(Received for publication, January 31, 1995; and in revised form, April 10, 1995)
From the
Serine hydroxymethyltransferase catalyzes the cleavage of a
variety of
The pyridoxal-5`-phosphate-dependent enzyme, serine
hydroxymethyltransferase (EC 2.1.2.1) catalyzes the reversible
interconversion of serine and glycine (Reaction
1).
The enzyme charges H
In
addition to catalyzing the transfer of a hydroxymethyl group from
serine to H
Figure 1:
A kinetic mechanism for cleavage of
Figure 2:
The proposed mode of reaction of serine
hydroxymethyltransferase with 4-chloro-L-threonine. A
base-catalyzed retroaldol cleavage of the substrate results in release
of chloroacetaldehyde product and formation of a quinonoid glycine
aldimine. E
Figure 3:
Outline of the synthesis of
4-chloro-L-threonine.
(2S,3R)-2-Amino-4-chloro-3-hydroxy-butanoic acid
(4-chloro-Lthreonine) (1) resulted from the
deprotection of 5. 0.1 g (0.26 mmol) of the protected
chlorohydrin (5) was suspended in 15 ml of 6 N HCl and
heated to 80 °C for 8 h. The solution was cooled and extracted with
diethyl ether, and the product was isolated by lyophilization of the
aqueous layer. The yield was 40 mg (94%) and decomposed at
140-145 °C. A single peak was observed for
4-chloro-L-threonine by ion exchange amino acid analysis and
postcolumn derivatization with ninhydrin. The retention time of
4-chloro-L-threonine was 56 min, and that of threonine was 59
min.
(2S, 3R)-2-Amino-3-hydroxybutanoic acid (L-threonine) (7) was prepared from 6 to
determine the stereochemistry of 5. Benzyl
(2S,3R)-2-{[(benzyloxy)-carbonyl]amino}-3-hydroxy-4-iodobutanoic
acid (6) was prepared from 5 by halide exchange using the
Finkelstein reaction(14) . The product 6 was dissolved
in 15 ml of methanol, and 30 mg of 10% Pd on carbon was added. The
suspension was placed in a Parr hydrogenator at 45 p.s.i.g. H
Figure 4:
Stimulation of the rate of
4-chloro-L-threonine cleavage by H
4-Chloro-L-threonine
inactivates serine hydroxymethyltransferase in both a time- and
concentration-dependent manner (Fig. 5). The inactivation
reactions are sufficiently slow relative to the rate of catalysis of
aldol cleavage that equilibrium is reached early in the inactivation
progress, particularly at concentrations of chlorothreonine below 10
mM. The equilibrium constant for formation of
chloroacetaldehyde and glycine from chlorothreonine is 5
Figure 5:
A, the inactivation of serine
hydroxymethyltransferase is time- and concentration-dependent. Rates of
inactivation of the enzyme were measured in the absence of
4-chloro-L-threonine (circles) and in the presence of
2.5 (diamonds), 6 (triangles) and 10 (squares) mM initial concentrations of
4-chloro-L-threonine. B, a plot of the apparent rate
of inactivation as a function of 4-chlorothreonine concentration.
