thre- presence of sulfate showed a pattern essentially similar to the
onine and the ␣-ketobutyrate formation is similar to the cor-
responding ratio of the kcat/Km values is considered to reflect
the fact that L-threonine and ␣-ketobutyrate are derived from
the same intermediate bound to the enzyme, i.e. the PLP-␣-
aminocrotonate aldimine (6).
reaction in the absence of both phosphate and sulfate, i.e. only
␣-ketobutyrate was formed without the formation of L-threonine
(Table 1).
The above results indicate that 6 derived from L-vinylgly-
cine is preferentially converted to L-threonine in the presence
of phosphate as efficiently as the corresponding reaction for
A chemical consideration predicts that besides the physio-
logical substrate OPHS, TS may also catalyze the formation of the normal catalysis starting from OPHS, and sulfate cannot
L-threonine from L-vinylglycine via 9 3 6 3 7 3 8. In this
context, the reaction of TS using L-vinylglycine was followed.
However, no formation of L-threonine was observed, whereas
the side reaction product ␣-ketobutyrate was formed with a
substitute for phosphate. To investigate in detail the effect of
phosphate ion on the catalytic steps after 6, we analyzed the
“reverse” reaction 1 3 8 3 7 3 6 3 1 starting from L-threo-
nine in the presence of phosphate or sulfate.
k
cat similar to that when OPHS was used as the substrate (Ta-
Reaction of TS with L-Threonine in the Presence of Phos-
phate or Sulfate Ion—The addition of L-threonine up to 50
mM to TS caused no spectral changes (supplemental Fig. S1, A
and B). The CD spectrum also showed no detectable changes
upon the addition of L-threonine to TS (data not shown).
ble 1). This indicates that the formation of 6 alone is not
enough for producing L-threonine.
During the reaction of TS with OPHS, a phosphate ion is re-
leased (4 3 5) before the formation of 6 (Scheme 1). To investi-
gate whether the released phosphate ion is involved in the cataly- That change in CD is a sensitive indicator of the formation of
sis, the reaction starting from L-vinylglycine was studied in the
presence of phosphate and its analog sulfate, which has a similar
shape and size as phosphate but has a much lower pKa value
(1.92 at 298 K). In the presence of 50 mM phosphate, L-threonine
was preferentially formed (Table 1). In both the kcat and kcat/Km
terms, the reaction specificity was 98%. This is the same pattern
the external aldimine (19) strongly suggests L-threonine alone
cannot form the external aldimine with PLP (8) in TS. On the
other hand, the addition of phosphate or sulfate to TS slightly
increased the 416-nm peak and concomitantly decreased the
absorption at around 340 nm (supplemental Fig. S1, C and D).
This indicates that the phosphate or sulfate ion binds to the
as that observed when OPHS was used as the substrate and again active site of TS, causing the PLP-Lys-61 aldimine to undergo
reflects the mechanism that L-threonine and ␣-ketobutyrate are
derived from the same intermediate (6) bound to the enzyme.
a tautomeric shift from enolimine to ketoenamine. From the
absorption change at 416 nm, the Kd for phosphate and sul-
The kcat values for the L-threonine and ␣-ketobutyrate formation fate ions were estimated to be 2.2 and 4.9 mM, respectively.
were similar to those when OPHS was used as the substrate (Ta-
ble 1). On the other hand, the kcat/Km values for the L-threonine
and ␣-ketobutyrate formation were several hundred-fold lower
than the corresponding values using OPHS as the substrate (Ta-
In the presence of both L-threonine and either of phosphate
or sulfate, distinct spectral changes in the TS (supplemental
Fig. S1) and ␣-ketobutyrate formation (Table 2) were ob-
served. However, neither OPHS nor O-sulfohomoserine was
ble 1). This indicates that L-vinylglycine is far more weakly bound detected in the reaction mixture (data not shown). This is
to TS than OPHS. According to the previous crystallographic
study of TS complexed with AP5 (8), the phosphate group of
OPHS is considered to be fixed by Thr-88, Asn-154, Ser-155,
Arg-160, and Asn-188 at the active site of TS. The low kcat/Km
values for L-vinylglycine (in the presence of phosphate as well as
in the presence of sulfate or in the absence of both phosphate
and sulfate; see Table 1) as compared with the corresponding
interpreted to reflect the fact that the step of phosphate elimi-
nation (4 3 5) is essentially irreversible, and neither phos-
phate nor sulfate can undergo addition to the C␥ of 5 due to
their low Lewis basicities. The kinetic parameters for the
␣-ketobutyrate formation are summarized in Table 2. The Kd
values for phosphate and sulfate kinetically obtained were
essentially consistent with the values obtained by the spectro-
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Product-assisted Catalysis of Threonine Synthase
scopic measurements described above. The similarity be-
tween the Kd values for phosphate and sulfate suggests the
similarity of the recognition of the two anions by TS. On the
other hand, the kcat and kcat/Km values in the presence of sul-
fate were about 7- and 2.4-fold higher, respectively, than
those in the presence of phosphate.
