(Received for publication, October 2, 1995; and in revised form, December 1, 1995)
From the
Previous studies showed that during the in vitro folding of Escherichia coli serine hydroxymethyltransferase at 4 °C, both monomer and dimer intermediates accumulated and were stable for periods of minutes to hours (Cai, K., Schirch, D., and Schirch, V.(1995) J. Biol. Chem. 270, 19294-19299). To obtain structural information on these intermediates, two of the three Trp residues in the protein were changed to Phe to generate a set of three single Trp mutant enzymes. These mutant enzymes were purified and characterized and shown to retain essentially all of the properties of the wild-type enzyme. The fluorescence and circular dichroism measurements of each mutant enzyme were studied under unfolding-refolding equilibrium conditions and during refolding. In addition, the sensitivity of the protein to digestion by subtilisin during refolding was investigated. The results of these studies show that the unfolded enzyme has two domains that rapidly fold to form a monomer in which the first 55 amino acids and a segment between residues 225 and 276 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate determining conformational change in which the unstructured segments assume their native state.
Escherichia coli serine hydroxymethyltransferase
(eSHMT) ()is a 94-kDa homodimer that catalyzes the
reversible interconversion of serine and glycine with tetrahydrofolate
serving as the one carbon carrier. Each subunit of the holoenzyme
contains PLP attached to Lys
as an aldimine. Removal of
the PLP generates the apoenzyme, which remains dimeric. The 417-residue
subunit contains 3 Trp residues at positions 16, 183, and
385(1) .
In a previous study we demonstrated that eSHMT could be reversibly refolded from 8 M urea after a 10-fold dilution at concentrations as high as 1 mg/ml of protein(2) . At 30 °C refolding was essentially complete in 6 min. This study also provided evidence for several intermediates and a mechanism as shown in . The most important information was obtained with refolding done at 4 °C, where distinct kinetic intermediates could be shown to exist for periods of several hours and complete refolding took as long as 20 h. U is the unfolded enzyme and rapidly folds in a few seconds to form a monomer M. This monomer forms dimers (labeled as D`) during 20 min at 4 °C. The dimer D` does not bind PLP but undergoes a slow rate-determining conformational change to a dimer (apoD) that can bind PLP to form native holoD. At 30 °C the process of converting D` to apoD takes a few minutes, but at 4 °C it takes many hours.
The purpose of this study was to obtain structural information
about M, D`, and apoD. Because these intermediates
exist for minutes to hours at 4 °C, a variety of physical
measurements can be made to aid in elucidation of their structure.
Important information can be obtained on the structure of intermediates
by monitoring the fluorescence properties of Trp residues. To simplify
the interpretation of results with eSHMT, we made single Trp mutants by
changing the remaining two Trp residues in this enzyme to Phe. We also
used protease digestion to determine what regions of the protein in
each intermediate had been folded into a protease-resistant form. The
results provide evidence for the rapid formation of two domains with an
NH-terminal region and a central region of the amino acid
sequence remaining largely in a random coil until the final
rate-determining step.
K and V
values
were determined at 30 °C with serine and tetrahydrofolate as
substrates (1) . Tetrahydrofolate was used at 0.15 mM in each assay, which is six times its K
value, and L-serine was varied in concentration between
0.2 and 3.2 mM.
Thermograms for the denaturation of each
protein were obtained with an MC-2 scanning calorimeter from Microcal,
Inc. (Amherst, MA). Protein samples were dialyzed for 24 h against
several changes of either 20 mM Tris-HCl, pH 7.5, 5 mM 2-mercaptoethanol, and 1 mM EDTA or the same buffer with
20 mM potassium phosphate replacing the Tris-HCl. Protein
concentrations were near 3.0 mg/ml for all samples. Data were analyzed
using the software supplied with the instrument. The T was the average value of three individual measurements for each
sample.
In this equation Q is acrylamide concentration, F and F are the fluorescence in the absence and the
presence of acrylamide, respectively, K
is the
collisional quenching constant, and V is the static quenching
constant.
