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THE JOURNAL OF BIOLOGICAL CEIEMISTRY
Vol. 248, No. 10, Issue of May 25, pp. 3631-3635, 19i3
Printed in U.S.A.
Serine Transhydroxymethylase
A KITETIC STUDY OF THE SYn’THESIS OF SEILI?\‘E IS THI4: A13SE:SCI1: OF TETl~AHYDBOFOLATE*
(Received for publication, 1l)ecember 5, 1972)
RIING
S.
CHEN AND
LAVERXE
SCHIHCH
From the Department of Chemistry, Blqjjlox College, BluJlorr, Ohio -45817
SUMMARY
Serine transhydroxymethylase was shown to catalyze the
synthesis of L-serine from glycine and formaldehyde in the
absence of tetrahydrofolate. Initial velocity, product in-
hibition, and exchange reaction studies showed that the
addition of substrates was ordered, with glycine adding first.
The effect of tetrahydrofolate on increasing the rate of serine
synthesis was correlated with the effect of this coenzyme on
the exchange of the a-hydrogen of glycine with protons of the
solvent. The data were interpreted to mean that formalde-
hyde is transferred from serine to 5, IO-methylene tetrahy-
drofolate through an intermediate enzyme-formaldehyde
complex.
Serine transhydroxymethylase (EC 2.1.2.1.) catalyzes the
reversible interconversion of serine and glyciue as shown in Rexc-
tion 1.
Aerine + tetrahydrofolate ti glycine (1)
+ 5,10-methylene tetrahydrofolate
Serine transhydroxymethylase is a pyridoxal 5-phosphatc-cou-
taining enzyme, and most of our previous communications have
dealt with the role of this coenzyme in the enzymatic reaction
(l-4). We assumed that the role of tetrahydrofolatc w-as to
accept or donate a l-carbon moiety at the oxidation level of form-
aldehyde. During the past several years we have found that
serine transhydroxymethylase will cleave several /%hydrosy
amino acids to glycine and an aldehyde. Most of these reac-
tions showed no requirement for tetrahydrofolate. These reac-
tions include the cleavage of L-threonine and allothreoniue to
glycine and acetaldehyde, and erythro- and three-P-phenylserine
to glycine and benzaldehyde (5). The cleavage of Lu-methyl-
serine, however, did show a requirement for tetrahydrofolate (2).
Our understanding of the involvement of tetrahydrofolate in the
reaction could not explain the above observations.
We have now demonstrated that serine transhydrosymcthylase
also catalyzes the interconversion of serine and glycine in the
absence of tetrahydrofolate. In this paper we present evidence
for the synthesis of serine from glycine and formaldehyde. A
* This work was supported by Natioual Science Foundation
Grant GB-31780.
study of the kiuct,ic properties of this reaction has permitted us
to write a mcchxuism which partially explains the substrate
specificity of the enzyme and the role of tetrahydrofolate.
EXPERIMEXTAL PROCEDURE
Jlaterials-Gl?-tine, scriue, and tctrahgdrofolate were pur-
chased from Sigma Chemical Co. Allothreonine and N, N-
bis(2-hydrosyethyl)2-amiuoethane sulfouic acid were obtained
from Nutritional Riochcmicals Corp. [I-14C]Glycine and [2-3H]-
glycine were purchased from International Chemical and Nu-
clear Corp. Serine transhydroxymethylase was purified as
previously described (6).
Purification of [%311]G/gcine-Tritiated glycine was adjusted to
~1-1 2 with I-ICI aud placed ou a Dowex 50-W-X12 column (0.5 x
2.0 cm) which had beeu washed with 6
M
HCl and HZ0 until the
eluate was pII 2. The loaded column was washed with 0.01 M
HCl until no radioactivity could be detected in the eluate. The
glycine was then cluted from the column with 6 M IICl. The
HCl was removed by lyophilization and the resulting residue
was dissolved iu ILO. The lyophilization procedure was re-
peated two more times.
Tritium Exchange Studies-The rate of enzymatic catalysis
of the exchange of the a-hydrogen of glycine with protons of the
solvent was determined as follows. To 80 ~1 of [2-3H]glycine
(220,000 cpm), 7.5
InM
in 0.01
M
N,N-bi@hydroxyethyl)2-
aminoethaue sulfonic acid, PI-I 7.2, were added 10 ~1 (56 pg) of
cuzyme. The reaction, which occurred at 37”, was stopped by
the addition of 10 ~1 of 12y0 trichloroacetic acid. The dena-
tured protein was removed by centrifugation and 80 ~1 of the
supernatant solution were placed on a Dowes SO-W12 column
(0.5
x
4.0 cm), pH 2.0. The loaded column was washed with
0.01 hr IICl, aud the eluate containing the exchanged tritium
was collected in three counting vials, 0.4 ml per vial. Each
vial was filled with 10 ml of Bray’s (7) solvent containing 0.5y0
2,5-bis-2-(5-tert-butylbeuzoxazolyl) thiophene and counted in a
Tri-Carb 3310 scintillation counter.
