(Received for publication, January 9, 1995; and in revised form, June 13, 1995)
From the
Escherichia coli serine hydroxymethyltransferase is a 94-kDa homodimer. Each subunit contains a covalently attached pyridoxal-P, which is required for catalytic activity. At which step pyridoxal-P binds in the folding pathway of E. coli serine hydroxymethyltransferase is addressed in this study. E. coli serine hydroxymethyltransferase is rapidly unfolded to an apparent random coil in 8 M urea. Removal of the urea initiates a complete refolding to the native holoenzyme in less than 10 min at 30 °C. Several intermediates on the folding pathway have been identified. The most important information was obtained during folding studies at 4 °C. At this temperature, the far-UV circular dichroism spectrum and the fluorescence spectrum of the 3 tryptophan residues become characteristic of the native apoenzyme in less than 10 min. Size exclusion chromatography shows that under these conditions the refolding enzyme is a mixture of monomeric and dimeric species. Continued incubation at 4 °C for 60 min results in the formation of only a dimeric species. Neither the monomer nor dimer formed at 4 °C bind pyridoxal phosphate. Raising the temperature to 30 °C results in the formation of a dimeric enzyme which rapidly binds pyridoxal phosphate forming active enzyme. These studies support the interpretation that pyridoxal phosphate binds only at the end of the folding pathway to a dimeric apoenzyme and plays no significant role in the folding mechanism.
The reversible unfolding-refolding of proteins in vitro is extensively used as a model to study the folding pathway of
proteins in vivo(1, 2) . Most folding studies
have been done with small monomeric proteins, but a number of studies
have looked at the folding pathway of the more complicated oligomeric
proteins(3) . However, relatively few studies have focused on
the effect of protein ligands on the rate of refolding. The most
extensive studies involving ligands have been done with NAD and pyridoxal 5`-phosphate (PLP) (
)requiring
enzymes(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
Many enzymes involved in amino acid metabolism require PLP bound as
an aldimine to the -amino group of a lysyl residue. More than 50
different PLP enzymes have been identified, and the crystal structures
have now been solved for several members of this group(17) . It
has been proposed that these enzymes can be grouped into three separate
classes based on sequence homology(17) . It is not yet clear if in vivo there is a common mechanism for folding of PLP enzymes
and to what extent PLP binds to early intermediates in the folding
pathway and help direct forming the native holoenzyme.
Escherichia coli serine hydroxymethyltransferase (eSHMT) is a PLP-containing dimeric enzyme of identical 47-kDa subunits (18) . The PLP can be easily removed to form the native dimeric apoenzyme. Addition of PLP to the apoenzyme results in the formation of fully active holoenzyme. We demonstrate in this study that eSHMT can be reversibly interconverted between an apparent random coil and native apoenzyme. Several intermediates on the folding pathway can be identified. This permits us to begin to address questions of how PLP adds to eSHMT during its initial folding in vivo. We specifically want to determine if PLP binding plays a role in the folding process. These results are compared with the limited studies previously done with other PLP enzymes which have addressed this question.
eSHMT
was purified from E. coli strain GS245 containing a high copy
plasmid expressing the glyA gene as described
previously(18) . Apo-eSHMT was prepared by adding L-cysteine (0.1 M) to 20 mg of holoenzyme. The
solution was made 40% in ammonium sulfate and added to a 1.5
10-cm phenyl-Sepharose column equilibrated with 40% ammonium sulfate
and 20 mM potassium phosphate, pH 7.2, containing 5 mM 2-mercaptoethanol and 50 mML-cysteine. The
absorbed enzyme was eluted with a linear gradient of 50 ml of
equilibrating buffer and 50 ml of 20 mM potassium phosphate,
pH 7.2, containing 2-mercaptoethanol. The apoenzyme elutes near the end
of the gradient. The L-cysteine in the high salt results in
removal of the bound PLP by forming a thiazolidine complex with the
PLP, which has a low affinity for the enzyme. The eluted apoenzyme was
precipitated with ammonium sulfate (75% of saturation), redissolved,
and dialyzed against the 20 mM potassium phosphate buffer with
5 mM 2-mercaptoethanol. The apoenzyme was stored at 0 °C
for no more than 3 days before use.