phate was reacted with L-threonine (Fig. 1A). The absorption
at around 330 nm decreased, and that around 410 nm in-
creased within the dead time (2.3 ms), indicating a rapid asso-
ciation of TS and L-threonine. Subsequently, two sharp peaks
emerged at 474 and 446 nm, and a broad absorption band at
around 330 nm increased. The sharp peaks at 474 and 446 nm
are clearly ascribed to the quinonoid intermediate. The ab-
sorbance at 331, 416, and 474 nm was plotted versus the
elapsed time after mixing (Fig. 2A). At any wavelengths, the
absorbance changes followed a double exponential process,
with a kapp of ϳ340 and ϳ40 sϪ1 at 250 mM L-threonine.
These results indicated that the spectral changes in the reac-
tion of TS with L-threonine in the presence of phosphate can
be analyzed by a model that includes two steps after the rapid
association of the enzyme (A) and the substrate (B):
Stopped-flow Spectroscopy of the Reaction of TS with
L-Threonine—To investigate the reaction steps from L-threo-
nine to ␣-aminobutyrate in detail, transient kinetic analyses
were carried out. A solution containing TS and 50 mM phos-
TABLE 2
Kinetic parameters for the ␣-ketobutyrate formation reaction from
L-threonine
TS was incubated with L-threonine in the presence of either phosphate or sulfate,
and the solutions were analyzed for ␣-ketobutyrate (see “Experimental
Procedures”).
Kd
-0 AB -
kϪ1
kϩ1
0 C -
kϪ2
kϩ2
|0 D
Coexistent anion Kd for phosphate/sulfate
kcat
s
k
cat/Km
A ϩ B
|
|
(Eq. 1)
Ϫ1
Ϫ1
mM
M
Ϫ1s
Phosphate
Sulfate
1.7 Ϯ 0.2
2.6 Ϯ 0.2
0.034 Ϯ 0.002 1.3 Ϯ 0.2
0.23 Ϯ 0.04 3.1 Ϯ 0.3
Ϫ1
As the rate of ␣-ketobutyrate formation (kcat ϭ 0.034 s
;
FIGURE 1. Time-resolved spectra of TS upon reaction with L-threonine in the presence of phosphate (A) and sulfate (B). TS (14 ␮M) in the presence of
50 mM phosphate or sulfate was reacted with 250 mM L-threonine at 298 K in 50 mM PIPES, 100 mM KCl, and 0.1 mM EDTA, pH 8.0, and the spectra were
taken at 2.30 (dead time), 3.84 ms, and every 2.56 ms up to 149.76 ms (phosphate) or 4097.28 ms (sulfate). In panel A all the spectra are shown. In panel B the
spectrum at 2.30 ms and those every 12.8 ms between 16.64 and 106.24 ms are shown as solid lines, and those every 128 ms between 106.24 and 3946.24
ms are shown as dotted lines. In both panels, the spectra with a larger absorption at around 470 nm indicate the spectra at the later times.
FIGURE 2. Time course of the absorbance changes in TS upon reaction with 250 mM L-threonine in the presence of phosphate (A) and sulfate (B).
The wavelength values in nm are indicated in the figure. The experimental values are shown by dots, and the theoretical double exponential curves fitted to
the experimental values are shown by solid lines.
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Product-assisted Catalysis of Threonine Synthase
FIGURE 3. Absorption spectra of the intermediate species obtained from the global analysis of the reaction of TS with L-threonine in the presence
of phosphate (A) and sulfate (B). The intermediate spectra were obtained from the global fitting analysis of the time-resolved spectra to Equation 1 (in the
presence of phosphate) and Equation 2 (in the presence of sulfate). The spectra were assigned to the intermediate species as follows: A: A, unliganded en-
zyme; AB (dotted line), Michaelis complex ϩ external aldimine; C, quinonoid intermediate; D, PLP-␣-aminocrotonate aldimine. B: A, unliganded enzyme; AB
(dotted line), Michaelis complex; C, external aldimine ϩ quinonoid intermediate; D, PLP–␣-aminocrotonate aldimine. See “Results” for details.
TABLE 3
Global Analysis of the Reaction of TS with L-Threonine—
Transient kinetic parameters for the reaction starting from
The time-resolved spectra of TS taken after the reaction with
L-threonine to form ␣-ketobutyrate
various concentrations (10, 20, 50, 80, 120, 160, 200, and 250
TS was reacted with various concentrations of L-threonine in the presence of
mM) of L-threonine were subjected to a global fitting analysis
either phosphate or sulfate, and the spectral changes were monitored by a
stopped-flow spectrophotometer. The time-resolved spectra were subjected to
using the models of Equation 1 (reactions in the presence of
global analysis based on the models of Equation 1 (phosphate) and Equation 2
phosphate) and 2 (reactions in the presence of sulfate). For
(sulfate), and the kinetic parameters were obtained. See “Results” for details.