Xerine and Threonine Synthesis-The synthesis of serine was
measured by the rate of couversion of [l-*4C]glycirie to [PC]-
serine. h typical expcrimcnt was performed as follows. To
40 ~1 of 0.01 M N, N-bis(2-hydroxyethyl)2-aminoethane sulfonic
acid (p1-T 7.2, aud 37”) were added [lJ*C]glycine and formalde-
hyde to final concentrations ranging from 1 to 8 mhf and 1 x lo5
to 8
x
IO5 cpm and 28 pg of enzyme. The reaction was stopped
by the additiou of 5 ~1 of 12% trichloroacetic acid. The dena-
tured proteiu was removed by centrifugation. -1liquots (10 ~1)
3631
This is an Open Access article under the CC BY license.
3632
were removed, spotted on sheets of Whatman No. 4 paper, and
subjected to high voltage electrophoresis (200 volts per cm) in
1.6
M
formic acid for 2 hours. Mixtures of glycine and serine
were used as markers. After they were sprayed with ninhydrin,
the areas corresponding to the serine marker were cut out and
placed in a counting vial with 20 ml of toluene containing 0.5%
2,5-his-2-(5.tert-butylbenzoxazolyl)thiophene. The radioactiv-
ity was counted as described above.
In several experiments acetaldehyde replaced formaldehyde.
Under these conditions the enzyme synthesizes threonine (or
allothreonine) (5). The extent of this reaction was determined
as with serine, except that threonine markers were used to locate
the product on the Whatman No. 4 sheets.
RESULTS
Se&e synthesis-Incubation of serine transhydroxymethylase
with glycine and formaldehyde was observed to produce a radio-
active compound which behaved as serine in electrophoresis
and paper chromatography experiments. This was concluded
from the following experiment. A reaction mixture of enzyme,
[l-1*C]glycine, and formaldehyde was incubated as described
under “Experimental Procedure.” Aliquots were spotted on
Whatman No. 4 paper and chromatographed, with pyridine-
water, 80:20, serving as the solvent system. The area of the
chromatogram corresponding to serine was found to contain a
measurable amount of a radioactive compound. This com-
pound was eluted from the paper and rechromatographed in
three solvents which totally or partially resolve serine and gly-
tine. These solvents were: butanol-acetic acid-water, 100 : 22 :
50; tert-butyl alcohol-acetone&pentanone-water-diethylamine,
40 : 20 : 20 : 20 : 4; phenol-water-acetic acid, 50 : 10 : 1. In each
case the radioactive compound migrated with serine. Because
the paper chromatography experiments are time-consuming, the
procedure used for separating glycine and serine for the kinetic
studies was high voltage electrophoresis in 1.6
M
formic acid.
With the experimental conditions employed, the synthesis of
serine was linear with time and enzyme concentration. Reac-
tion times were 30 min. The counts per min for the no-enzyme
control compound did not exceed 10% of the reaction counts
per min. Duplicate experiments were consistently within the
range of &2%.
The use of formaldehyde resulted in two limitations on reaction
conditions. First, formaldehyde inhibited the reaction at con-
cent.rations above 15 mM, which limited the range of concentra-
tions which could be studied. Second, since amino acids react
with formaldehyde (8), we avoided experiments in which the
concentrations of amino acid was more than one order of mag-
nitude greater than the formaldehyde concentration.
Initial Velocity Studies-The initial rate of serine synthesis
was determined at several concentrations of glycine and form-
aldehyde. Duplicate reaction mixtures were used for each con-
centration of substrate. Three 10.~1 aliquots were used from
each reaction mixture in the electrophoresis system. In Fig. 1
is shown a double reciprocal plot with glycine as the variable
substrate and formaldehyde as the changing fixed substrate.
From the crossover point the dissociation constant for glycine is
calculated to be 7
mM
which is in good agreement with the 6
InM
value obtained from spectral measurements (5). Fig. 2 shows
slope and intercept replots of the data in Fig. 1 versus the recipro-
cal of the formaldehyde concentration. From the intercept
replot the limiting Michaelis constant for formaldehyde was
calculated to be 5.6 mM. The slope replot appears to go through
the origin, indicating that at infinite formaldehyde concentra-
iHCHOJ mM /
(IIGLY CINE) x lO-2 M-’
FIG.