CD spectra were taken on a Jasco J-500A spectropolarimeter as described previously(1) . The concentration of apo-eSHMT was 0.15 mg/ml for both fluorescence and CD experiments except where otherwise indicated.
Y is the observed value of the
fluorescence signal at a particular urea concentration in the region
where the protein is unfolding. The fluorescence values for Y
(native apoenzyme) and Y
(unfolded apoenzyme) were obtained by linear extrapolation of the
base lines for native and unfolded protein into the region where the
protein was unfolding(8, 9) .
The equilibrium unfolding-refolding plots were fit to either a two-state or three-state model. In a three-state model an intermediate (I) accumulates as shown in . For a two-state model only U and N are present. The equations used to fit each model are as described by Matthews and Crisanti (9) .
K values for serine were
essentially identical for all of the single Trp mutants and wild-type
enzymes (Table 1). Each mutant enzyme exhibited considerable
catalytic activity as well as the enzyme, which had all three Trp
residues replaced by Phe. The single Trp mutants with the least
activity were the Trp
and Trp
enzymes, which
exhibited relative specific activity values of 0.86. The properties of
the mutant eSHMTs, as recorded in Table 1, suggest that each
mutant enzyme has only minor structural differences compared with the
wild-type enzyme.
The solvent exposure of the Trp residue in each
single Trp mutant apoenzyme was estimated by analyzing its fluorescence
spectra(7) . The Trp fluorescence emission maxima at both 4 and
30 °C for the Trp, Trp
, and Trp
mutant apoenzymes were 338 nm, 325 nm, and 334 nm, respectively (Fig. 1). Evidence that the individual Trp residues in the
mutant proteins are very close to their environment in the wild-type
enzyme is suggested by the combined fluorescence spectra of the mutant
enzymes. If the Trp residues in the mutant apoenzymes are in the same
environment as they are in the native apoenzyme, then their combined
emission spectra should be the same as the emission spectrum of the
wild-type apoenzyme. As shown in Fig. 1, the sum of the
fluorescence spectra for the three Trp mutant apoenzymes is about 5%
lower than the fluorescence spectrum of the wild-type apoenzyme. Errors
in calculating protein concentrations for the mutant enzymes could
easily account for the 5% difference. The sum of the three emission
spectra of the mutant apoenzymes has the same 333 nm wavelength maximum
observed for the native enzyme (Fig. 1).
Figure 1: Fluorescence emission spectra of apo forms of wild-type and single Trp mutant eSHMTs. Each enzyme was 0.15 mg/ml in Tris buffer, pH 7.5. Excitation wavelength was 290 nm. The dashed line labeled calculated is the sum of the three single Trp forms of eSHMT in comparison with the emission spectrum of the wild-type enzyme.
Figure 2:
Equilibrium unfolding-refolding curves for
apo forms of wild-type and single Trp mutants of eSHMT as monitored by
CD and fluorescence. Symbols for enzyme forms for both A and B are: filled circles, wild type; open
diamonds, Trp; open triangles,
Trp
; open squares, Trp
. A,
CD signals of 0.15 mg/ml solutions of enzymes were monitored at 220 nm.
The data are plotted as the fraction folded as described under
``Experimental Procedures.'' The solid lines are the
fit of the data points to a two-state transition. B,
fluorescence emission signals of 0.15 mg/ml solutions of enzymes
monitored at 335 nm. The data are plotted as the fraction folded as
described under ``Experimental Procedures.'' The solid
lines are the fit of the data points to either a two-state (open diamonds and triangles) or three-state
transition (closed circles and open
squares).