Because enzyme turnover rapidly results in equilibration of
chlorothreonine with glycine and chloroacetaldehyde, the equilibrium
concentrations of chlorothreonine, rather than the initial
concentrations, are plotted. The values for K of 1.7 ±
0.5 mM and k
Both serine and glycine
protect against inactivation by 4-chloro-L-threonine (data not
shown). The enzyme incubated with 5 mML-serine (a
concentration significantly above its K
The protection by alcohol
dehydrogenase and
The data obtained in this study document a linear free energy
relationship between the rate of cleavage of
Figure 6:
Linear free energy plot of log k
Figure 7:
Mechanism proposed by Ulevitch and
Kallen (8) for general base-catalyzed aldol cleavage of
We thank Professor James K. Coward for the use of
equipment and space in his laboratory and for the helpful advice he and
his students John R. Lakanen and Mark R. Burns provided. We thank James
T. Drummond for helpful discussions and Professor Leodis Davis for the
gift of serine hydroxymethyl transferase.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-hydroxy-L-amino acids to form glycine and
aldehyde products. 4-chloro-L-threonine has been synthesized
and shown to be both a substrate and a mechanism-based inactivator of
serine hydroxymethyltransferase. k
values for
the formation of glycine in the absence of tetrahydrofolate were
determined for 4-chloro-L-threonine and other
-hydroxyamino acid substrates; an inverse relationship between the
rate of cleavage of the amino acid and the electrophilicity of the
product aldehyde was demonstrated. 4-Chloro-L-threonine
inactivates serine hydroxymethyltransferase in a time- and
concentration-dependent manner and exhibits saturation of the rate of
inactivation at high concentrations. Our evidence suggests that
4-chlorothreonine undergoes aldol cleavage, and generation of
chloroacetaldehyde at the active site of the enzyme results in
inactivation. Serine or glycine protect the enzyme against inactivation
by chlorothreonine, while tetrahydrofolate does not. The enzyme is also
protected from inactivation by 2-mercaptoethanol or by alcohol
dehydrogenase and NADH. These studies suggest that halothreonine
derivatives that generate electrophilic aldehyde products will be
effective inhibitors of serine hydroxymethyltransferase and might be
potentially useful chemotherapeutic agents.
folate
(
)with a one-carbon unit that is used by enzymes
involved in the de novo synthesis of deoxythymidylate and
purines and in the methylation of homocysteine to form methionine.
Because deoxythymidylate and purines are required for DNA synthesis,
and thus for cell division, serine hydroxymethyltransferase activity
may limit the growth rate of cells. Several studies demonstrating
higher serine hydroxymethyltransferase activity in hepatoma and myeloma
cells than in untransformed cells indicate that the enzyme may be an
appropriate target for anti-cancer chemotherapeutic agents, since
rapidly dividing tumor cells rely more heavily on the de novo biosynthesis of DNA and hence require more serine
hydroxymethyltransferase activity than slowly dividing
cells(1, 2) . Therefore, specific inhibitors of serine
hydroxymethyltransferase may be useful in cancer chemotherapy.
folate, serine hydroxymethyltransferase also
reacts with
-hydroxyamino acids in the absence of
H
folate, converting them to aldehydes and
glycine(3, 4) . Of these
-hydroxyamino acid
substrates, only L-serine and
-methyl-L-serine,
which are the slowest reactants in the absence of H
folate,
require H
folate as a cosubstrate for efficient
catalysis(5, 6) . The H
folate-independent
cleavage of L-serine to form glycine and formaldehyde is more
than 10
-fold slower than the reaction in the presence of
H
folate(6) . For substrates such as threonine and
-phenylserine that do not require H
folate for
efficient catalysis, the reaction is thought to proceed by a retroaldol
cleavage, and the accepted mechanism is shown in Fig. 1(7) . An important mechanistic question is whether
the cleavage of the physiological substrate, serine, also involves
retroaldol cleavage.
-hydroxyamino acids in the absence of H
folate. In step
1, the
-hydroxyamino acid binds the internal aldimine form of the
enzyme (EIA), and the external aldimine is formed by
transimination (EEA). In step 2,
C
-C
bond cleavage takes place to
form a resonance-stabilized quinonoid form of the glycine carbanion (Equin), and the aldehyde product is released. In step 3, the
quinonoid form of the glycine aldimine is protonated (Egly).
In step 4, transimination takes place to release glycine and regenerate
the internal aldimine form of the enzyme.
In this work we describe the synthesis of a
serine analog, 4-chloro-L-threonine, and characterization of
its properties as a substrate and inhibitor for serine
hydroxymethyltransferase. We show that chlorothreonine is a substrate
for serine hydroxymethyltransferase and that it is cleaved to form
chloroacetaldehyde and glycine (Fig. 2), and we provide evidence
that the chloroacetaldehyde generated at the active site is responsible
for enzyme inactivation. The rate of cleavage of chlorothreonine is
intermediate between the rates of cleavage of serine and threonine.