1. Double reciprocal plots of the initial rate of serine
synthesis as a function of glycine concentration at the indicated
concentrations of formaldehyde. Each reaction mixture con-
tained 28 pg of enzyme. Details are described under “Experi-
mental Procedure.”
“0
-
x
w
n
0
d
6
-i
m
4
L
0 2 6 6
(IC-lkiO~ x 10-2M-’
IO 12
FIG. 2.
Slope and intercept replots from Fig. 1. Slope replot,
0-O;
intercept replot, A---A.
tion the requirement for glycine disappears. This initial velocity
pattern is characteristic of equilibrium-ordered mechanisms
(9)
Fig. 3 shows the double reciprocal plot of the initial velocity,
with formaldehyde as the variable substrate and glycine as the
changing fixed substrate. The observation that all lines in-
tersect at the vertical axis is in agreement with the interpretation
of the data in Fig. 2 that, at infinite formaldehyde conccntra-
tion, the reaction is independent of the glycine concentration.
Product Inhibition-Figs. 4 and 5 show that serine is a com-
petitive inhibitor of glycine and a noncompetitive inhibitor of
formaldehyde in the synthesis of serine. These experiments
indicate that the addition of the substrates is ordered, with gly-
tine combining with the enzyme before formaldehyde. This
mechanism is different from the one indicated in the initial ve-
locity studies in Figs. 2 and 3 which supported an equilibriunl-
ordered addition. This mechanism predicts compet,ition by
serine with both glycine and formaldehyde (9).
Formaldehyde was also tested as an inhibitor of allothreonine
degradation to glycine and acetaldchyde. This reaction was
8
0 2 3 6 8 13 12
(I/HCHO) x IO“ M-’
FIG.
3. Double reciprocal plots of the initial rate of scrine syn-
thesis as a function of formaldehyde concentration at the indi-
cated concentrations of glycine. Details are described under
“Experimental Procedure.”
I I
0 2 4 6 8
(I/GLYCINE) x IO-*M-I
FIG.
4. Inhibition of serine synthesis by serine as a function of
glycine concentration. Each reaction mixture contained 40 fig
of enzyme and G rnM formaldehyde.
-I Q I 2 3 4 5
II-HCHO) x 10-2M-’
FIG.
5. Inhibition of serine synthesis by serine as a function of
formaldehyde concentration. Each reaction mixture contained
40 fig of enzyme and 1.8 mM glycine. Details are described under
“Experimental Procedure.”
assayed by converting the acetaldehyde to ethanol with DPNH
and alcohol dehydrogenase (5). Formaldehyde is a poor sub-
strate for alcohol dehydrogenase, thus the concentration range
that could be used in the inhibition studies was limited. We
found no inhibition of the allothreonine reaction by 1 mM form-
aldehyde at allothreonine concentrations as low as 0.25 m&I.
This finding suggests that formaldehyde cannot bind to the free
enzyme and it is in agreement with the ordered addition stated
above.
From the data in Fig. 4 the Zii for L-scrine was calculated to
3633
TABLE 1
E$ect of tetrahydrofolate on serine and threonine synthesis and
exchange of or-hydrogen of glycine with protons of the solvent
Reaction’”
-
I
Serine synthesis.
...............
Serine svnthesis. ...............
Glycine a-hydrogen exchange. .
Glycine or-hydrogen exchange.
Threonine synthesis. .
Threonine synthesis.
ntoles/min/mole
+ 80
- 0.5
+ 120
1.2
+ 10
- 7.0
a See “Experimental Procedure” for reaction conditions.
Tetrs-
hydrofolate Turnover number
be 0.8 mM, which is the same value we found previously by dif-
ferent methods (5). From the inhibition studies we calculated a
Km of 7.7
mM for formaldehyde.
Tritium Exchange Studies-We
have previously shown that
serine transhydroxymethylase catalyzes the exchange of the
a-hydrogen of glycine with protons of the solvent (3) in the
absence of an aldehyde. The rate of this exchange reaction
was enhanced severalfold by the addition of tetrahydrofolate.
