SHMT_BBA_1994

et Biophysica Aftra
ELSEVIER Biochimica et Biophysica Acta 1209 (1994) 40-50 , ,
Interactions of L-serine at the active site of serine
hydroxymethyltransferases: induction of thermal stability
Brahatheeswaran Bhaskar a, V. Prakash b, Handanahal S. Savithri a, N. Appaji Rao a,.
a Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
b Department of Protein Technology, Central Food TechnologicalResearch Institute, Mysore 570 013, India
Received 11 April 1994; revised 28 June 1994
Abstract
Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad substrate and reaction specificity. In addition to cleaving many
3-hydroxyamino acids to glycine and an aldehyde, the enzyme also catalyzed the decarboxylation, transamination and racemization of
several substrate analogues of amino acids. To elucidate the mechanism of interaction of substrates, especially L-serine with the enzyme, a
comparative study of interaction of L-serine with the enzyme from sheep liver and Escherichia coli, was carried out. The heat stability of
both the enzymes was enhanced in the presence of serine, although to different extents. Thermal denaturation monitored by spectral
changes indicated an alteration in the apparent Tm of sheep liver and E. coli SHMTs from 55 + I°C to 72 + 3°C at 40 mM serine and
from 67 + I°C to 72 + I°C at 20 mM serine, respectively. Using stopped flow spectrophotometry k values of (49 + 5). 10 -3 s- 1 and
(69 + 7)" 10 -3 S- 1 for sheep liver and E. coli enzymes were determined at 50 mM serine. The binding of serine monitored by intrinsic
fluorescence and sedimentation velocity measurements indicated that there was no generalized change in the structure of both proteins.
However, visible CD measurements indicated a change in the asymmetric environment of pyridoxal 5'-phosphate at the active site upon
binding of serine to both the enzymes. The formation of an external aldimine was accompanied by a change in the secondary structure of
the enzymes monitored by far UV-CD spectra. Titration microcalorimetric studies in the presence of serine (8 mM) also demonstrated a
single class of binding and the conformational changes accompanying the binding of serine to the enzyme resulted in a more compact
structure leading to increased thermal stability of the enzyme.
Keywords: Serine hydroxymethyltransferase; Serine interaction; Thermal stability; Aldimine, internal and external
1. Introduction
Serine is a versatile amino acid with many functions. In
addition to being a part of the protein structure, it is also a
gluconeogenic amino acid, provides one-carbon fragments
for the biosynthesis of purines and methyl group for
thymidine and methionine [1]. Serine hydroxymethyltrans-
ferase, EC 2.1.2.1 (SHMT), which is a key enzyme in the
pathway for interconversion of folates, has attracted
Abbreviations: Serine hydroxymethyltransferase, SHMT; pyridoxal
5'-phosphate, PLP; ethylenediaminetetraacetic acid, EDTA; dithiothreitol,
DTI'; thiosemicarbazide, TSC; nicotinamide adenine dinucleotide, NAD +;
2-mercaptoethanol, 2-ME; 5,5-dimethyl-l,3-cyclohexane-dione, dime-
done; 2,5-diphenyloxazole, PPO; carboxymethyl-Sephadex, CM-Sep-
hadex C-50.
* Corresponding author. E-mail: bcnar@bi°chem'iisc'ernet'in' Fax:
+ 91 80 3341683.
0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0167-4838(94)00135-9
widespread attention as a model pyridoxal 5'-phosphate
(PLP) protein and as a possible target for cancer chemo-
therapy [2]. For these reasons, several laboratories have
examined different facets of the structure-function rela-
tionship of the enzyme, including elucidation of the pri-
mary structure [3-5], interactions at the active site [6-9]
and cloning and expression of the enzyme from various
sources [10-15]. Although details of the interactions of the
inhibitors such as D-cycloserine [16], O-amino-D-serine [7]
and methoxyamine [17] at the active site have received
extensive attention, the role of substrates and substrate
analogues in protecting the enzyme against thermal inacti-
vation has not been investigated extensively, except for the
tetrameric rabbit liver and the dimeric Escherichia coli
enzymes. [18-20]. In this communication, we report the
results of experiments aimed at understanding the mecha-
nism by which serine stabilizes the enzyme against thermal
denaturation.

B. Bhaskar et al. / Biochimica et Biophysica Acta 1209 (1994) 40-50 41
2. Experimental procedures
2.1. Materials
The following biochemicals were obtained from Sigma,
St. Louis, MO, USA: 2-mercaptoethanol (2-ME), DL-di-
thiothreitol (DTT), ethylenediaminetetraacetic acid
(EDTA), pyridoxal 5'-phosphate (PLP), L-serine, glycine,
L-threonine, D-alanine, 5,5-dimethyl-l,3-cyclohexane-di-
one (dimedone), 2,5-diphenyloxazole (PPO), and car-
boxymethyl (CM)-Sephadex C-50. Sephacryl S-200 was
purchased from Pharmacia, Uppasala, Sweden. Tetrahy-
drofolate (H4-folate) was prepared by the method of Hatefi
et al. [21]. L-[3-14C]Serine (specific radioactivity 53
mCi/mmol) was purchased from Amersham, Bucks, UK.
All other chemicals were of analytical reagent grade.
2.2. Methods
Bacterial cultures. The bacterial strain used for the
isolation of SHMT was GS245, a derivative of E. coli
K12 and is pheA905 araD139 81acU169 8glyA strA thi.
Host bacteria were transformed with plasmid pGS29 by
the CaC12 procedure of Mandel and Higu [22]. pGS29 is a
derivative of pBR322 and contains the E. coli glyA gene
on a 3.3 kb SalI-EcoRI fragment [10]. Transformant bacte-
ria were screened for ampicilin resistance and complemen-
tation of glyA deletion. Bacterial cultures were maintained
as Luria-Bertani (LB) agar stabs and glycerol cultures
containing 100/xg/ml ampicilin at 4°C. Host bacteria and
plasmid DNA were kindly supplied by Dr. George Stauffer
of Iowa University, Iowa City, IO, USA.