Fig. 2B compares the fluorescence signals of Trp residues at equilibrium as a function of urea concentration. The data points were fit to either a two-state or a three-state model and are represented by the solid lines. A three-state model suggests that an intermediate (I) exists at a significant concentration between the unfolded state U and the folded state N as shown in . The wild-type apoenzyme (solid circles) is fit by a three-state model, suggesting that the fluorescence of Trp residues in the intermediate are different from either the fully folded or the unfolded state. Fig. 3shows how the relative concentrations of N, I, and U vary with increasing urea concentration. In general, between 1 and 2 M urea the predominate species are N (solid circles) and I (triangles), and between 2 and 4 M urea the predominate species are I and U (open circles). I is the predominate protein species at 2 M urea. An estimate of the free energy changes for N to I and I to U () in the absence of urea was calculated by fitting the three-state equilibrium curve for the wild-type apoenzyme(9) . These values are recorded in Table 2. Although there is considerable error in these estimations, the results suggest that the free energy of denaturation for this protein consists of two similar transitions.
Figure 3: Analysis of the equilibrium unfolding of apo-eSHMT by urea gradient gel electrophoresis and equilibrium fluorescence. Upper panel, urea gradient gel of apoeSHMT (75 µg) electrophorized for 2 h in a polyacrylamide gel made with a 0-8 M linear urea gradient. Lower panel, analysis of the equilibrium unfolding-refolding fluorescence data for wild-type eSHMT as shown in Fig. 2(closed circles). The data points were fit to a three-state equation (). The solid circles represent the fraction of native enzyme N; the open circles represent the fraction of unfolded enzyme U; and the open triangles represent the fraction of the intermediate I.
The equilibrium fluorescence properties
of the three single Trp mutant apoenzymes are very different (Fig. 2B). Because these mutant apoproteins all have
similar stabilities, the differences in Trp fluorescence with
increasing urea concentration are not the result of differences in the
stability of the intermediate (I) for each mutant apoenzyme
but reflect the differences in the fluorescence properties of the
individual Trp residue in the intermediate. Inspection of the curves
suggests that most of the change in fluorescence for the Trp apoenzyme occurs in the N to I transformation,
whereas for Trp
apo-eSHMT virtually all of the change in
fluorescence occurs in the I to U transition. This
shows that in the intermediate Trp
is nearly fully exposed
and Trp
is still fully buried. The Trp
fluorescence change occurs in both reactions, suggesting that in
the intermediate Trp
experiences an environment that is
different than in either the native state or the unfolded state. The
equilibrium fluorescence curve for Trp
eSHMT did not fit
either a two-state or three-state model. It is shown in Fig. 2B as a two-state fit only for visual effect.
Each refolding curve was determined over a 10-fold concentration
range of enzyme. Only the Trp mutant apoenzyme showed any
concentration dependence. A 25-fold increase in protein concentration
shifted the midpoint of the urea unfolding curve from 1.1 to 1.6 M urea. This suggests that changes in the fluorescence of Trp
apo-eSHMT may be associated with the formation of dimers in the
equilibrium unfolding pathway.
The multimeric state and the size of a protein as a function of urea concentration can be investigated by electrophoresis in a urea gradient gel. The photograph of a urea gradient gel with wild-type apo-eSHMT is shown in Fig. 3. The native dimer migrates only slightly faster at 0 M urea than the fully unfolded monomer at 6-8 M urea. In the region of 1-4 M urea, there is an increase in size with a sharp band that runs considerably slower than the native dimer. There is also a background smear of protein that occurs between the two bands. A possible interpretation of this pattern is given under ``Discussion.'' No reproducible differences could be found between wild-type and single Trp mutant apo-eSHMTs in these urea gradient gels, further supporting the conclusion that the mutations have not significantly altered the unfolding-refolding mechanism.
As shown previously, the rate of refolding at 4 °C is dramatically different than at 30 °C. For the wild-type enzyme less than 10% of the catalytic activity returns within 100 min after initiation of refolding. Furthermore, the previous study showed that for wild-type apo-eSHMT refolded at 4 °C, all of the secondary structure had returned within 2 min and that the enzyme had completely formed dimers after about 100 min(2) . This dimer could not bind PLP and is listed as structure D` in . When the single Trp mutant enzymes were tested for refolding at 4 °C, the return of activity was the same slow rate as observed for the wild-type enzyme. Although we did not check for the rate of dimer formation, the results of urea gradient PAGE suggests that the mutations have not altered the unfolding and separation of dimers. We assume that each mutant enzyme forms dimers at close to the same rate as observed previously for the wild-type enzyme(2) . These results suggest that the rate-controlling step in each single Trp mutant enzyme is the conversion of D` to apoD as shown in .