These studies showed that the rate of exchange could be cor-
related with the presence of an enzyme-glycine complex which
absorbs at 495 nm (3, 4). Since tetrahydrofolate greatly ac-
celerates both the rate of synthesis of serine and the rate of ex-
change of the a-hydrogen of glycinc, it was of interest to com-
pare the effect of this compound on the two reactions. The
reactions were initiated as described under “Experimental Pro-
cedure.” The concentrations of reactants were the same for
both studies: glycine, 7.5 mnf; formaldehyde, 7.0 rnnf; and tet-
rahydrofolate, 0.2 mM. Since the formaldehyde concentration
in the serine synthesis reaction in the absence of tetrahydrofolate
is at its
K,
value, we multiplied the observed serine synthesis
rate by 2 so that we could compare the maximum rate with the
tritium exchange reaction rate which does not involve form-
aldehyde. The results are recorded in Table I and show that
the rate of exchange of the a-hydrogen of glycine is faster than
the rate of serine synthesis. This shows that the breaking of
the a-hydrogen-carbon bond of glycinc is not the rate-limiting
step in serine synthesis. Tetrahydrofolate enhanced the tritium
exchange and serine synthesis reactions by 100- and 160-fold,
respectively. The a-hydrogen exchange reaction probably ex-
hibits an isotope effect, and thcrc is no assurance that it is the
same in the presence and absence of tctrahydrofolate. However,
the enhancement of the rates of the two reactions is sim-
ilar enough to indicate that the role of tetrahydrofolate in the
two reactions may be due to the same effect.
The effect of tetrahydrofolate on the cleavage and synthesis
of allothreonine was also tested. We found that tetrahydrofolate
had no effect on the rate of cleavage of allothreonine but did
increase the rate of synthesis from glycine and acetaldehyde by a
factor of 1.4 (Table I). The rate of allothreonine synthesis in
the absence of tetrahydrofolate is 14 times greater than the rate
of serine synthesis (Table I).
Glycine-Serine Exchange-Even though L-serine is cleaved to
glycine and formaldehyde very slowly in the absence of tetra-
hydrofolate, it would be possible that the enzyme could catalyze
a rapid exchange between carbon atoms 1 and 2 of glycine and
serine. Reaction mixtures containing L-serine and glycine, at
concentrations equal to their
Km
values, and enzyme were incu-
bated at 37” for periods up to 4 hours in the presence of [VC]-
3634
glycine. So exchange between glycinc and serine could be
c
detected at enzyme levels which would show serine synthesis
from glycine and formaldehyde in a 5-min incubat,ion. These
data are consistent with an ordered mechanism.
DISCUSSIOS
The role of t,etrahydrofolate as a carrier of l-carbon groups is
well established. However, the very stable formation of the
tetrahydrofolate-formaldehyde complex, 5,10-metlrylene tct-
rahydrofolate, does not explain its apparent absolute rcquirc-
meut in the serine transl~ydroxymetl~ylase reaction (10). Pre-
vious studies which showed that tetrahydrofolate greatly accel-
erated the enzymic catalyses of the exchange of the a-protons
of glycine with the solvent produced the first evidence that this
compound may have another role in addition to the one of acting
as the formaldehyde carrier (4). The observation that serine
transhydrosymethylase would cleave several P-hydroxy amino
acids in the absence of tetrahydrofo1at.e added to the mystery
of the role of this coenzyme in the serine-glycine reaction (5).
We have now shown that serine transhydroxymethylase also
catalyzes the reversible interconversion of serine and glycine in
the absence of tetrahydrofolate. The reaction rates are very
slow, but the synthesis of serine was sufficiently fast to permit a
kinetic study of this reaction. We feel that the data help to
explain the role of tetrahydrofolate in the serine transhydrosy-
methylase reaction.
One of the key questions concerning tetrahydrofolnte
is whether it act,s as the direct l-carbon acceptor from serine or
whether formaldehyde is transferred through an intermediate
enzyme-formaldehyde complex. These two mechanisms are
shown schematically in Scheme 1 (S, serine; G, glgcine; F, form-
aldehyde; THF, tetrahydrofolate; 5,10-THF, 5, lo-methylene
tetrahydrofolatc). Scheme la shows a direct transfer of the
l-carbon moiety between serine and tetrahgdrofolate, while
Scheme lb shows the transfer through an intermediate enzyme-
formaldehyde complex.
1 THF S j + l&GiG - -iq (la)
hiF I
ii1
+ I THF k --cl * 1 5, IO-THF I /
G1 (I@
ScrreME 1
The observation that tetrahydrofolate is not absolutely required
in the reactions catalyzed by serine transhydroxymetl~ylase
favors Scheme lb as the mechanism. Scheme lb also is more
consistent with data presented in this paper.