Ulevitch and Kallen (8) previously demonstrated a relationship
between the rate of cleavage of substituted -phenylserine
substrates and the electrophilicity of the product aldehydes. Our work
extends this relationship to include threonine, chlorothreonine, and
serine, establishing that the mode of cleavage of all
-hydroxyamino acid substrates, whether natural or unnatural
substrates, is likely to be the same. While H
folate
accelerates the rate of cleavage of serine more than 200,000-fold, it
accelerates the rate of cleavage of 4-chloro-L-threonine less
than 4-fold. These studies suggest that halothreonine derivatives that
are cleaved to produce highly electrophilic aldehyde products will be
effective inhibitors of serine hydroxymethyltransferase that may be
useful as chemotherapeutic agents.
B is an amino acid side chain that serves as the
general base in this reaction.
Materials
Benzyl-(2S)-2-{[(benzyloxy)-carbonyl]amino}
butanoic acid (or N-Cbz-L-glutamic acid--benzyl
ester) was purchased from Bachem, Inc. Glucose-6-phosphate
dehydrogenase and alcohol dehydrogenase were purchased from Sigma.
Other chemicals used in the synthetic procedures were reagent grade or
higher quality and were obtained from Aldrich or Sigma. The following
buffers were used: Buffer A (50 mM HEPES, pH 7.5), Buffer B
(50 mM potassium phosphate, pH 7.2, 0.1 mM pyridoxal-5`-phosphate, 0.3 mM EDTA and 2.0 mM
2-mercaptoethanol), Buffer C (100 mM sodium acetate, pH 5.1),
Buffer D (70% acetonitrile, 0.10 M sodium acetate, pH 4.4),
Buffer E (50 mM HEPES, pH 7.5, 25 mM
Na
SO
, 1 mM EDTA), and Buffer F (0.10 M potassium phosphate, pH 7.0).
General Methods
H and
C
NMR spectra were recorded on a Bruker AM 360 (Fourier transform) or
Bruker AM 300 (Fourier transform) instrument as indicated. Chemical
shifts were referenced to the residual HOD resonance at 4.63 ppm (
H NMR), a tetramethyl silane resonance at 0.00 ppm (
H NMR), or the central peak of CDCl
at 77 ppm (
C NMR). Mass spectra were recorded on a 7070E J G
analytical organic mass spectrometer. Elemental analyses were performed
by Atlantic Microlabs, Inc., and by the University of Michigan
Department of Chemistry Elemental Analysis Facility. Optical rotation
was measured on a Perkin Elmer 241 Polarimeter. Protein concentrations
were determined using the Bio-Rad protein assay(9) . Enzyme
assays were performed on a Beckman DU Spectrophotometer outfitted with
modern electronics and a recorder. All enzyme incubations and assays
were performed at 30 °C in Buffer A unless otherwise noted.
Stereoselective Synthesis of
4-Chloro-L-threonine from
N-Cbz-L-glutamate-
The
synthesis of 4-chloro-L-threonine from N-Cbz-L-glutamic acid--benzyl ester(2
-benzyl ester (2)
is shown in Fig. 3, and a detailed description of the synthesis
is presented in the Ph.D. dissertation of Webb(10) . Benzyl
(2S)-2-{[(benzyloxy)carbonyl]amino}-3-butanoic
acid (3) was prepared by the method described by Hanessian and
Sahoo(11) , and benzyl
(2S,3R)-2-{[(benzyloxy)-carbonyl]amino}-3,4-epoxybutanoic
acid (4) was prepared by a method similar to that described by
Shaw et al.(12) . 16.7 g (48 mmol) of 50% m-chloroperoxybenzoic acid in CH
Cl
was
prepared as described in (13) . This solution was then
transferred into a flask containing 3.28 g (9.6 mmol) of 3 and
stirred for 120 h at 25 °C under nitrogen. The reaction was cooled
on ice and extracted with 10% NaSO
and then with Buffer F.
The CH
Cl
layer was dried with MgSO
,
and the solvent was removed under vacuum. The product was crystallized
from an 8.5:1.5 mixture of hexane:ethyl acetate.
Benzyl
(2S,3R)-2-{[(benzyloxy)carbonyl]amino-3-hydroxy-4-chlorobutanoic
acid (5) was prepared by a regiospecific ring opening of the
epoxide (4). 0.12 g (0.32 mmol) of the protected epoxide (4) was placed in a dried round bottom flask. 7.0 ml (2 eq) of
0.1 M LiCuCl
in dry tetrahydrofuran
was added to the flask, and the reaction was stirred at room
temperature for 18 h. The reaction was neutralized with 25 ml of Buffer
F and then extracted with diethyl ether. The diethyl ether layer was
dried with MgSO
and was removed under vacuum. The product
was crystallized from a 4:1 mixture of hexane:ethyl acetate.
and shaken for 1 h at room temperature. The suspension was then
filtered through Celite, and the methanol was removed under vacuum.