The rates of change in fluorescence was investigated
with each of the mutant apoenzymes at 30 °C after initiation of
refolding. Both the Trp and Trp
mutant
apo-eSHMTs showed a rapid change in fluorescence spectra to native
values following a 10-fold dilution into refolding buffer at 30 °C (Fig. 4, A and B, solid lines).
However, the Trp
apoenzyme showed a much slower return of
the fluorescence signal, taking about 500 s to reach 90% of the native
enzyme value (Fig. 4C). The rate of return of
native-like fluorescence for the Trp
enzyme was
concentration-dependent increasing about 3-fold from 0.004 to 0.1 mg/ml
enzyme. Above this concentration there was no further increase in rate.
Figure 4: Rate of return of fluorescence emission during refolding of apo forms of wild-type and single Trp mutants of eSHMT. Excitation was at 290 nm, and emission was monitored at 335 nm. Each enzyme was diluted 10-fold from an 8 M urea solution into Tris buffer, pH 7.5. The solid lines are for data obtained at 30 °C, and the dashed lines are for data obtained at 4 °C. The lines do not conform to any model. Panels A-C are the single Trp mutant eSHMTs and panel D is the wild-type enzyme with 3 Trp residues.
The fluorescence emission properties for each apo-eSHMT during
refolding at 4 °C are shown as dashed lines in Fig. 4. For wild-type apo-eSHMT, refolding at 4 °C in the
presence of PLP results in almost no active enzyme being formed in the
1000-s time period recorded in this figure. For the Trp and Trp
mutant apoenzymes, the fluorescence signal
returned to native values by 120 s (Fig. 4, A and B, dashed lines). However, the return of native
fluorescence for the Trp
mutant eSHMT took as long as 15 h
to reach final native values. As shown in Fig. 4C about
50% of the fluorescence change has returned in 20 min after refolding.
As observed at 30 °C, only Tp
eSHMT showed any
observable concentration dependence. The fluorescence emission
properties of the wild-type eSHMT during refolding at 4 °C as shown
in Fig. 4D is a composite of the properties of the
mutant enzymes.
We had previously shown that in the refolding of wild-type apo-eSHMT the CD spectrum returned rapidly even at 4 °C(2) . In this study, we determined the CD spectrum from 200 to 240 nm after initiation of refolding for each mutant apo-eSHMT at 4 °C. Within the 120 s it took to record the spectra, the CD signals were the same as for the native apoenzymes, confirming that for each mutant enzyme the secondary structure returns very rapidly (data not shown).
To further characterize the environments of the three
Trp residues during refolding at 4 °C, the susceptibility of each
mutant enzyme to acrylamide quenching was investigated (Fig. 5).
The results are presented as the collisional quenching constant (K) and the static quenching constant (V) as recorded in Table 3. K
is
obtained from the initial slope of the quenching curve, and V is obtained by applying (7) . The collisional
quenching constant values show that each Trp is partially
solvent-exposed with values ranging from 1.6 M
for Trp
eSHMT to 5.8 M
for Trp
eSHMT, suggesting that Trp
is
the most buried and Trp
is the most solvent exposed of the
three Trp residues. The collisional quenching constant values are in
agreement with the values of the fluorescence emission maximum for each
mutant enzyme as shown in Fig. 1. In general, the shorter the
wavelength of maximum emission the more buried the Trp residue
(Trp
, 325 nm; Trp
, 334 nm; and
Trp
, 338 nm) with fully exposed Trp residues in the
unfolded state having an emission wavelength of about 354 nm.