Enzyme purification. Sheep liver SHMT was purified as
described by Baskaran et al. [7]. E. coli SHMT was
purified as described by Schirch et al. [23] with minor
modifications in the final step of purification.
Enzyme assay. The enzyme activity was determined as
described by Manohar and Appaji Rao [19] using L-[3-
14C]serine as substrate [24]. Protein concentration was
determined by the method of Lowry et al. [25] using
bovine serum albumin (BSA) as the standard.
Absorption spectroscopy. All spectral measurements
were carried out in 50 mM phosphate buffer (pH 7.2) with
1 mM EDTA and 1 mM DTT at 25 __+I°C. Absorption
spectra were recorded in a Shimadzu UV-240 Graphicord
double beam spectrophotometer. Enzyme solutions were
extensively dialyzed against the buffer mentioned above
before spectral measurements.
Heat inactivation. Sheep liver (3.4 mg/ml) or E. coli
SHMT (2 mg/ml) in 50 mM potassium phosphate buffer
(pH 7.2) was kept in a thermostatically controlled water
bath at different temperatures. At different time intervals
(0-15 min) aliquots of 20 /zl were withdrawn and diluted
to 1 ml and chilled in ice. Aliquots (20/.tl) of this solution
were assayed at 37°C for residual enzyme activity after
adding the remaining components of enzyme assay mix-
ture [19]. Results of heat inactivation experiments were
expressed as percent activity remaining compared to the
control value obtained at zero time of incubation.
Thermal denaturation studies. The thermal denaturation
of sheep liver SHMT or E. coli SHMT in the absence and
in the presence of the ligands L-serine, glycine, folate,
D-alanine, L-threonine and thiosemicarbazide (TSC) was
carried out by measuring absorbance changes at 287 nm in
a Gilford Response II spectrophotometer from Ms. Ciba
Coming, USA. A clear solution of 0.3 mg/ml of the
protein in 50 mM potassium phosphate buffer (pH 7.2)
containing 1 mM EDTA and 1 mM DTF was prepared.
About 250 /zl of protein with the ligand was used with
appropriate blanks in the thermal quartz cuvettes and
equilibrated to 25°C in the instrument to obtain the base-
line. The samples were heated from 25°C to 95°C at the
rate of l°C/min using the software available with the
instrument. The absorbance change in each case was moni-
tored at 287 nm and data were averaged from three
experiments. The first derivative of the denaturation profile
was used to evaluate the apparent transition temperatures
(Tm) using the software supplied along with the instru-
ment.
The results were analyzed according to the method
suggested by White and Olsen [26] in which the fraction of
protein in the denatured state (F D) is given by
Al -A N
F D -- - - (1)
A D -A N
where A N is the absorbance of protein solution at 20°C,
A D is the absorbance of protein solution of the plateau
region (in this case 80°C and A1 is the absorbance of
protein solution at different temperatures between 20°C
and 80°C. The apparent denaturation temperature (app. Tm)
was defined as the temperature at which the value of F o
was 0.5.
Fluorescence spectroscopy. Fluorescence excitation and
emission spectra were recorded in a Shimadzu RF-500
spectrofluorophotometer. All the fluorescence measure-
ments were made using quartz cuvettes (3 ml) with 1 cm
path length at 25 _ 1°C.
In the fluorescence titration experiments, the titrant was
delivered in 3 /xl aliquots into the sample cuvettes. The
concentrations of the protein used were 0.7/zM for sheep
liver SHMT and 1.5 /xM for E. coli SHMT. The sample
solution was mixed well inside the cuvette holder which
had a magnetic stirrer attached. At least 5-10 min time
was given for stabilization of the reading. Appropriate
corrections were made for dilution of the protein sample
upon addition of the ligand. The protein was excited at its
excitation maximum of 285 nm and emission monitored at
338 nm.
Circular dichroism (CD) spectra. CD measurements
were made in a Jasco J-500A automated recording spectro-
polarimeter. The spectropolarimeter was continuously

42 B. Bhaskar et al. / Biochimica et Biophysica Acta 1209 (1994) 40-50
purged with nitrogen before and dur!ng the experiments.
Slits were programmed to yield 10 A bandwidth at each
wavelength.
The enzyme CD-spectra were plotted as molar elliptic-
ity values assuming a relative Mr of 213 000 for sheep
liver SHMT and 97 000 for E. coli SHMT, respectively
with mean residue weights (mrw) of 110.24 for sheep
enzyme and 116.30 for E. coli enzyme, respectively and O
was calculated using the following equation [27]:
[O]mrw = [O] × mrw/lO × l× c (2)
where O is the observed ellipticity in degrees, I is the
optical path length in cm and c is the concentration of
enzymes in mg/ml. All CD spectra were recorded at
22 + I°C in 50 mM phosphate buffer (pH 7.2) containing
1 mM EDTA and 1 mM DTI" using the same buffer as
blank. The protein concentrations used were 7.0 /xM and
15.0 /xM for obtaining far UV-CD spectra and 14.0 /xM
and 30.0 /,M for visible CD spectra in the case of sheep
liver and E. coli enzymes, respectively.
Sedimentation velocity. Sedimentation velocity experi-
ments were performed in a Beckman Model E analytical
ultracentrifuge equipped with an RTIC unit. Experiments
were carried out in a Kel F coated aluminium single sector
centre piece with quartz windows, at 25 + I°C and at
59780 rpm. Schlieren patterns were recorded on Agfa
Parachrome films. The S20,w values were calculated ac-
cording to the standard procedure [28].