[
]
(c = 1,
H
O) reported for 7 was -27° and observed
was -25°(15) .
Identification of the Breakdown Product of
4-Chlorothreonine
Incubation of 4-chloro-L-threonine in
Buffer A or incubation of chloroacetaldehyde and
[U-C]-glycine with buffered enzyme results in
formation of an amine breakdown product from
4-chloro-L-threonine with an approximate half-time of 12 h.
Mass spectroscopy studies demonstrated that
4-chloro-L-threonine had lost its chlorine. The NMR spectrum
was similar to that of threonine, except there were only two
protons and there was a down-field shift for the
and
protons. These results are consistent with cyclization of
4-chloro-L-threonine with the elimination of chlorine to
produce 2-amino-3-hydroxy-5-lactone. During incubation in Buffer A, the
half-time for breakdown of 4-chloro-L-threonine to form the
lactone is approximately 12 h, and no glycine formation is detected.
Enzyme Purification and Assays
The cytosolic
isoenzyme of serine hydroxymethyltransferase was purified from pig
liver using the method described in Matthews et al.(16) and was given to us by Professor Leodis Davis. All
incubations and activity assays were performed after exchange of the
enzyme into Buffer A. A unit of enzyme activity is defined as 1
µmol benzaldehyde formed min from 100
mMDL-threo-
-phenylserine in Buffer E (17) ; the formation of benzaldehyde was monitored at 279 nm. A
second assay method was developed based on amino acid sequencing
methods and involves conversion of the substrate and amino acid
products in the assay mixture to phenylthiohydantoin derivatives and
separation of the derivatized amino acids by reversed-phase HPLC.
Samples containing
20 µg of amino acid were removed at timed
intervals from an incubation mixture of enzyme and
-hydroxyamino
acid substrate in Buffer A. The enzyme in the sample was denatured at
95 °C, and the sample was dried using a Speed-Vac vacuum pump. The
amino acid samples and standards were then derivatized with
phenylisothiocyanate as described by Shively(18) . The samples
were dissolved in 1.0 ml of Buffer C, and 80 µl portions were
injected onto an HPLC with a 4.6 mm
25 cm Beckman Ultrasphere
ODS 5-µm column. Products were detected by absorption at 254 nm.
The phenylthiohydantoin derivatives of the amino acids were separated
isocratically with 90% Buffer C and 10% Buffer D at a flow rate of 1
ml/min. The retention time of glycine phenylthiohydantoin is 9.3 min.
Glycine concentrations ranging from 10 pmol to greater than 150 pmol
gave a linear response, and the amount of glycine formed was calculated
by comparison with a standard curve. No significant loss in recovery
was observed due to the presence of enzyme at the levels used in these
assays. The retention time for the phenylthiohydantoin derivative of
4-chloro-L-threonine is 10.5 min, but when
4-chloro-L-threonine is heated to 95 °C and derivatized in
buffer it decomposes and elutes as three peaks at 8.6, 13.6, and 15
min. Measurements of
-hydroxyamino acid cleavage in the presence
of H
folate were performed under argon in Buffer A with
0.025 M
-mercaptoethanol.
Inactivation Assays
4-Chloro-L-threonine
was prepared and stored as the hydrochloride salt. Prior to use for
enzyme inactivation, 4-chloro-L-threonine was dissolved in
0.05 M HEPES and 0.1 M NaOH, yielding a final
solution of 0.1 M 4-chloro-L-threonine, 0.1 M NaCl, 0.05 M HEPES, pH 7.5. The concentration of
chloride ion in the inactivation reactions, 1-10 mM, has
little effect on enzyme activity measured at pH 7.5. The percentage of
residual enzyme activity was determined by comparison with the initial
activity in the same reaction. The enzyme was prepared for inactivation
studies by exchanging into Buffer A. The inactivation incubation
mixtures contained enzyme and freshly diluted
4-chloro-L-threonine at varying concentrations. Samples were
taken at timed intervals and tested for activity using the
spectrophotometric and the HPLC assays. The inactivation assay
performed in the presence of alcohol dehydrogenase contained 2
µM enzyme active sites, 10 mM 4-chlorothreonine,
50 mM HEPES, pH 7.5, 15 µg of alcohol dehydrogenase, 0.4
mM NADH, 7.5 units of glucose-6-phosphate dehydrogenase, and
3.3 mM glucose-6-phosphate in a total volume of 150 µl.