Reaction
(E ؉ phosphate) ؉
(E ؉ sulfate) ؉
the initial values of the kinetic parameters, the apparent rate
constants obtained from the double exponential fitting (Fig. 2)
were divided by 2, assuming the equilibrium constant of 1 for
[AB]/[C] and [C]/[D], and were used for kϩ1, kϪ1, kϩ2, and
system
L-threonine
L-threonine
K
d (mM)
380 Ϯ 2
92 Ϯ 0.4
34 Ϯ 1
270 Ϯ 4
Ϫ1
k
k
k
k
k
ϩ1 (s
Ϫ1 (s
ϩ2 (s
Ϫ2 (s
ϩ3 (s
)
)
)
)
)
19 Ϯ 0.5
Ϫ1
Ϫ1
Ϫ1
Ϫ1
16 Ϯ 0.1
210 Ϯ 4
66 Ϯ 1
1.1 Ϯ 0.1
0.053 Ϯ 0.030
0.43 Ϯ 0.03
k
Ϫ2. For the initial values of Kd, the Km values for L-threonine
calculated from the kcat and kcat/Km values (Table 2) were
used. After the fitting, the spectra of TS and the intermedi-
ates, i.e. A, AB, C, and D, and the kinetic parameters were
obtained, each summarized in Fig. 3 and Table 3, respectively.
For both reactions in the presence of phosphate and sulfate,
the spectra of the intermediate D showed a large absorption
band at 450 nm and a smaller one at around 330 nm. These
spectra of D resemble those of the PLP-␣-aminoacrylate aldi-
mine reported for several enzymes in the absorption maxima,
strength, and shape (20–22). Considering the same conjugate
system between the PLP–␣-aminoacrylate aldimine and the
PLP-␣-aminocrotonate aldimine, we can conclude that D is
the PLP-␣-aminocrotonate aldimine (6), whose ketoenamine
and enolimine tautomers absorb at 450 and 330 nm,
Table 2) is much slower than the kapp values of the spectral
changes, the regeneration of A from D was not incorporated
in this model (Equation 1).
The reaction of TS with L-threonine in the presence of 50
mM sulfate was followed in the same way (Fig. 1B). In contrast
to the reaction in the presence of phosphate, no significant
accumulation of the quinonoid intermediate was observed.
The absorption at 416 nm first increased followed by a slow
red shift in ␭max and an appearance of a shoulder at around
480 nm. The time course of the absorption changes followed a
double exponential process (Fig. 2B) with kapp values of ϳ30
and ϳ0.85 sϪ1. The kapp values for the slower spectral
changes were of the same order as that of the ␣-ketobutyrate
formation (kcat ϭ 0.23 sϪ1; Table 2). In this regard, the accu-
mulation of the spectra of the intermediates was apparently
lower than that in the presence of phosphate (Fig. 1, A and B).
These results indicate that, contrary to the case in the pres-
ence of phosphate, the regeneration of the free enzyme can-
not be neglected. Therefore, the spectral changes in the reac-
tion of TS with L-threonine in the presence of sulfate should
be analyzed according to the model
respectively.
For the reaction in the presence of phosphate, the interme-
diate C showed the typical spectrum of the quinonoid inter-
mediate (Fig. 3A) and was attributed to 7. Accordingly, the
spectrum of the intermediate AB, which was similar to that of
A, was considered to be that of the Michaelis complex with
L-threonine (not shown in Scheme 1 but exists between 8 and
1), the external aldimine (8), or both. On the other hand, for
the reaction in the presence of sulfate ion, the spectra of the
intermediate C showed a large absorption band at 420 nm and
a faint absorption band at around 480 nm (Fig. 3B). The shape
and the position of the spectrum showed that the intermedi-
ate C is largely the PLP aldimine with no double bond in the
Kd
-0 AB -
kϪ1
kϩ1
0 C -
kϪ2
kϩ2
|0 D
kϩ3
¡ A
A ϩ B
|
|
O
(Eq. 2)
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Product-assisted Catalysis of Threonine Synthase
substrate moiety conjugated to the imine. As compared with
the absorption band of AB, which was again similar to A, the
absorption band of C was red-shifted (ϳ8 nm) and had a
larger intensity at 420 nm and a smaller one at 330 nm. This
indicates that AB and C are both PLP aldimines but are
clearly distinguished. The most probable interpretation is that
AB and C are the Michaelis complex with L-threonine and the
external aldimine (8), respectively. Spectral changes similar to
this are observed in the transaldimination process of a num-
ber of PLP enzymes (19). Assuming that the Michaelis com-
plex and the external aldimine in the reaction in the presence
of phosphate have similar absorption spectra as those in the
reaction in the presence of sulfate, the absorption spectrum of
AB in the presence of phosphate indicates that it is mainly
composed of the Michaelis complex.