Stopped-flow spectrophotometry. The stopped-flow ex-
periments were performed in a Union-Giken RA 401
stopped-flow spectrophotometer equipped with a 10 mm
cell. The data were collected using a NEC 9801E computer
interfaced to the spectrophotometer. The solutions were
mixed under nitrogen pressure (4 kg/cm2). The dead time
of the instrument was 40 msec and the slit width was set at
1.4 nm in all the experiments. All the reaction curves
presented were the average of at least 5 sets of experi-
ments. The k values calculated using the specific software
agreed with the values calculated manually by the proce-
dure of Hiromi [29].
Titration calorimetry. The titration of sheep liver SHMT
(27.2 /,M) with L-serine (8 mM) was carried out in a
Microcal Omega Ultrasensitive calorimeter (Microcal,
Northampton, MA). A window-based software package
(ORIGIN version 1.1) also supplied by Microcal was used
to analyze and plot the data. The reaction cell was equili-
brated at 15°C using insulated constant temperature circu-
lating water bath. Internal calibration was performed for
cell constants and other parameters of the microcalorime-
ter. L-Serine (8 mM) was taken in 250/zl injection syringe
and 18 injections of 15 /,1 each at 3 min interval with a
delivery time of 15 s was programmed in titrating the
protein against the ligand using the instrument software.
The data analysis was done according to the method of
Wiseman et al. [30].
3. Results
The conformational and functional features of sheep
liver and E. coli SHMTs were examined by monitoring:
(a) thermal inactivation of the enzymes in the absence and
presence of serine and other ligands; (b) interaction of
serine with both the enzymes measured by changes in its
fluorescence and circular dichroism; (c) alterations in the
sedimentation coefficient of the enzymes; (d) fast reaction
kinetics of the interaction of serine with the enzymes by
stopped-flow spectrophotometry and (e) heat capacity
changes as a result of interaction of the enzyme with serine
by titration microcalorimetry.
3.1. Thermal stability of SHMT-L-serine complex
It is well known that substrates and effectors either
increase or decrease stability of enzymes. Sheep liver
SHMT (3.4 mg/ml) was incubated separately at 62°, 65°
and 67°C for different periods of time and the residual
enzyme activity estimated. It can be seen from Fig. 1A that
although the sheep liver enzyme was stable at 60°C, it lost
85% of its activity within 10 min when the temperature
was increased to 65°C. When a similar experiment was
carried out using E. coli SHMT, it was observed that
increasing temperatures above 60°C inactivated the en-
zyme and more than 90% activity was lost at 75°C (Fig.
1C). When the sheep liver SHMT was incubated at 65°C in
the presence of either, 1 or 10 mM serine, considerable
protection (65% and 73%, respectively) of enzyme activity
was observed during 15 min (Fig. 1B). In the case of E.
coli SHMT, at 65°C only 35% activity was lost which
could be prevented by the addition of serine. On the other
hand at 75°C, 95% of the enzyme activity was lost in the
absence of serine which could be prevented to the extent
of 25% in the presence of 10 mM serine (Fig. 1D). These
results suggested that serine protected both sheep liver and
E. coli SHMTs against heat inactivation.
In addition to serine, SHMT interacted with a number
of amino acids, nucleotides and folate derivatives [20]' It
was, therefore, of interest to study the effect of these
ligands on the temperature induced denaturation of the
enzyme. The inactivation of enzymes in the presence of
different concentrations of serine, 10 mM glycine and 10
mM NAD + is given in Table 1. It is evident from the table
that increasing concentrations of serine brings about in-
creased protection of both sheep liver and E. coli SHMTs
and almost complete protection is observed at 40 mM
serine (Table 1). Glycine (10 raM) protected the enzyme to
the extent of 60%. Similarly, NAD + also protected the
enzymes, but the protection was not as significant as that
of serine. Several other ligands such as folic acid, Cibacron
blue F3GA, L-threonine, D-alanine and thiosemicarbazide
(TSC) even at high concentrations failed to protect either
of the enzymes significantly (data not shown). As serine

maximally protected the enzymes against heat inactivation
detailed investigations were carried out with this ligand.
3.2. Thermal denaturation of SHMT
Fig. 2 (A and B) show the effects of temperature on
absorbance changes of the enzymes at 287 nm. In Fig. 2A
and B (insets) representative first derivative plots of sheep
liver and E. coli SHMT, respectively, are shown. In the
case of sheep liver SHMT, in buffer alone the apparent Tm
was 55 + I°C. Upon the addition of 10 mM serine, a shift
in the transition curve was observed. An apparent Tm of
69 + 2°C at 10 mM serine and an apparent Tm of 72 + 3°C
at 40 mM serine suggested that the enzyme was stabilized
by serine against thermal denaturation.
I0C
6o
0 0 0 60
5 10 15
TIME (rnln)
~ I00
2C
[] .
t I L l I i Ii
5 10 15
TIME (min)
IO0
g 20
0 0 0 6(
,,.
5 10 15
TIME(rnln)
6s'c
-- 10
0 5 I0 15
TIME(rain)
Fig. 1. Heat inactivation of SHMT in the absence and presence of
L-serine. (A) Sheep liver SHMT (3.4 mg/ml) in 200 /xl of 50 mM
potassium phosphate buffer (pH 7.2) was incubated at 60, 62, 65 and
67°C, respectively.Aliquots (20 /zl) were withdrawnat time intervals
indicated in the figure and rapidly chilled in ice. At the end of time
periods indicated,aliquotswere assayedfor the residualenzymeactivity
as describedin Section2 [19,24].(B) SheepliverSHMT(3.4 mg/ml) in
200/xl of 50 mM potassiumphosphatebuffer (pH 7.2) was incubatedat
65°Cin the presenceof 1 mM or 10 mM serine.Aliquotswerewithdrawn
at regular intervals as indicated in the figure and assayed for residual
enzyme activity [19]. (C) In a separate experiment E. coli enzyme (2
mg/ml) in 200 /xl of 50 mM potassiumphosphatebuffer (pH 7,2) was
incubatedat 60, 65, 70 and 75°C, respectively.Aliquots (20 /zl) were
withdrawn at time intervals indicated in the figure and assayed for
residualactivity[19]. (D) E. coli SHMT(2 mg/ml) in 200 #1 of 50 mM
potassiumphosphatebuffer(pH 7.2) was incubatedat 65 and 75°Cin the
presence of 10 mM serine, Aliquots (20 /zl) were withdrawn at time
intervalsindicatedin the figureand assayedfor the residualactivity[19].