Synthesis of 4-Chloro-L-threonine
The
stereoselective synthesis of 4-chloro-L-threonine from the
-benzyl ester of N-Cbz-L-glutamic acid (2) is shown in Fig. 3. Parts of the synthesis described
in this paper draw on the work of the groups of Hanessian (11) and Rapoport(12) . This route begins with
oxidative decarboxylation of protected glutamic acid 2,
resulting in the vinyl glycine derivative (3). The vinyl glycine
derivative 3 is then converted in high yield to the epoxide 4 using m-chloroperoxybenzoic acid. Unlike previous work
with the
-methyl ester(12) , which yielded a 4:1 ratio of
the threo to erythro diastereomers, the epoxidation
of the benzyl ester-protected amino acid results in the formation of
>95% of the threo diastereomer; the erythro form
is undetectable by either
C or
H NMR. Using
the general method described by Bell and co-workers(19) , the
epoxide ring (4) was then regiospecifically opened using
Li
CuCl
to form the protected chlorohydrin (5). The benzyl-protected chlorohydrin was deprotected by acid
hydrolysis to form the stable hydrochloride salt.
4-Chlorothreonine Is a Substrate
The steady state
kinetics for several -hydroxyamino acid substrates were determined
in the absence of H
folate using the same enzyme source,
buffer, and temperature (Table 1). The rate of glycine formation
was measured for each substrate tested, so that different assays for
different substrates were avoided and a direct comparison of k
values is possible. For substrates that had
previously been studied, the values obtained in our studies compare
well with values we calculated from published data, despite the fact
that earlier studies employed enzyme from different sources and used
different assays and conditions(8, 17, 20) . K
values for the amino acid substrates
decrease as the k
values decrease, as noted
previously(8) .
4-Chloro-L-threonine serves as a
substrate for serine hydroxymethyltransferase with a k value that is intermediate between serine and threonine. Glycine
production was observed by HPLC analysis of the amine products in the
incubation mixture before and after incubation of
4-chloro-L-threonine with serine hydroxymethyltransferase. A k
value of 0.033 ± 0.001 s
and a K
value of 1.6 ± 0.7
mM were determined for 4-chloro-L-threonine (Table 1). The production of glycine from
4-chloro-L-threonine by the enzyme was corroborated by HPLC
and thin layer chromatographic analysis of the mixture before and after
incubation with the enzyme.
H
The rates of turnover of L-serine and 4-chloro-L-threonine were measured at
varying concentrations of Hfolate Increases the Rate of
4-Chlorothreonine Cleavage
folate with resulting values for k
of 21 ± 5 s
and 0.19
± 0.03 s
, respectively. These measurements
were performed under argon in the presence of 25 mM
-mercaptoethanol to minimize the oxidation of H
folate
and enzyme inactivation by the chloroacetaldehyde product. The presence
of this concentration of
-mercaptoethanol does not significantly
affect k
for serine cleavage in the absence of
H
folate but changes the observed k
value for 4-chloro-L-threonine cleavage from 0.033 to
0.046 s
. As previously observed(20) , k
for serine was more than 5 orders of magnitude
greater in the presence of H
folate than in its absence,
with a k
of 21 ± 5 s
with H
folate as compared with 0.0001 s
without H
folate. The K
for (6R,S) H
folate with serine as the
second substrate is 350 µM. The rate constant for
4-chloro-L-threonine turnover, on the other hand, shows only a
4.1-fold increase in the presence of H
folate with a k
value of 0.19 s
and a K
value for H
folate of 87
µM (Fig. 4). This effect of H
folate is
not due to protection of the enzyme against inactivation by
chloroacetaldehyde, which occurs very slowly compared with turnover.
folate. The solidcircles show observed rate constants for the
cleavage of 4-chlorothreonine as a function of the concentration of
H
folate. Each point represents a k
value obtained from a Michaelis-Menten plot of data obtained at a
series of concentrations of 4-chlorothreonine at the indicated
concentration of H
folate. In the presence of saturating
H
folate, k
is 0.19
s
, which is a 4.1-fold increase over the k
for 4-chloro-L-threonine measured
under the same conditions in the absence of
H
folate.