FIGURE 4. Free energy profiles of the catalytic reaction of TS after the
PLP-␣-aminocrotonate aldimine in the presence of phosphate (solid
line) and sulfate (dashed line). The energy levels of the two profiles are
adjusted at the PLP-␣-aminocrotonate aldimine. The kinetic parameters
obtained from the reaction of TS with L-threonine in the presence of
phosphate or sulfate were used for calculating the free energy levels. The
equations used are: k ϭ (kBT/h)exp((Ϫ⌬G‡)/RT) for the transition states
The presence of a small absorption at around 480 nm in the
spectrum of C in the presence of sulfate suggests the presence
of a trace amount of the quinonoid intermediate (Fig. 3B).
Using the ⑀ value at 476 nm of C in the presence of phosphate
(Fig. 3A), the fraction of the quinonoid intermediate in C in
the presence of sulfate was estimated to be 6.7%. We could
not resolve C by global fitting based on the model of a three-
step process after the Michaelis complex. This indicates that
in the presence of sulfate the concentrations of the external
aldimine and the quinonoid intermediate change in parallel,
showing that the process of the interconversion between the
external aldimine (8) and the quinonoid intermediate (7) is
considerably fast when compared with the other processes.
Comparison of the Steady-state and Transient Kinetic Pa-
rameters for L-Threonine—The steady-state and the transient
kinetic parameters are related to each other by the following
equations:
Keq ϭ exp(Ϫ⌬G/RT) for the free energy difference between the two states in
equilibrium. The energy levels of E ϩ L-threonine are those when [L-threo-
nine] ϭ 1 mM, and those of KB are arbitrary. The curved portions of the lines
indicate that these energy levels are not quantized. For the free energy pro-
file in the presence of phosphate, the energy level of the transition state (‡)
between EA and MC is lower than that between Q and EA, and the energy
level of EA is significantly higher than that of MC. For the free energy profile
in the presence of sulfate, the energy level of ‡ between Q and EA is signifi-
cantly lower than that between EA and MC. Abbreviations are: KB, TS ϩ
NH3 ϩ ␣-ketobutyrate; AC, PLP-␣-aminocrotonate aldimine; Q, quinonoid
intermediate; EA, external aldimine of TS with L-threonine; MC, Michaelis
complex of TS with L-threonine.
Free-energy Profile of the Steps of the Latter Part of the TS
Catalytic Reaction—Using the microscopic kinetic parame-
ters obtained above, the free energy profile of the reaction
steps between L-threonine and ␣-ketobutyrate could be ob-
tained. The energy profiles in the presence of phosphate and
sulfate are shown in Fig. 4. The profiles were drawn according
to the direction of the normal catalytic steps shown in
Scheme 1. Uncertainties about the energy levels of several
intermediates and transition states arising from the inability
to dissect the reaction steps are also shown. The profiles indi-
cate that, in the presence of phosphate as compared with sul-
fate, the transition state between the PLP-␣-aminocrotonate
aldimine (6) and the quinonoid intermediate (7) and that be-
tween the external aldimine (8) with L-threonine and the
Michaelis complex with L-threonine are stabilized (Fig. 4). For
the transition state between the PLP–␣-aminocrotonate aldi-
mine (6) and the quinonoid intermediate (7), the stabilization
energy was calculated to be 17.7 kJꢁmolϪ1, corresponding to
the rate enhancement of about 103.
1
k
cat ϭ
1
kϪ1
1
k
Ϫ1 kϪ2
1
1
kϪ2
1
1
ϩ
ϩ
ϩ
ϩ
ϩ
kϩ1
k
ϩ1 kϩ2
k
ϩ1 kϩ2 kϩ3 kϩ2
kϩ2 kϩ3 kϩ3
(Eq. 3)
kcat
Km
1
ϭ
(Eq. 4)
1
kϪ1
1
k
Ϫ1 kϪ2
1
ϩ
ϩ
K
d
ͩ
ͪ
kϩ1
k
ϩ1 kϩ2
k
ϩ1 kϩ2 kϩ3
where the microscopic kinetic parameters are those shown in
Equations 1 and 2. For the reaction in the presence of phos-
phate, the kϩ3 value was the only undetermined value. This
was calculated to be 0.049 sϪ1 using Equation 3 and the ob-
served kcat value for the reaction with L-threonine in the pres-
ence of phosphate.