Results in all the above experimentsare expressed as percent activity
remainingoverthe control.
Table 1
Heat inactivationof (SHMT)in presenceof differentligands
Ligand Percentactivityremaininga
sheep liver E. coli
(65°C) (70°C)
Enzymealone 16 36
Enzyme+ 0.5 mMserine 50 46
Enzyme+ 1.0 mM serine 46 60
Enzyme+ 5.0 mM serine 78 70
Enzyme+ 40.0 mMserine 82 78
Enzymealone 15 33
Enzyme+ 10.0mMglycine 60 68
Enzymealone 25 38
Enzyme+ 10.0mM NAD+ 55 45
a Theseare independentmeasurementsof activityat the end of 15 minat
the specifiedtemperatureand ligandconcentration.
SheepliverSHMT(4 mg/ml) in 50 mM potassiumphosphatebuffer(pH
7.2) containing1 mM EDTA and 1 mM DTIr was incubatedat 65°Cin
the absenceand in the presenceof 0.5 mM, 1 mM, 5 mM, and 40 mM
L-serine, 10 mM glycine or 10 mM NAD+, respectively.After 15 min
incubationthe residualenzymeactivitywas determined.A similarexperi-
mentwas carriedout with the E.coli enzyme(2 mg/ml) but inactivation
was carriedout at 70°Cboth in the presenceand absenceof the ligands
mentionedabove.
In the case of E. coli SHMT, the native enzyme in
buffer had an apparent Tm of 67 _+ 1°C, which is 12°C
higher compared to the apparent Tm for the sheep liver
enzyme. However, the addition of serine increased the
apparent T~ for the E. coli enzyme by 5 + 1°C (from 67
to 72°C, see Fig. 2B). A comparison of denaturation curves
both in convoluted and deconvoluted states showed a well
defined hump in the control enzymes, which became more
pronounced with increasing serine concentration. How-
ever, the reason for the hump is not very clear.
An analysis of data on the denaturation of the enzymes
in the presence of different concentrations of serine and
glycine and at a single concentration of o-alanine, L-
threonine and TSC (Fig. 3A and B) showed that compared
to serine, glycine was a poor protector of both sheep liver
and E. coli enzymes. L-Threonine, o-alanine and TSC
which interacted at the active site of the enzyme like serine
or glycine, did not increase the thermal stability of the
enzymes. On the other hand, the two enzymes in the
presence of these ligands were more susceptible to heat
denaturation. A common feature of interaction of all these
ligands including serine was that they interacted with PLP
at the active site and generated characteristic intermediates.
In spite of this commonality, the external aldimine formed
with serine and to a lesser extent with glycine appeared to
induce changes in the enzyme structure leading to the
formation of a more stable enzyme, whereas the formation
of a similar complex with other ligands led to the genera-
tion of a less stable structure indicating that the equilib-
rium between different structures of enzymes was affected
by the presence of ligands at the active site. From Fig. 3, it
is evident that in the concentration range of 1 to 40 mM,

the apparent Tm increased linearly in the presence of
L-serine. On the other hand, in the presence of 1 to 10 mM
folic acid the apparent Tm decreased sharply (Fig. 3B)
suggesting destabilization of the sheep liver enzyme.
Higher concentrations of folic acid (above 10 mM) could
not be used due to its limited solubility at pH 7.2.
3.3. Effect of serine on fluorescence spectra of SHMT
The results thus far described suggested that binding of
serine probably brought about subtle changes in the struc-
ture of the enzymes. It was, therefore of interest to deter-
mine whether conformational changes were a prerequisite
to binding of serine to the enzyme. Intrinsic fluorescence
changes provided a convenient handle to examine this
question. Sheep liver enzyme contained 1 tryptophan
residue per subunit amounting to 4 residues per mole of
2.C )3 -.- , _._....j.~
6~=C 1 ~'~
7I ,:, tA I / //
, V ,:t J/ tk I ,' 7 I.: • i
w : - " /
<1.o .~1 i i , i ~1/ / /
O! i I I I
25 36 47 58 69 80
TEMPERATURE(°C)
2.0
67°C l
/'/X~.3 , /
/t~ : - - : Iil 2
? JS~3
25 36 47 58 69 80 ./ j / -
-o.2 .... i
25 36 47 58 69 80
"TEMPERATURE(°C)
Fig. 2. Representative thermal denaturation profiles of SHMT determined
using Gilford Response II Tm spectrophotometer at 287 nm. About 250
/zl of a clear solution of 0.3 A280 nm per ml of the protein in 50 mM
potassium phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM
DTT with or without serine prepared in the same buffer along with
appropriate blanks were taken in thermal quartz cuvettes which were
equilibrated to 25°C in the instrument. The samples after equilibration
were heated from 25° to 95°C at the rate of I°C per rain. The profiles
were obtained after smoothening. (A) Sheep liver SHMT: 1, enzyme
alone; 2, enzyme + 10 mM serine; and 3, enzyme + 40 mM serine. (B) E.
coli SHMT: 1, enzyme alone; 2, enzyme + 5 mM serine, 3; and enzyme +
20 mM serine. Insets of (A) and (B) show first-derivative plots analyzed
to evaluate transition temperatures (app. Tm).