Figure 5:
Acrylamide quenching curves for apo forms
of single Trp mutants of eSHMT at 4 °C as a function of refolding
time. Each mutant enzyme was diluted 10-fold from an 8 M urea
solution into 2 ml of Tris buffer, pH 7.5. 10-µl aliquots of an 8 M acrylamide solution were added, and the fluorescence was
monitored at 335 nm at various times after initiation of refolding. It
took about 5 min to record each quenching curve. The times on each
graph represents the time at which the quenching curve had been
started. The symbols are as follows: squares, Trp eSHMT; triangles, Trp
eSHMT; circles, Trp
eSHMT. The panel labeled Native shows the quenching curves obtained at 4 °C for the native
single Trp mutant enzymes in 0.8 M urea.
The
quenching by acrylamide was used to follow each Trp residue during
refolding at 4 °C to determine what the status of each Trp residue
was during the formation of intermediates M, D`, and
apoD (). Quenching by acrylamide was followed for each
single Trp mutant enzyme as a function of time after initiation of
refolding and compared with the quenching properties of the native
mutant enzymes as shown in Fig. 5D. For the Trp apoenzyme, acrylamide quenching was native-like within the 5 min
it took to determine a quenching curve (Fig. 5A and Table 3). After 5 min the quenching of Trp
apo-eSHMT was close to native values, but both the values of K
and V were larger than the native
enzyme. By 5 min the CD spectra of these proteins show that all of the
secondary structure has returned and the fluorescence emission of both
Trp
and Trp
have returned. Previous studies
using size exclusion chromatography show that the enzyme is mostly the
monomer (M) after 5 min of refolding. These results suggest
that both Trp
and Trp
are close to their
native environments. However, Trp
is far from its native
value, as indicated by the large value of 0.93 for the static quenching
constant (V) (Table 3). This shows that Trp
is largely solvent exposed in structure M. After 2 h of
refolding the enzyme will be mostly D` with some apoD. Both
Trp
and Trp
apoenzymes have values for the
quenching constant that are the same as the native enzyme. Trp
apo-eSHMT may still be slightly more solvent exposed than the
native enzyme.
Figure 6: SDS-PAGE of proteolytic digestion during the refolding of wild-type apo-eSHMT. Unfolded enzyme was diluted 10-fold into Tris buffer at 4 °C, and at various times aliquots were removed and incubated with subtilisin for 2 min at 4 °C. The digestion was stopped by the addition of phenylmethanesulfonyl fluoride. These digestions were then submitted to SDS-PAGE. Lane 1 is subtilisin. Lanes 2-6 are refolding solutions that were incubated from 0, 0.17, 1.7, 17, and 60 min, respectively, before incubation with subtilisin. Lane 7 is a sample that had been refolded for 60 min at 4 °C, and then the temperature was increased to 30 °C for 10 min before protease digestion. Lane 8 is native apoenzyme and subtilisin incubated at 4 °C for 2 min. Lane 9 is native enzyme without protease. Lane 10 shows the molecular mass markers.
The major bands at 23 and 17 kDa were eluted
from the gel and analyzed for their amino-terminal and
carboxyl-terminal sequences. The 17-kDa band showed a NH terminus sequence of KEAME-, which is consistent with residues
277-281. Carboxypeptidase Y digestion released Ala, Tyr, and Val,
consistent with the sequence of the carboxyl terminus of the enzyme.
NH
-terminal sequencing of the 23-kDa fragment was
consistent with the sequence AEGYP-, which suggests protease cleavage
at Tyr
. Carboxypeptidase Y digestion was complex,
releasing rapidly two equivalents of Val, two equivalents of Leu, and
one equivalent each of Thr, Ile, and Tyr. These results suggest that
subtilisin had cleaved the protein about equally in at least two
positions. The major sites were Thr
and
Leu
. The origin of the single Tyr is not clear.
Several other researchers have cited the many advantages of using single Trp mutants to study the mechanism of protein folding(12, 13, 14, 15) . E. coli SHMT offers several unique advantages for using single Trp mutants. First, this enzyme has three Trp residues that are widely spaced in the sequence of the enzyme. There is no conservation of any of the Trp residues in a list of 14 different SHMT sequences from a variety of sources, suggesting that they do not play a critical catalytic role in this enzyme(16) . Second, eSHMT refolds rapidly at high protein concentration. Third, monomeric and dimeric intermediates accumulate at 4 °C for periods that allow physical and chemical probes to be used.