Crystal Structure of TS Complexed with a Structural Analog
for the PLP-␣-Aminocrotonate Aldimine—10 (Fig. 5B), which
is a condensation product of PLP and pyruvic acid, has been
used as an analog for the PLP-␣-aminoacrylate aldimine in
serine dehydratase (11). Because of the structural similarity of
the PLP-␣-aminoacrylate aldimine and the PLP-␣-aminocro-
tonate aldimine as described above, we expected that 10 can
also be used as an analog for the PLP-␣-aminocrotonate aldi-
mine (6) in TS. The apoenzyme of TS was reconstituted with
10 and crystallized in the presence of phosphate. Crystalliza-
tion of the relevant structures, such as TS, TS complexed with
Now that the microscopic kinetic parameters have been
obtained, we can estimate the steady-state kinetic parameters
k
cat and kcat/Km using Equations 3 and 4. The kcat/Km value
was 1.1 MϪ1sϪ1 in the presence of phosphate, and kcat ϭ 0.23
s
Ϫ1 and kcat/Km ϭ 4.0 MϪ1sϪ1 in the presence of sulfate. The
calculated values excellently matched the observed values
(Table 2), showing the validity of the kinetic analysis de-
scribed above.
2780 JOURNAL OF BIOLOGICAL CHEMISTRY
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Product-assisted Catalysis of Threonine Synthase
FIGURE 5. A, the overall structure of TS obtained by x-ray crystallography is shown. Left, overlay of TS complexed with 10 (gray) and the unliganded holoen-
zyme of TS (magenta; PDB code 1UIM, 8). Right, overlay of TS complexed with 10 (gray) and TS complexed with AP5 (green; PDB code 1V7C, 8). In both struc-
tures, only 10 and the bound phosphate ion are shown in the sphere models for clarity. B, the Fo Ϫ Fc omit map of TS complexed with 10, contoured at 5␴.
The chemical structure of 10 is shown with the atomic numbering.
TABLE 4
10, and the apoenzyme of TS either in the phosphate- or sul-
Statistics of data collection and crystallographic refinement
fate-bound form has been attempted. However, only the crys-
tals of the apoenzyme of TS complexed with sulfate (apoTS-S)
were obtained so far. The structures of TS-10-P and apoTS-S
were determined by x-ray crystallography at 2.10 and 1.92 Å
resolutions, respectively (Table 4). The overall structure of
TS-10-P is shown in Fig. 5A and compared with the struc-
tures of TS and TS complexed with AP5 (TS-AP5) (Fig. 5A).
The structure of apoTS-S was superimposable on that of TS,
showing that the enzyme does not undergo any global confor-
mational change upon the binding of PLP (supplemental Fig.
S2A). However, the binding of the substrate analog AP5 to the
holoenzyme induces a large conformational change from the
open to the closed form in which the small domain of TS
moves as a rigid body to close the active site (8). This closed
structure was maintained in TS-10-P (Fig. 5A), indicating that
TS is in the closed form in the enamine and the PLP-␣-ami-
nocrotonate aldimine intermediates. These data support the
catalytic model that TS is in the closed form, whereas the sub-
strate and the intermediates are bound to the enzyme.
apoTS-S
TS-10-P
Data collection
Temperature (K)
Wavelength (Å)
100
0.9
100
0.9
Space group
P3221
P3221
Unit cell dimensions, a, b, c (Å)
No. of observations
No. of unique reflections
Multiplicity
113.0, 113.0, 150.3 115.2, 115.2, 99.7
929,780
164,186
5.7 (5.3)
525,702
92,864
5.7 (5.2)
d
max Ϫ dmin (Å)
50-1.92 (1.95-1.92) 50-2.05 (2.09-2.05)
Overall completeness (%)
100 (100)
6.3 (50.6)
100 (100)
8.3 (72.1)
Overall Rmerge (%)a
Refinements statistics
d
max Ϫ dmin (Å)
48.9-1.92
26.6-2.10
89.2
Residues in the core ␸␺ region (%) 90.3
No. of solvent atom
Root mean square deviation from
ideal values
599
272
Bond lengths (Å)
0.005
1.2
0.006
1.44
21.6
25.3
5
Bond angles (degrees)
Residual R (%)b
22.7
26.0
5
Free residual R (%)c
Fraction of data in Rfree set (%)
a Rmerge ϭ ⌺h⌺i͉Ihi Ϫ ͗Ih͉͘/⌺h⌺iIhi, where Ihi is the intensity value of the ith mea-
surement of h, and ͗Ih͘ is the corresponding mean value of Ih for all i
measurements.
bR ϭ ⌺ʈFo͉ Ϫ ͉Fcʈ/⌺͉Fo͉.
c Free residual R is an R factor of the CNS refinement evaluated for 5% of reflec-
tions that were excluded from the refinement. Values in parentheses refer to the
highest resolution shell.