0
~-2
-4
r.-,e,. ...... • L Cb)
- t'-O
1 ~ I I I
-1.o o 1.o
LOG[ 5ERINE], mM
A
B
0 --
¢...1
I i I r ~ I
0 0.5 1.0 1.5 2.0
LOG [ LIGAND ], mM
Fig. 3. A logarithmic plot of apparent midpoint of transition of thermal
denaturation vs. ligand concentration. Apparent Tm values obtained from
first-derivative analyses after heat denaturing the enzyme in the presence
of different concentrations of ligands were plotted against the concentra-
tion of ligands. (A) in the presence of L-serine. (B) In the presence of
other ligands. T~= app. Tm set to maximum at the highest ligand concen-
tration. To = app. Tm at specified concentration of the ligand. (a) Sheep
liver SHMT + L-serine (0.1-40 mM), (b) E. coli SHMT + L-serine (0.5-
20 mM), (c) Sheep liver SHMT+ folic acid (1-10 mM), (d) Sheep liver
SHMT+glycine (1-40 mM), and (e) E. coli SHMT+glycine (1-32
mM).
the tetramer [5], whereas the E. coli enzyme has 3 trypto-
phan residues per subunit amounting to 6 residues per
mole of the dimer [10].
Fig. 4 (Inset) shows fluorescence emission spectra of
both sheep liver and E. coli SHMT at different concentra-
tions of serine. Upon progressive addition of L-serine
significant quenching was observed in both the cases.
However, there was no marked difference in the extent of
quenching with increasing concentrations of serine. The
data were analyzed by Lehrer and Fasman's method [31]
for the binding parameters (Fig. 4A and B). The K a values
were 26.7 + 3.5 M -1 and 97.6 -t- 10 M -1 and AG values
were -1.88 + 0.15 kcal/mol and -2.69 ± 0.11
kcal/mol, respectively for the sheep liver and E. coli
enzymes. These values suggest that the interactions of
L-serine with the enzyme may not have specifically altered
the environment around the tryptophan residues during the
formation of premediated or postmediated complex. How-
ever, the energy transfer between tryptophan residues and
bound pyridoxal phosphate cofactor cannot be the cause
since the apoenzyme also showed similar quenching phe-
nomenon upon titrating with increasing concentrations of
serine (Bhaskar, unpublished data). This does not exclude
conformational changes in regions devoid of tryptophan.

3.4. Effect of serine on the velocity sedimentation of SHMT A B
Any conformational change upon binding of serine in
different domains of the enzyme can alter the shape result-
ing in increase or decrease of the frictional coefficient of
the enzyme. Results of velocity sedimentation of both
sheep liver and E. coli enzymes in the absence and
presence of serine is shown in Fig. 5. Sheep liver SHMT
had an S20,w value of 8.0 + 0.2, whereas in the presence of
50 mM serine it decreased to 7.8_ 0.2. These results
suggested that upon binding of serine, the enzyme molecule
had an increased frictional coefficient thereby decreasing
the S20,w value of the protein. On the other hand, the S20,w
value of E. coli enzyme which was 5.0_ 0.15 did not
change upon binding of 50 mM serine. These changes in
the sedimentation coefficients are not very significant due
to error bars involved in the calculation and measurements.
Fig. 5. Sedimentation velocity patterns of SHMT in the absence and
presence of L-serine. The photographs were taken after reaching two
thirds maximum speed. The time at which photographs were taken are
shown againsteach frame. (A) Sheep liver SHMT:upper trace, enzyme+
50 mM serine (71 min);lower trace, native enzyme (71 min). (B) E. coli
SHMT: upper trace, native enzyme (49 min); lower trace, enzyme+ 50
mM serine (49 min).
0.4
m.
0.2
oo zu
25
0 10
1
o ----.L_2_._L_
300 350 4C0
20 30 50
FSERINE] f tee
I /oOO1.5 B
• o/ue
0.5 7/"
/.
I
0 10
~o
da:
0 J I i
300 350 400
WAVELENGTH(rim)
I I j~t
20 30 50
[SERINE'] free
Fig. 4. The effect of serine on the fluorescence emission spectrum of
sheep liver and E. coli SHMT. The enzyme (0.15 A280/ml) in 50 mM
potassium phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM
DTT was excited at 285 nm and fluorescence emission spectrum was
recorded in a Shimadzu RF-500 spectrofluorometer attached with a
recorder. To the enzyme 3 /xl aliquots of 500 mM L-serinewere added,
mixed well inside the chamberwhich had a magnetic stirrer attached to it
and the emission spectrum was recorded in the range 300-400 nm and
emission maximum at 338 nm was recorded. The data was then analyzed
by Lehrer and Fasman method [31].The graph shown in this figure gives
a plot of /3/1-fl vs. [serine]fre¢,where /3 is the fractional change
occuring in fluorescence upon addition of L-serine. (A) Sheep liver
SHMT, and (B) E. coli SHMT. Insets of (A) and (B): fluorescence
emission spectrum of sheep liver and E. coli SHMT recorded after the
addition of each aliquot of L-serine(3 Ixl of 500 mM L-serine).
3.5. Effect of serine on the CD spectra of SHMT
Earlier results on the interaction of serine with the
enzyme showed that it produced an external aldimine at
the active site of the enzyme [1]. The spectral properties of
PLP-Schiff's base provided a convenient probe to monitor
the changes at the active site of the enzyme. It can be seen
from Fig. 6A that the addition of serine caused a very
significant decrease in molar ellipticity of PLP at the
active site of the enzyme. Similar changes were also seen
in the case of E. coli enzyme (Fig. 6B); however, the
extent of change was less as compared to the sheep liver
enzyme. These results indicated that the decreased positive
band at 430 nm was due to the alteration in the orientation
of PLP with respect to the neighbouring groups as a result
of the interaction with L-serine. These results also indi-
cated the extent of conformational change at the active site
of the enzyme.