In Scheme 2 is depicted a mechanism which we feel best fits
the experimental data published in this and previous manuscripts
(l-6). In t’his mechanism, L-serine forms a Schiff base complex
with the enzyme (Structure 1) which is cleaved to an enzyme-
formaldehyde-glycine complex (Structure 2). The next step
in the reaction is the dissociation of formaldehyde, which we
now know occurs at an extremely slow rate, i.e. turnover number
SCHEME 2
of about 0.004. Since glycine cannot dissociate prior to form-
nldehydc dissociation it cannot equilibrate with glycine in solu-
tion, thus explaining the failure to observe exchange of L-serine
with [‘“C]glycinc. When allothreonine or threonine replaces
L-serine, lnt~ermediate 2 would have enzyme-bound acetaldehyde
which, because of either its difference in chemical reactivity or
its shape, can dissociate from the enzyme, leaving a dissociable
enzymeglycine complex. fl-Phenylserine would generate an
enzyme-benzaldehyde complex in Structure 2. This large bulky
structure apparently dissociates from the enzyme rapidly, since
this compound is cleaved by the enzyme even more rapidly than
serine (5).
Scrinc synthesis was detected because k, is large enough that
the reaction of the enzyme-glycine complex with formaldehyde
occurs at a measurable rate. The mechanism is more complex
than that shown in Scheme 2 since at least four enzyme-glycine
compleses have been identified spectrally (6) and kinetically
(II). In Scheme 2 we show only two of the complexes with
formaldehyde binding to the enzyme-glycine complex in which
the glycine has lost its a-proton. There is no evidence that
formaldehyde binds to this particular intermediate, however.
The breaking of the oc-proton-carbon bond of glycine might be
expected to be the rate-limiting step, but the data in Table I
indicate that this is not so since serine synthesis is slower than
tritium exchange. This suggests that the rate-limiting step
somehow involves formaldehyde.
Cheng and Haslam (11) have recently shown that the forma-
tion of the enzyme-glycine complex has a rate constant of about
104. This would be several orders of magnitude faster than
the rate of synthesis of serine in the absence of tetrahydrofolate.
It is not surprising, then, that the initial velocity patterns sug-
gest that the enzyme-glycine complex is at thermodynamic equi-
librium. As Cleland (9) points out, under these conditions
the product serine should behave as a competitive inhibitor of
both glycine and formaldehyde even though the reaction is or-
dered. In this system we find that serine is a noncompetitive
inhibitor of formaldehyde. This means that at saturating levels
of formaldehyde t,he concentration of free enzyme is not zero.
This would be true in Scheme 2 if the addition of formaldehyde
to the enzyme-glycine complex was slow and the enzyme was
not saturated with glycine. The data for Figure 5 were obtained
at 1.8 IBM glycine, which is well below the
K,
value of 6 mM for
glycine.
Wllen
we repeated this experiment at 20 mM glycine
and 10 mnr serine, the reciprocal plots deviated from linearity.
The deviation could be explained by assuming a nonenzymatic
reaction of glycine and serine with formaldehyde to the extent
that it changed the formaldehyde concentrations. We could
not minimize this effect by raising the formaldehyde concentra-
tions since this would have led to formaldehyde inhibition.
The observation that tetrahydrofolate accelerates only the
serine-glycine reaction suggests that its role is to catalyze the
removal or addition of formaldehyde from the active site. Since
the enzyme in the absence of tetrahydrofolate degrades allo-
threonine and P-phenylserine at rates comparable to serine in the
presence of tetrahydrofolate, it is apparent that the role of this
coenzymc is not crucial to the breaking of the LY-/3 carbon bond
of the @-hydroxy amino acid (5). Formaldehyde is known to
react readily with primary and secondary amines to form stable
hydroxymethylamines (8). If in the cleavage of serine such
an intermediate was generated, it would tend not to break down
to free formaldehyde and enzyme. One way of removing the
formaldehyde from the enzyme would be to transfer the form-
aldehyde to an even more stable form. The formation of 5, lo-
3635
methylene tetrahydrofolate with an equilibrium constant of
3.2 x lo4 would serve this function very well (10). One unan-
swered question concerning tetrahydrofolate is the relationship
of the effects of this coenzyme on the tritium exchange reaction
and the serine synthesis reaction. It is intriguing to speculate
that both effects are attributable to a single chemical interaction
between tetrahydrofolate and the enzyme. Also, it should be
possible to determine which of the four known enzyme-glycine
complexes acts as acceptor of formaldehyde.
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