Phosphate Ion Bound to the Active Site—The Fo Ϫ Fc omit
map clearly showed an electron density of 10 in the active site
(Fig. 5B). The modeled structure of 10 was almost completely
planar, reflecting that all the C atoms of 10 except for C2Ј and to confirm that this is not a sulfate ion that has been used to
C5Ј are sp2-hybridized. In addition to this electron density, a
distinct electron density having a tetrahedral shape was ob-
served. A phosphate ion could be excellently fit to this den-
sity, indicating that a phosphate ion derived from the solvent
prepare the apoenzyme. The solution used for crystallization
contained less than 5 pmol of sulfate ion (1 ␮M ϫ 5 ␮l, see
“Experimental Procedures”). The number of the cells in a sin-
gle crystal is estimated to be 1.5 ϫ 1012 as calculated from the
is bound to the active site of TS-10-P (Fig. 5B). It is important size of the crystal and the unit cell dimensions. Because there
JANUARY 28, 2011•VOLUME 286•NUMBER 4
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Product-assisted Catalysis of Threonine Synthase
FIGURE 6. Overlay of the structures of TS complexed with 10 and phosphate ion (TS-10-P) and AP5 (TS-AP5). The carbon atoms of TS-10-P and TS-AP5
are shown in gray and green, respectively. The ␣-carbon atoms of the active site residues shown in this figure of subunit B of TS-10-P and subunit C of TS-
AP5 were fitted by root mean square minimization. For details of the hydrogen bond and other interactions, refer to “Results.”
were at least 10 crystals of a size similar to this, the “amount”
of the active sites in the crystals are calculated to be more
TS-10-P (supplemental Fig. S2B). A third sulfate ion forms
hydrogen bonds with the side chains of Thr-88, Arg-160, and
than 50 pmol. Therefore, even if all the sulfate ions remaining Asn-188 and a water molecule. These residues are involved in
in the solution are bound to the crystals, it can occupy less
than 10% of the active sites. From these calculations we con-
sider that the observed tetrahedral electron density comes
from the bound phosphate ion.
the binding of the phosphate ion in TS-10-P, and as the result,
the sulfate ion is located at a position close to that of the
phosphate ion in TS-10-P (supplemental Fig. S2B). The devia-
tion in the position of the sulfate ion is considered to be due
to the open conformation of the enzyme protein. Although
the crystal structure of TS complexed with 10 and the sulfate
The active site structure of TS-10-P was superimposed on
that of TS-AP5 (Fig. 6). The PLP moiety of TS-AP5 and that
of TS-10 reside in the same plane. However, in contrast to the ion has not been obtained, these data strongly suggest that the
planar conformation of 10, C4Ј and N␣ of TS-AP5 are sp3-
sulfate ion binds to the same position as the phosphate ion
hybridized. As a result, the C␣ of 10 of TS-10-P deviates from during the course of catalysis by TS.
the corresponding C␣ of TS-AP5 by 0.4 Å, with a clockwise
DISCUSSION
21° rotation (viewed from the Re face of AP5, i.e. the Si face of
10, C␣ being taken as the prochiral center) of the three bonds
around C␣ about the axis passing through C␣ and perpendic-
ular to the plane of 10. Consequently, the position of the car-
boxylate group is substantially different from that of AP5.
Nevertheless, the carboxylate group of 10 and that of AP5
Reaction Specificity of PLP Enzymes and TS—PLP catalyzes
versatile reactions of amino acids, such as racemization,
transamination, elimination, replacement, decarboxylation,
aldol cleavage, etc. This indicates that a PLP enzyme must be
organized to carry out a specific type of reaction. An impor-
make hydrogen bonds with the same neighboring groups (Fig. tant mechanism that accounts for the reaction specificity of
6), i.e. the main chain NH groups of Thr-85, Asn-87, and Thr- the PLP enzymes has been provided by the Dunathan hypoth-
88, the side chain OH group of Ser-84, and a water molecule.
The phosphate ion in TS-10-P is located at exactly the
same position as the phosphono group of AP5 in TS-AP5 and
is stabilized by interaction with the same groups, i.e. the side
chains of Lys-61, Thr-88, Asn-154, Ser-155, Arg-160, and
Asn-188 (Fig. 6). The only difference between the phosphate
ion and the phosphono group is the possible presence of a
esis (23), which suggests that the conformation of the external
aldimine at the active site determines the reaction type; if the
bond perpendicular to the plane of the imine-pyridine ring is
Ϫ
C-H, C-COO , or C-CHOH-, then ␣-deprotonation, decar-
boxylation, or aldol cleavage occurs, respectively. Although
this crucially determines the reaction during the early step of
the catalysis, this alone cannot explain all of the reaction spec-
hydrogen bond between the carboxylate group of 10 and an O ificity of the PLP enzymes. For example, there are many types
atom of phosphate, which is replaced by a C atom in AP5
(Fig. 6).
of reactions that start with ␣-deprotonation, such as racemi-
zation, transamination, ␤-elimination, ␤-replacement, ␥-elim-
ination, ␥-replacement, etc. The individual PLP enzymes cata-
lyzing only one of those reactions must control the proton
transfer processes to realize their reaction specificity.