The single Trp mutant forms of the
enzyme used in this study appear to fold by the same mechanism and to
form essentially the same structure as previously observed for the
wild-type enzyme. Both equilibrium and kinetic folding studies suggest
that at 4 °C intermediates accumulate on the refolding pathway.
Kinetic studies suggest that upon dilution of unfolded enzyme at 4
°C, two domains rapidly fold to structures characteristic of the
native state. These are listed as domains 1 and 2 in Fig. 7.
This rapid folding is supported by the fluorescence studies that show
that Trp, which is in domain 1, is rapidly buried within
a few seconds and exhibits characteristics of fluorescence emission, CD
spectrum, and acrylamide quenching of its environment in the native
enzyme. Almost the same conclusions can be stated for
Trp
, which is a part of domain 2. Only the acrylamide
quenching studies suggest that the enzyme does not completely reach its
native environment by 5 min. Protease digestion studies show that these
two domains have become resistant to digestion immediately upon
dilution of the unfolded enzyme into refolding buffer, further
supporting the view that two domains exist in a largely condensed
state.
Figure 7:
Model showing the structure of an early
intermediate in the folding of apo-eSHMT. Protease digestion
experiments show that folding intermediates representing both monomer
and dimer forms of apo-eSHMT are digested at Tyr,
Thr
, Leu
, and Leu
. A
23-kDa domain is resistant to protease digestion. This domain
contains Trp
(circle), which is the most buried
of the three Trp residues. Also, a
17-kDa domain at the carboxyl
terminus of the protein containing Trp
is resistant to
protease digestion. Trp
remains exposed to solvent during
the early stages of refolding and appears to reach its native state
only during the rate-determining step of forming native apoenzyme.
Lys
, which is the PLP binding site, also remains
protease-sensitive until the final stages of forming native
enzyme.
Kinetic studies show that Trp is outside of
domains 1 and 2 and appears not to be part of any ordered structure
during the first stages of folding. It is removed by protease treatment
and is solvent exposed as indicated by fluorescence emission and
acrylamide quenching ( Fig. 4and Fig. 5). It appears to
reach its native state only when the enzyme reaches its catalytically
active form. The fact that the equilibrium refolding studies and the
rate of burial, as determined by fluorescence emission of this residue
in the Trp
eSHMT, both show some concentration dependence
suggests that this residue is in a different environment in the dimers D` and apoD as compared with the monomer intermediate M (). The observation that the CD signal of all mutant
eSHMTs are fully formed after a few seconds and that the equilibrium
refolding curves are nearly the same by this technique (Fig. 2)
suggests that most of the secondary structure of eSHMT is present in
domains 1 and 2.
Protease digestion studies also suggest that an
amino acid segment between domains 1 and 2 is unordered, being
accessible to protease digestion during the period when D` is
the dominant intermediate. This segment includes the active site
Lys, which binds PLP (Fig. 7). In our previous
study we concluded that the rate-determining step in forming active
eSHMT involved the formation of the active site that includes
Lys
(2) . The protease digestion studies confirm
that this section of the protein remains in a nonnative state until the
rate determining final isomerization of D` to apoD (). Unfortunately there is no Trp residue in this segment
of the molecule to monitor the environment during folding studies.
However, we have used NaCNBH
to reduce the bound PLP of
holo-eSHMT to form a stable secondary amine to Lys
. This
now places a fluorescent probe in this region of the protein.
Preliminary studies show that the PLP is not buried in its native state
until late in the folding process. (
)
Equilibrium
unfolding-refolding studies are in agreement with the model developed
from the kinetic refolding studies as shown in Fig. 7.