4-Chloro-L-threonine Inactivates Serine
Hydroxymethyltransferase
Criteria for mechanism-based inhibitors (21) include (a) inactivation in a time- and
concentration-dependent manner that saturates at high concentrations of
the inhibitor, (b) protection against enzyme inactivation by
the physiological substrate, (c) inactivation at a rate no
faster than the rate of substrate consumption, (d)
irreversible covalent bond formation, with no return of activity after
removal of the inhibitor, and (e) no release of an activated
species prior to enzyme inactivation.
10
M, as determined by measurement of the
amount of glycine formed from chlorothreonine at equilibrium. The rate
of inactivation saturates at high concentrations of
4-chloro-L-threonine, and the apparent K
of 1.7 ± 0.5 mM is consistent with the
formation of a Michaelis complex between the enzyme and
4-chloro-L-threonine. At saturating levels of chlorothreonine, k
was found to be (3.0 ± 0.2)
10
s
. This rate of inactivation
is significantly slower than the rate of glycine formation from
4-chloro-L-threonine of 3.3
10
s
. The partition ratio was calculated from the
ratio of k
to k
and
found to be 940; at different concentrations of chlorothreonine the
same partition ratio is observed.
of (3.0 ± 0.2)
10
s
were calculated as
described by Kitz et al.(24) using a nonlinear fit to
the data and the KaleidaGraph plot fitting program (Synergy Software,
Reading, PA).
The enzyme is apparently
permanently inactivated by 4-chlorothreonine. There is no observable
return of activity when 4-chloro-L-threonine is removed by
either gel filtration or ultrafiltration. Incubation of completely
inactivated enzyme with pyridoxal-5`-phosphate and
-mercaptoethanol after the removal of
4-chloro-L-threonine does not result in a return of enzyme
activity. These results indicate that loss of cofactor is not the mode
of inactivation. There is no discernible change in the apparent subunit
molecular weight as determined by SDS-polyacrylamide gel
electrophoresis; thus gross modification of the enzyme due to covalent
cross-linking of subunits does not occur.
)
and 5 mM 4-chloro-L-threonine (near its K
) showed no detectable loss of activity
over 400 min. Glycine also protects against inactivation, and this
protection is maximal at glycine concentrations above 1 mM.
When the enzyme is incubated with 10 mM
4-chloro-L-threonine and 10 mM glycine, the rate of
inactivation is 46% of that seen for incubations with
4-chloro-L-threonine alone. These results are consistent with
4-chloro-L-threonine competing with serine and glycine for
binding to the active site. In contrast, H
folate did not
protect serine hydroxymethyltransferase against inactivation by
4-chloro-L-threonine but instead accelerated the rate of
inactivation (data not shown).
-Mercaptoethanol and alcohol
dehydrogenase also protect serine hydroxymethyltransferase against
inactivation by 4-chloro-L-threonine. Enzyme incubated with 10
mM 4-chloro-L-threonine and 10 mM
-mercaptoethanol shows no significant loss of activity over 7
h. There is no detectable modification of 4-chloro-L-threonine
by
-mercaptoethanol as observed by thin layer chromatographic
analysis. The presence of alcohol dehydrogenase and NADH also prevents
inactivation of the enzyme by 4-chloro-L-threonine. The
predicted product, chloroacetaldehyde, is known to be reduced by
alcohol dehydrogenase (22) .