The crystal structure of apoTS-S showed that the cavity
formed by the removal of PLP is filled with three water mole-
cules and two sulfate ions derived from the solvent (supple-
mental Fig. S2B). One of the sulfate ions is located at the same
TS is a unique PLP enzyme undergoing ␥-elimination and
position, albeit with an ϳ30° rotation, as the phosphate group ␤-addition reactions. Therefore, TS shares the reaction path-
of 10 in TS-10-P and that of PLP in the holoenzyme. Another
way 1–6 with ␥-lyases, and this distinguishes them from
sulfate ion interacts with the ⑀-amino group of Lys-61, a water ␥-synthases, which do not catalyze the step between 5 and 6,
molecule, and the main chain NH group of Asn-87, which is
the proton transfer between C4Ј and C␥. However, TS is dif-
ferent from the ␥-lyases in that it preferentially catalyzes the
the residue that interacts with the carboxylate group of 10 in
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Product-assisted Catalysis of Threonine Synthase
addition of water at the C␤ of 6 as compared with the
transaldimination of 6 to yield ␣-ketobutyrate (Table 1).
Therefore, it is important to know how TS controls these
steps to carry out the specific biosynthetic reaction.
SCHEME 2
Phosphate Ion Promotes the Reaction Steps from 6 to
L-Threonine—In this study it was shown that L-vinylglycine,
which has been known to be an alternate substrate for the
␥-synthase (24, 25) and 1-aminocycleopropane-1-carboxylate
synthase (26) reactions, can be incorporated into and pro-
cessed at the active site of TS (Table 1). In the presence of
phosphate ion, TS forms L-threonine from L-vinylglycine with
a kcat value and a reaction specificity comparable with those
when OPHS was used as the substrate (Table 1). However, in
the absence of phosphate ion or in the presence of sulfate ion,
only ␣-ketobutyrate was formed (Table 1). These results indi-
cated that the phosphate ion accelerates the reaction from 6
to the L-threonine formation, and this function is related to a
property of the phosphate ion, which is absent in the sulfate
ion.
group(s) are considered to be required to protonate/deproto-
nate the atom. Using the kinetic parameters obtained in this
study, we estimated the effect of phosphate on this step as
follows.
A simple mechanism for the reaction starting from L-vinyl-
glycine is Scheme 2, where E, S, and AC denote the enzyme,
L-vinylglycine, and the PLP-␣-aminocrotonate aldimine (6),
respectively. Using the above simple mechanism, the kcat val-
ues for L-threonine and ␣-ketobutyrate formation are ex-
pressed as,
kakb
kLc-atthreonine ϭ
ka ϩ kb ϩ kc
(Eq. 5)
kakc
Phosphate as a Base Catalyst for the Addition of Water at
the C␤ of 6—To clarify the function of the phosphate ion in
the L-threonine formation reaction in detail, the reverse reac-
tion starting from L-threonine was kinetically studied in the
presence of phosphate or sulfate. The result showed that the
phosphate ion lowers the energy barrier for the addition of
the water molecule at the C␤ of 6 and the transaldimination
to form L-threonine, as compared with the sulfate ion (Fig. 4).
Therefore, we structurally studied how the phosphate ion is
involved in the reactions starting from 6 by solving the crystal
structure of TS complexed with an analog for 6 (Fig. 5). The
phosphate ion was found to bind to the site originally occu-
pied by the phosphate group of OPHS and was supposed to
interact with the attacking water molecule in the step 6 3 7.
The energy barrier for this step in the absence of phosphate
or sulfate was not obtained because of the extremely low af-
finity of L-threonine in the absence of phosphate or sulfate.
However, the finding that the phosphate ion lowers the en-
ergy barrier more effectively than the sulfate ion, which has a
much lower basicity (pKa ϭ 1.92 versus 7.21; values for the
second dissociation at 298 K), strongly indicates that the
k␣ca-kt etobutyrate ϭ
ka ϩ kb ϩ kc
(Eq. 6)
The ratio of these kcat values is equal to kb/kc and was calcu-
lated to be 47 in the presence of phosphate (Table 1). There-
fore, using the value of kc ϭ 0.049 sϪ1 (kc is equal to k
which in the presence of phosphate is estimated to beϩ03.,049
s
Ϫ1; see the calculation shown after Equations 3–4), ka was
estimated to be 0.85 sϪ1 from Equation 5.
In the absence of phosphate, on the other hand, Equation 6
is simplified to
kakc
k␣ca-kt etobutyrate ϭ
ka ϩ kc
(Eq. 7)
This has a value of 0.015 sϪ1 (Table 1). As the PLP-␣-aminoc-
rotonate aldimine was not detected in the reaction of TS with
L-vinylglycine (supplemental Fig. S3B), ka is apparently
smaller than kc and should be almost equal to the kcat value,
0.015 sϪ1. Therefore, we can consider that ka is increased 57-
fold in the presence of phosphate.