In Fig. 7 the far UV-CD spectra of the enzymes from
sheep liver and E. coli in the absence and presence of 50
mM L-serine is shown. The data was analyzed by the CD
Estima method of Fasman (program courtesy of Prof. G.D.
Fasman, Brandeis University, Waltham, MA, USA). In the
case of sheep liver enzyme upon the addition of serine, the
/3-pleated sheet decreased by 50% of its value in the native
enzyme with a concomitant increase in /3-turns. However,
in the case of E. coli enzyme the decrease in the/3-pleated
sheet was much more drastic from 25% to 6% which was
accompanied by an increase in fl-turns. However, the
other secondary structural parameters of ct-helix and
aperiodic components were not significantly altered by the
addition of L-serine in both cases. Hence it was apparent
that the regions of fl-pleated structure were altered in both
the enzymes upon the addition of L-serine. Conformational
change was thermodynamically stabilized by the binding
of serine to the enzyme irrespective of its source, (either
sheep liver or E. coli) suggesting similar mechanisms
might be responsible for the change.

3.6. Spectroscopic changes upon the addition of L-serine to
SHMT
(A) Visible absorption spectroscopy. Fig. 8 shows the
visible absorption spectra of SHMT from sheep liver as
well as E. coli (Insets IA and IIA) with the absorption
maximum at 425 nm due to lysine-PLP-Schiff's base. An
increase in absorption at 425 nm was observed as a
function of serine concentration with a concomitant de-
crease at 343 nm and no change was observed at 280 nm.
(B) Stopped-flow spectrophotometry. The visible spec-
troscopy studies dearly suggested an increase in absorp-
tion at 425 nm occurred duc to the interaction of serine
with PLP bound to the enzyme. Serine-PLP-Schiff's base
had an absorption maximum at 425 nm like the lysine-
PLP-Schiff's base in both enzymes (Fig. 8). It was ob-
served in the visible spectrum that within one min after the
addition of L-serine at 50 mM concentration there was
already a large increase in the absorbance at 425 urn. This
raised the question of existence of a rapid reaction compo-
nent in the protein-ligand complex formation which could
4C
-o
E
2o
o
A ENZYME
rnM SER
I i I i I I I
350 400 4 50 500
WAVELENGTH (nm)
25.0 -B
ENZYME
~15.0
e,,E $ER
u
oo
-o 5.0
!
0
-5.0 --
I I [ I [ I [
350 400 ,,50 500'
WAVELENGTH (rim)
Fig. 6. The effect of L-serine on the visible CD-spectrum of sheep liver
and E. coli SHMT. The enzyme (3 mg/ml) in 50 mM potassium
phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM D'IT was
used to record the CI) spectrum in the range 350 to 500 nm in a Jasco
J-500A spectropolarimeter equipped with a DP-501N data processor and
Kenwood Oscilloscope CO-1530A. To the enzyme solution L-serine (50
/zL of 1000 mM) was added, mixed well and CD spectrum was recorded
again. The time taken for each scan was 8 min. Each spectrum given in
the figure is an average of 4 scans. (A) Sheep liver SHMT, and (B) E.
coli SHMT.
0--
%u
~-4.0
~-~-8.0
I
200
v(-~z+somMSER/-
~, /(
LENZYME
I i ]
220 240 260
WAVELENGTH (rim)
T
"6
E 0
oJ"
E,j
L
~'- 4.0
1D
%
x
-8.0

ENZ+ 50mM SER /,/~
~EHZYME
I I ]
200 220 240
WAVELENGTH (nm)
Fig. 7. The effect of L-serine on the far UV-CD spectrum of sheep liver
and E. coli SHMT. The enzyme (1.5 mg/ml) in 50 mM potassium
phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM DTT was
used to record the CD spectrum in the range 193 to 250 nm in a Jasco
J-500 A spectropolarimeter equipped with a DP-501N data processor and
Kenwood Oscilloscope CO-1530A. To the enzyme solution L-serine (50
p.! of 1000 mM) was added, mixed well and the CD spectrum was
recorded again. The time taken for each scan was 8 min. Each spectrum
given in the figure is an average of 4 independent scans. (A) Sheep liver
SHMT, and (B) E. coli SHMT.
be best studied by stopped-flow spectrophotometry. In
order to analyze the kinetics of the fast reaction, the
change in absorption of the enzyme-serine complex, the
reaction was monitored at 425 nm under pseudo-first order
conditions by adding a large excess of L-serine. Represen-
tative curves are shown in Fig. 8 insets IB and IIB. Fig. 8
insets IC and IIC show derivative plots from which rate
constants were calculated. A comparison of k values of
the two enzymes at 50 mM serine concentration gave a
value of (49 + 5). 10 -3 s-1 for sheep enzyme compared
to the value of(69 + 7)- 10 -3 s -1 for the E. coli enzyme.
These results showed that kinetics of interaction of sheep
enzyme with serine was slower compared to E. coli
enzyme.
At lower concentrations of L-serine, namely at 1 mM
the k values were (19+2)-10 -3 s-1 and (16.5+1).
10 -3 s-l; and at 10 mM the values were (33.5 + 4)- 10 -3
s -1 and (27+3). 10 -3 s -] for sheep liver and E. coli

B. Bhaskar et al./ Biochimica et Biophysica Acta 1209 (1994) 40-50 47
enzymes, respectively. The lower k value for the sheep
enzyme was probably due to the difference in accessibility
of serine to PLP between the two enzymes. The nature of
reaction being fast implied that the reaction was probably
electronic in nature as compared to the slow reactions in
many other systems due to long range factors and Van der
Waals' forces stabilizing such interactions [32,33].
3. 7. Titration calorimetry.