-mercaptoethanol might indicate that
chloroacetaldehyde leaves the active site before inactivation, since a
large enzyme has access to the product. Indeed, chloroacetaldehyde has
been shown to inactivate serine hydroxymethyltransferase(22) ,
and we have determined that the apparent rate of inactivation is first
order in chloroacetaldehyde between 0 and 10 mM concentrations. However, no lag phases are seen in the
inactivation progress experiments shown in Fig. 5A, and
in fact points on each of the lines actually suggest a burst phase
early in the inactivation process. Such observations are only
consistent with the assumption that inactivation occurs before
chloroacetaldhyde leaves the active site of the enzyme, with the burst
phase of inactivation being associated with the change in concentration
of chlorothreonine as equilibrium is approached. It is possible that
the active site of serine hydroxymethyltransferase is accessible to a
large protein. Studies of Stover et al.(23) have
established that native serine hydroxymethyltransferase converts from
an open to a closed form on formation of the external aldimine, but it
is not known whether the binding of substituted
-hydroxyamino
acids also results in closure of the active site.
-hydroxyamino acid
substrates by serine hydroxymethyltransferase in the absence of
H
folate and the hydration equilibria of the product
aldehydes, thus demonstrating that cleavage of these substrates is
accelerated by electron-donating substituents on the
-carbon. The
rate-limiting step in cleavage of
-phenylserine substrates in the
absence of H
folate is associated with
C
-C
bond cleavage and aldehyde
release (Fig 1, step2), and this study establishes
that the same step is rate-limiting for all
-hydroxyamino acid
substrates tested. Such information is useful in designing active
site-directed inhibitors for serine hydroxymethyltransferase, and one
inhibitor, 4-chloro-L-threonine, has been characterized in
this study.
There Is a Linear Free Energy Relationship between the Rate
Constants for Cleavage of
Ulevitch and Kallen studied the rates of
cleavage of substituted -Hydroxyamino Acids and the Hydration of
the Product Aldehydes
-phenyl serine substrates of serine
hydroxymethyltransferase(8) . They demonstrated a linear
relationship between the logarithm of k
for
cleavage and the Hammett
value for substituent, which indicated
that the rate of cleavage of substituted
-phenylserines is
accelerated by electron-donating substituents on the phenyl ring. These
nonphysiological substrates are cleaved rapidly by serine
hydroxymethyltransferase in the absence of H
folate. Our
examination of the k
values for cleavage of
nonaromatic
-hydroxyamino acid substrates by serine
hydroxymethyltransferase suggested a correlation between these values
and the electrophilicity of the product aldehydes formed, a correlation
that extends from the substituted
-phenylserines to include
threonine, chlorothreonine and the physiological substrate, serine. The
logarithms of k
values for
-hydroxyamino
acids cleaved in the absence of H
folate were found to be
linearly related to the pK
values for hydration
of the aldehyde product over 5 orders of magnitude (Fig. 6). A
simple linear free energy relationship implies that a single transition
state dictates the rate for each of the reactions obeying the
relationship. The strong linear relationship seen in Fig. 6is
indicative of a common rate-determining step for all of the substrates
tested, from serine to the
-phenylserines. Furthermore, the slope
of 0.93 indicates that the
-substituent has a large effect on the
rate-determining step.
for
-hydroxyamino acid cleavage in the
absence of H
folate versus the
pK
of the product aldehyde. The k
values are those listed in Table 1. From left to right the substrates are serine,
4-chlorothreonine, threonine and allo-threonine, p-nitro-erythro-
-phenyl serine,
3,4-dichloro-erythro-phenylserine, m-chloro-erythro-
-phenylserine, p-chloro-erythro-
-phenylserine, erythro-
-phenylserine and threo-
-phenylserine, and p-methoxy-erythro-
-phenylserine. Opencircles indicate values for k
calculated from work by others (references given in Table 1), while solidcircles are shown for
data measured in this study. When there is more than one value listed
for a single compound, the k
determined in this
study was used. The K
values are from the
following sources: formaldehyde(25) ;
chloroacetaldehyde(26) ; acetaldehyde(27) ;
benzaldehyde(28) ; and p-nitrobenzaldehyde, p-chlorobenzaldehyde, m-chlorobenzaldehyde, and p-methoxybenzaldehyde(29) .
What Is the Rate-limiting Step?