In the presence of sulfate, kc ϭ 0.43 sϪ1 (equal to kϩ3; Table
Ϫ1
phosphate acts as a base catalyst during the attack of water on 3). Therefore, ka is almost equal to the kcat value of 0.0034 s
the C␤ of 6. From the difference in the pKa values and the
rate constants, the Brønsted ␤ was estimated to be 0.59, indi-
cating that the proton is in an intermediate position between
the O atoms of the attacking water molecule and the phos-
phate or sulfate at the transition state.
(Table 1). This is even lower than the ka in the absence of
phosphate or sulfate (0.015 sϪ1; see above), indicating that the
sulfate ion, unlike the phosphate ion, does not have the ability
to enhance ka.
Altogether, these calculations indicate that phosphate pro-
motes the step 9 3 6, probably by functioning as an acid-base
catalyst. The rate enhancement (57-fold) is moderate as com-
pared with the value (103-fold) for the addition of water at
C␤. The remaining reaction in the absence of phosphate may
be ascribed to the catalysis by water molecule(s) near C␥.
The step 9 3 6 is a nonphysiological reaction that exists on
In contrast to the effect on the step of the addition of water
at C␤, the effect of phosphate ion on the transaldimination
8 3 1 is more difficult to understand. We will return to this
problem later.
Possible Involvement of the Phosphate Ion in the Protona-
tion/Deprotonation at C␥—That the phosphate ion locates
near the C␥ of 6 and is the base catalyst for the addition of the the way from the unnatural substrate L-vinylglycine. However,
water molecule to C␤ raises the possibility that this phosphate considering the similarity in the reaction (protonation at C␥),
ion is also involved in the protonation/deprotonation at C␥,
as we have suggested in a previous crystallographic study on
TS complexed with AP5 (8). This conjecture is attractive as
the ⑀-amino group of Lys-61 is too distant from C␥ and other
we can expect that the phosphate ion functions in the same
way in the step 5 3 6 during the normal catalysis.
Effect of Phosphate Ion on the Transaldimination Steps—
One of the problems concerning the reaction specificity of TS
JANUARY 28, 2011•VOLUME 286•NUMBER 4
JOURNAL OF BIOLOGICAL CHEMISTRY 2783

Product-assisted Catalysis of Threonine Synthase
is the two transaldimination processes starting from 6 and 8.
Ideally, the former should be inhibited, and the latter should
be promoted during the catalytic reaction of TS. It is, then,
important to know the effect of the phosphate ion on these
steps. However, as the rate constant for neither of these steps
in the absence of phosphate or sulfate could be obtained, the
effect of the phosphate ion can be discussed only in compari-
son with the effect of the sulfate ion.
group at C␥. However, as shown in this study, the released
phosphate in turn functions as the catalyst in the subsequent
steps, and this mechanism explains how the reaction is con-
trolled in TS at the branching point (6) of the catalysis divid-
ing the ␤-synthase and the ␥-lyase. Although this is a crucial
element that warrants the reaction specificity of TS, the cata-
lytic reaction of TS contains other branching points that are
subject to side reactions unless there are mechanisms to con-
trol the pathway. These points should be studied in the future.
The rate constant for the transaldimination from 6 is 9-fold
higher in the presence of the sulfate ion (Table 3 and the esti-
mated value of kϩ3 ϭ 0.049 sϪ1 in the presence of phosphate),
whereas the rate constant for the transaldimination from 8 is
higher in the presence of phosphate. The difference between
the structure of 6 and 8 is the presence (8) or absence (6) of
the ␤-OH group and the hybridization at C␣ (sp2 for 6 and
sp3 for 8). As the phosphate ion can interact with both the
␤-OH group and the ⑀-amino group of Lys-61 and the latter is
the group that directly involved in the transaldimination
process, the presence/absence of the ␤-OH group may affect
the transaldimination process. As a simple hypothesis, assum-
ing that the ␤-OH group and the ⑀-amino group of Lys-61
compete with each other for binding to the phosphate ion, we
can expect that the ⑀-amino group forms a hydrogen bond
with the phosphate ion in 6, as observed in the crystal struc-
ture of TS-10-P (Fig. 5) but is liberated from the phosphate
ion in 8. This may result in attenuating the transaldimination
of 6 without affecting the transaldimination of 8. The reverse
effect of the sulfate ion may be ascribed to the absence of pro-
tons in the sulfate ion.
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2784 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 4•JANUARY 28, 2011

Enzymology:
Product-assisted Catalysis as the Basis of
the Reaction Specificity of Threonine
Synthase
Takeshi Murakawa, Yasuhiro Machida and
Hideyuki Hayashi
J. Biol. Chem. 2011, 286:2774-2784.
doi: 10.1074/jbc.M110.186205 originally published online November 17, 2010
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This article cites 27 references, 6 of which can be accessed free at
http://www.jbc.org/content/286/4/2774.full.html#ref-list-1