Fig. 9 shows a calorimetric titration of 2.2 ml (5.8
mg/ml) of sheep liver SHMT with a ligand solution of 8
0.100
0.099
Et,.-
~ o.o98
Z
II1
¢Y
~ o.o97
0.096
0.102
7 <
350 450 550 5 10 15 20
WAVELENGTH(nm) TIME (see)
I ~ I ~ I ~ I
20 60 100 140
TIME (s¢c)
I1 {s)
o.loo
¢J
Z
<(
m 0.098
"" "~0 -
,,° -.;,
..... .I J,, .... I *-_,--: X.0.096 0.0625 ___j_ , ,,, ,..,., . ,=,..it.
350 450 550 5 10 15 20
0.094 WAVEL GTH (rim) TIME(Jet)
20 60 100 140 180
T I ME (see)
Fig. 8. The effect of L-serine on the absorption properties of sheep liver
and E. coli SHMT. Insets IA and IIA show the visible absorption spectra:
the sheep liver SHMT (0.5 mg/ml) was taken in 50 mM potassium
phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM DTI" and
its spectrum was recorded in the range 200 to 550 nm in a Shimadzu
UV-visible recording spectrophotometer UV-240 Graphicord. To the
enzyme serine to a final concentration of 50 mM was added, mixed well
and again the spectrum was recorded. Insets IB and IIB depict the rapid
reaction kinetics. The enzyme (0.3 mg/ml) in 50 mM potassium phos-
phate buffer (pH 7.2) containing 1 mM EDTA and 1 mM DTI" was taken
in reservoir A and L-serine (100 raM) prepared in the same buffer in
reservoir B of the Union-Giken RA 401 stopped-flow spectrophotometer.
The solutions were mixed and the absorbance change was recorded at 425
nm. Insets IC and IIC show the first order plots were constructed for the
absorbance changes measured at 425 nm. The tracings shown in the
figure are an average of 5 different experiments.
-20
o~
-40
1
o
I I
4 8 12
INJECTION NUMBER
I I
16 20
Fig. 9. Titration calorimetry of binding of L-sefine to SHMT. Plot of
processed data in the derivative format obtained for 18 automatic injec-
tions, each of 15 /xl of 8 mM I,- serine into the sample cell containing
sheep liver SHMT solution at a concentration of of 0.0272 raM. Other
conditions used were 28°C 50 mM potassium phosphate buffer (pH 7.2)
containing 1 mM EDTA and 1 mM D'Iq'. The total duration of the
experiment was 74 rain.
mM L-serine in the same buffer. Using the instrument
software, 18 injections of 15 /xl each of 8 mM serine at 3
rain interval with a delivery time of 15 s was programmed
in titrating the protein against the ligand. Immediately
following the injection of serine an initial exothermic
phase was observed which increased upon each successive
addition of serine reaching a plateau after 15 injections.
The energy value at the plateau region was 25 + 3
kcal/mol of serine. An analysis of data using the o~6[N
software revealed a single class of binding. Our earlier
data indicated that conformational changes induced in the
enzyme by serine was occurring due to its interaction at
the active site (Figs. 6 and 8).
4. Discussion
The stability of an enzyme and kinetics of the reaction,
especially its specificity is profoundly affected by the
presence of ligands. Serine, one of the substrates of SHMT
protects the enzyme from various sources against heat
inactivation [18-20,34]. The results (Figs. 1-3 and 6 and
Table 1) described in this paper showed that the interaction
of serine with the enzyme enhanced the stability of the
enzyme due to the formation of external aldimine with
PLP at the active site of the enzyme. Earlier work with
aspartate aminotransferase and SHMT from rabbit liver
and E. coli [20,35] suggested that the reaction specificity
was profoundly regulated by the presence of substrates. In
addition to the transfer of hydroxymethyl group of serine

48 B. Bhaskar et al./ Biochimica et BiophysicaActa 1209 (1994) 40-50
to form 5,10-methylenetetrahydrofolate, a physiologically
important reaction, the enzyme also catalyzed several other
reactions such as decarboxylation and transamination with
amino acid analogues [1,36,37]. Although E. coli and
sheep liver enzymes have different subunit structures they
react similarly in the presence of serine. In the present
investigation changes in absorbance values as a function of
temperature were used to determine the denaturation status
of the SHMTs in the absence and presence of ligands. This
method was earlier used in the case of lysozyme [38].
A second method of monitoring ligand binding is by
measuring catalytic activity of the enzyme and also moni-
toring alterations in a reporter group present at the active
site. In this study, unlike in the earlier work of Schirch et
al. [20], changes in stability of the enzyme consequent to
binding of serine was monitored by activity measurements.
Activity is the most sensitive parameter to measure the
integrity of catalytic centre of enzymes. It is evident from
data presented (Fig. 1 and Table 1) that serine protected
both sheep liver and E. coli enzymes very significantly
but to different extents. In fact, upon heating the sheep
liver enzyme in the absence of serine for 10 min at 65°C
almost complete loss of activity was observed, however, in
the presence of serine, nearly 80% of the activity was
present (Fig. 1B). Similarly in the case of E. coli enzyme
serine protected the enzyme (Fig. 1D), whereas glycine --
the other substrate -- was partly effective (Table 1). On
the other hand, other ligands which were shown earlier to
bind to the enzyme, were least effective in protecting the
enzyme against heat inactivation (data not shown). In
general, E. coli enzyme was found to be more stable than
the sheep liver enzyme as evident from the apparent Tm
values of 67 _+ l°C and 55 +_ I°C, respectively (Fig. 2).
Addition of serine to a final concentration of 40 mM led to
a large increase (from 55 _+ 1°C to 72 _+3°C) in the appar-
ent Tm value of sheep liver enzyme. The increase was not
as large in the case of E. coli enzyme (from 67 _+1°C to
72 _+ I°C) (Fig. 3).