Fig. 1describes a kinetic mechanism that delineates the
major steps in turnover of a generic -hydroxyamino acid in the
absence of H
folate(8) . Steps 1, 3, and 4 from Fig. 1can be eliminated as possible rate-limiting steps. Kinetic
studies of the condensation of glycine and formaldehyde (aldol
condensation to form serine) were performed by Chen and Schirch (20) , indicating that the reaction was ordered, with glycine
binding preceding that of formaldehyde. Thus, steps 3 and 4 in the
forward direction would not be expected to show any substituent effect,
since they would occur after release of the product aldehyde. The
rate-determining step is also not the binding step, step 1, since
stopped-flow experiments have shown that formation of the external
aldimine occurs in the instrument dead time with
-phenylserine(8) . The enzyme-serine aldimine is also
completely formed before measurements can be made in a recording
spectrophotometer at 10 °C, which indicates a rate of formation of
the serine aldimine that is much faster than k
for the reaction with serine (6) . These two substrates,
-phenylserine and serine, exhibit two extremes in rate, and
neither rate is limited by substrate binding. Thus, steps 1, 3, and 4
are not the rate-limiting steps in cleavage of
-hydroxyamino
acids. This leaves step 2 as the rate-limiting step as described by
Ulevitch and Kallen(8) .
What Can We Infer about the Mechanism of
From the free energy relationship
shown in Fig. 6, we can conclude that the rate of cleavage
increases as the substituent on -Hydroxyamino Acid Cleavage in the Absence of
H
folate?
-hydroxyamino acid substrates
becomes more electron-donating, indicating either an increase in
positive charge or a decrease in negative charge as the reaction
approaches the transition state. Based on their measurements with
substituted
-phenylserines, Ulevitch and Kallen (8) interpreted this observation as indicating a rapid removal
of the proton from the
-hydroxy group, followed by a rate-limiting
C
-C
bond cleavage (Fig. 7). Approach to the transition state for this
base-catalyzed aldol cleavage would involve a decrease in negative
charge. The strong correlation between rate (k
)
and hydration equilibrium of the product suggests that the transition
state for this reaction is very late and resembles the aldehyde, with
very little residual negative charge on the carbonyl, so that the
reaction is disfavored by electron-withdrawing substituents that
stabilize the alkoxide. H
folate, which accelerates the
reaction with the slowest substrates, must either alter the reaction
mechanism or selectively destabilize the alkoxide or stabilize the
nascent aldehyde during aldol cleavage.
-phenylserine substrates in the absence of H
folate.
Deprotonation of the
-hydroxyl group was assumed to occur in a
rapid step prior to the rate-limiting
C
-C
bond
cleavage.
According to this
hypothesis, -hydroxyamino acid substrates with
electron-withdrawing substituents that give rise to highly
electrophilic aldehydes will be very effective reversible inhibitors of
serine hydroxymethyltransferase, because they will react at extremely
slow rates and will thus block the reaction of the enzyme with serine
and/or glycine. 4-Chloro-L-threonine represents a
synthetically accessible example of this type of potential inhibitor,
but 4-fluorothreonine and 4-trifluorothreonine would be expected to be
even more potent inhibitors. Clearly, the efficacy of halothreonine
substrates as inhibitors would be compromised if their rate of cleavage
was substantially accelerated in the presence of H
folate.
However, in contrast to serine, which is cleaved more than 200,000-fold
faster in the presence of H
folate than in its absence ( Table 1and (20) ), H
folate only accelerates
the rate of cleavage of 4-chloro-L-threonine
4-fold (Fig. 4). Thus, even in the presence of H
folate,
halothreonine inhibitors may be expected to retain their potency.
Mechanism of Inactivation
The observed kinetics of
inactivation of serine hydroxymethyltransferase are consistent with
conversion of 4-chlorothreonine to an active site-directed affinity
label, chloroacetaldehyde. This inactivation probably occurs as a
result of partitioning of chloroacetaldehyde formed at the active site
between release and alkylation of the protein.
4-Chloro-L-threonine is probably unsuitable for chemotherapy,
because it leads to the release of a reactive electrophile into
solution following reaction with serine hydroxymethyltransferase.
However, fluorinated derivatives such as
4-trifluoro-L-threonine or the corresponding allo-threonine derivative would be expected to react extremely
slowly with serine hydroxymethyltransferase and might thus be suitable
for chemotherapy.
folate,
tetrahydrofolate; CH
-H
folate,
N
,N
-methylenetetrahydrofolate; HPLC, high
performance liquid chromatography; Cbz-, benzyloxycarbonyl-.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.