Equilibrium fluorescence studies of the wild-type enzyme containing all
three Trp residues suggest that an intermediate structure I dominates the population of protein molecules at 2 M urea (Fig. 3). Equilibrium fluorescence studies of the single Trp
mutants show that Trp becomes disordered at a much lower
urea concentration than the other two Trp mutants. This would suggest
that the conversion of the native form N to the intermediate I may not represent a significant energy barrier. However, as
shown in Table 2,
G
for the conversion
of N to I is significant and in the same range as the
conversion of the intermediate I to the unfolded form U that takes place at a much higher urea concentration. The
relationship of
G to the concentration of urea is given
by (17) . For the conversion of the native
apoenzyme N to I, the value of m is
-3.1
kcal
mol
M
,
and its value for the conversion of I to the unfolded form U is -1.6
kcal
mol
M
.
When plots of
G versus [urea] are extrapolated
to zero urea concentration to obtain
G
, the
two transitions give nearly the same values for
G
(Table 2).
However, it is clear that in I the amino-terminal
segment is largely disordered and Trp is solvent-exposed.
It is not clear from the fluorescent studies if the residues between
the two domains are also being exposed and disordered between 1 and 2 M urea. The urea gradient gel suggests that two distinct sizes
of molecules do exist in the range of stability where I is
formed (Fig. 3). The band at the left of the gel (0 M urea) represents the migration of the dimer apoD, and the band
above 6 M urea represents the migration of the unfolded
monomer (U). There is not much difference in the size of these
two species. In the range where the intermediate I is stable
(1.5-3.0 M urea), there is a transition between these
two extremes. In this range of urea concentration, we observed for all
apoenzyme forms a distinct band that migrated much more slowly than
either native or unfolded enzyme (Fig. 3). We suggest that this
could be an expanded dimer in which the NH
-terminal
sequence and the section between the two domains have become
disordered. The smear of protein between the upper and lower bands in
the 2-4 M urea range of the gel suggests that there is
equilibrium between species of different sizes during the 2 h it took
to run the gel.
Protease digestion by subtilisin gives some evidence about the structure of D`. Protease digestion fails to distinguish between M and D` as evidenced by the same pattern of forming 17- and 23-kDa fragments from the first few seconds of refolding until 1000 s after refolding. By 1000 s the apoenzyme is a dimer(2) . These results suggest that the dimer D` is greatly expanded, having disordered segments at the amino terminus and between domains 1 and 2. For most oligomeric proteins, forming dimers occurs only after the formation of nearly native monomers(18, 19) . This does not seem to be the case with apo-eSHMT.
Subtilisin will probably not digest the full length
of the amino acid residues between domains 1 and 2. Only the portion of
the sequence that is readily accessible to the protease and meets its
specificity requirements will be digested. It is likely that the
unfolded segment starts at the sequence PNPVP- and
extends to -PEP
(16) . This portion of the amino
acid sequence contains Lys
, which binds PLP and 8 of the
20 Pro residues in eSHMT. The lack of native structure of this part of
the molecule helps explain why D` does not bind
PLP(2) . A highly conserved sequence in all SHMT enzymes lies
next to the active site Lys
. In almost all sequences
there are 4 Thr and 2 Val residues between Val
and
Thr
(16) . This stretch of 6
-branched amino
acids would be predicted to exist in an unordered state. The rate
determining step may involve the folding in of this stretch of amino
acids to form the active site of the apoenzyme.
Several studies have suggested that eSHMT has the same fold as several other PLP enzymes, including aspartate aminotransferase(16, 20) . One of these studies aligned the amino acid sequence of eSHMT with aspartate aminotransferase(16) . The three-dimensional structure of aspartate aminotransferase has been solved and shown to consist of two domains. These two domains coincide with domains 1 and 2 of eSHMT as determined from the protease digestion studies. In mitochondrial aspartate aminotransferase the large domain starts at residue 47 and the small domain starts at residue 329. These are only a few residues different than the digestion sites found for our subtilisin digestion of eSHMT. It has been shown previously that the large domain of aspartate aminotransferase can be expressed and folded independently of the small domain(21) . This large domain also bound PLP but did not form dimers and showed no catalytic activity. Our studies suggest that the small domain (domain 2 in Fig. 7) may also fold by itself.