Does this mean that thermal denaturation of the enzyme
was initiated at a single point and the protein cooperatively
denatured rapidly? It could be postulated that a conforma-
tional change upon ligand binding resulted in this site of
interaction becoming inaccessible and inactivation started
from a different site leading to increased or decreased
stability. When substrates were used as ligands it was
necessary to distinguish between binding of substrates at
the active site and possibility of binding elsewhere and
causing a conformational change resulting in protection
against heat inactivation. These two possibilities could be
examined by probing changes at the active site and/or
monitoring gross conformational changes.
The conformational changes in the protein were moni-
tored by using intrinsic tryptophanyl fluorescence as a
probe (Fig. 4). It can be seen that the fluorescence emis-
sion spectra of the enzyme in the presence and absence of
serine for both sheep liver and E. coli (Fig. 4) were
similar suggesting that conformational change induced by
serine did not involve changes in the microenvironment
around the tryptophanyl residue. This was also reflected in
the magnitude of K a values determined for both the
enzymes in the presence of serine. The conformational
analyses of the enzymes in the far UV-CD spectra indi-
cated a decrease in /3-sheet and an increase in 0-turns
upon binding of serine for both the enzymes. All these
studies clearly indicated that the binding of serine to the
enzyme leading to increased stability was accompanied by
a generalized change in structure of the protein.
In order to pinpoint the origin of structural changes
which could be occurring at the active site, interactions
with PLP-Schiff's base were monitored by a variety of
methods. The absorbance in the region at 425 nm was
increased when serine was added to the enzyme (Fig. 8, IA
and IIA). This change was attributed to the formation of an
external aldimine of serine with the enzyme [1,39]. This
absorbance change was accompanied by a slight shift in
absorption maximum to around 430 nm (Fig. 8). It was
suggested [1,39]) that there was initial formation of a
geminal diamine from an internal aldimine which was
subsequently converted to an external aldimine. The kinet-
ics of these steps were monitored by stopped flow spec-
trophotometry. The initial fast phase of the reaction lead-
ing to the formation of geminal diamine was accompanied
by a decrease in absorbance at 422 nm with a concomitant
increase at 343 nm [40]. However, this reaction was very
rapid in the case of sheep liver enzyme and could be
observed only at 8°C when stoichiometric amounts of the
enzyme and ligand were used. The very rapid rate of the
reaction which occurred very close to the dead time of the
instrument made it difficult to evaluate the kinetic con-
stants accurately for this phase of the reaction. It was
evident from Fig. 8 that there was a reasonably rapid
change in absorbance at 425 nm upon the addition of
serine to either of the enzymes. The reaction reached a
near stationary phase within 20 s. The rate constants of the
reaction for both enzymes were calculated and it was
found that the rate constants increased with increasing
concentrations of serine. Qualitatively this increase was
similar to the increased protection against heat inactivation
afforded by high concentrations of serine (Figs. 1 and 2
and Table 1). The reaction was relatively rapid with E.
coli enzyme compared to the sheep liver enzyme (Fig. 8,
IIB) indicating that the constraints in the structure of the
sheep liver enzyme slowed down the rate of reaction of
this enzyme with serine. These results suggested that the
rapid kinetic step of formation of external aldimine of
serine with PLP at the active site of the enzyme was a
prerequisite for the final conformational change of the
enzyme molecule upon binding of serine.
Yet another specific method for monitoring changes at
the active site of SHMTs was the visible CD spectrum of
the enzymes. The structure of the active site causes an
asymmetric orientation of PLP and the external aldimine

formed in the presence of serine is still bound to the active
site residues and this property provides a convenient tool
for measuring changes in the PLP environment. It can be
seen from Fig. 6A that serine caused a large change in
visible CD spectrum of the sheep liver enzyme. Similar
change was also seen with the E. cold enzyme (Fig. 6B).
The titration microcalorimetry experiments presented in
this paper show that binding of serine to SHMT resulted in
an exothermic reaction with a single class of binding
leading to conformational changes that induced stability
against thermal denaturation.
Brandts [41] showed that negative heat capacity changes
were associated with burial of hydrophobic amino acids.
The data obtained in Fig. 9 were obtained at a single
protein concentration and at one temperature (22°C). In
order to arrive at the various thermodynamic parameters
experiments have to be conducted at different protein
concentrations and different temperatures. The limited
availability of the enzyme has precluded these experi-
ments. In SHMT it was clearly shown that the enzyme
molecule had an altered conformation upon binding of
serine and also had a compact structure indicated by
sedimentation velocity experiments. The noninvolvement
of the lone tryptophan residue in the protein was con-
firmed by fluorescence spectroscopy and the heat capacity
changes that have arisen must be derived from other
hydrophobic amino acids other than tryptophan. These
results comprehensively showed that conformational
changes accompanying binding of serine led to the forma-
tion of a more compact structure. This could explain the
exothermic phase in the two kinetic pathways detected
using rapid kinetic measurements.
The existence of SHMT in 'open' and 'closed' forms
was postulated by Schirch et al. [20] based on their studies
with rabbit liver enzyme using differential scanning
calorimetry. Similar 'open' and 'closed' structures were
proposed in case of aspartate aminotransferase [35], trypto-
phan synthase [42] and triosephosphate isomerase [43]. Our
results from thermal stability parameters, rapid kinetic
measurements, spectroscopic studies and activity measure-
ments of both sheep liver and E. cold enzymes clearly
demonstrate that this phenomenon is more widespread and
is a common feature of SHMT from several sources and
may be a common phenomenon in case of several PLP
enzymes.
Acknowledgments
We thank the Department of Science and Technology
for the financial assistance. The technical assistance of Ms.
Seetha Murthy, Mr. Ramesh Kumar, Mr. Sudhindra Rao,
Mr. Muralidhar and Mr. Srinivasulu in carrying out some
of the experiments described in this paper is gratefully
acknowledged.
This work was supported by the Department of Science
and Technology, Government of India, New Delhi, India.
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SHMT_BBA_1994

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