Equilibrium unfolding and refolding experiments were performed by adding a concentrated solution of either native apo- or holo-eSHMT or the enzyme in 8 M urea to a series of urea concentrations (0.2-8 M) in the Tris-HCl buffer described above. These solutions were incubated for 2 h at 30 °C or 20 h at 4 °C before analysis by CD and fluorescence spectrometry and determination of catalytic activity.
The goal of these studies was to determine if in the folding of eSHMT there are folding intermediates and which of these intermediates bind PLP. A second question was whether PLP assists in forming the native enzyme during folding. Both equilibrium and kinetic studies were done using fluorescence properties of the three native Trp residues, the far-UV CD spectral properties of the secondary structure of the polypeptide, and the visible CD signal of the bound PLP. Experiments were done at both 30 and 4 °C. The low temperature studies are important in determining the binding of PLP to intermediates on the folding pathway.
Figure 1: CD and fluorescence properties of native and unfolded eSHMT. A, far-UV CD spectra of native holo-eSHMT (curve 1), apo-eSHMT (curve 2), and the enzyme in 8 M urea (curve 3). B, visible CD spectra of native holo-eSHMT (curve 4), apo-eSHMT (curve 5), and enzyme in 8 M urea (curve 6). Protein concentration was 0.15 mg/ml for the far-UV CD spectral studies and 0.6 mg/ml for visible CD studies. Both were in 20 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 7.5, buffer at 30 °C. C, fluorescence emission spectra of native holo-eSHMT (curve 7), native apo-eSHMT (curve 8), and eSHMT in 8 M urea (curve 9). Excitation was at 290 nm and protein concentration was 0.05 mg/ml in 20 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 7.5, at 30 °C.
The fluorescence of Trp residues decrease in both apo- and holo-eSHMT with unfolding, and the maximum emission wavelength increases from 335 to 352 nm (Fig. 1C). This shift in emission wavelength is characteristic of exposing buried Trp residues to an aqueous environment(19) . The fluorescence of the apoenzyme is significantly greater than the native holoenzyme, suggesting that enzyme-bound PLP is quenching Trp fluorescence. This was verified by showing that the decrease in Trp fluorescence coincides with PLP binding to the apoenzyme as determined by forming catalytically active holoenzyme. Both the visible CD signal at 420 nm and Trp fluorescence quenching at 335 nm can be used to monitor the binding of PLP to the apoenzyme.
The unfolding of eSHMT is reversible, since complete enzymatic activity and the native CD spectrum can be recovered after refolding. In addition, the same equilibrium transition profile of Trp fluorescence emission at different urea concentrations was obtained starting from either unfolded or native eSHMT (data not shown).
The rate of unfolding of apo-eSHMT was followed by both far-UV CD and fluorescence measurements. When native apoenzyme was diluted 20-fold into a solution of 8.3 M urea at 4 °C 90% of the change in optical activity at 220 nm occurred in the 10-15 s it took to obtain a measurement. After 30 s in the 8 M urea solution, the optical activity at 220 nm was the same as fully denatured eSHMT (data not shown). In a similar experiment the rate of decrease in fluorescence was monitored when apo-eSHMT was added to an 8 M urea solution at 4 °C. The fluorescence decreased to the same level as fully denatured eSHMT in less than 100 s (Fig. 3, curve 3). The ability to denature the protein rapidly at low temperature is an important factor, since studies to be described later suggest that cis-trans isomerization of some of the 19 proline residues in the enzyme slows rapid refolding to a fully active protein. This problem can be circumvented by rapid unfolding at 4 °C and starting refolding before proline isomerization can occur to a significant extent.
Figure 3: Changes in fluorescence properties during unfolding and refolding of apo-eSHMT. Final protein concentration was 0.05 mg/ml, excitation was 290 nm, and emission measurements were taken at 335 nm. The refolding reaction was a 10-fold dilution of an unfolded apo-eSHMT (0.5 mg/ml equilibrated in 8 M urea for 2 h at 30 °C) into 20 mM Tris-HCl, pH 7.5 (curve 1), and in the presence of 5 µM PLP (curve 2). A sample of 0.05 ml/mg unfolded enzyme in 8 M urea is shown as the zero time point. Curve 3 is the unfolding reaction that was initiated by diluting 20 µl of a 2.6 mg/ml native apo-eSHMT solution to 1 ml of 8 M urea in a Tris-HCl buffer, pH 7.5, at 4 °C. A sample of 0.05 ml/mg native apoenzyme is shown as the zero time point.
Figure 2:
Urea-induced unfolding of eSHMT. Either
native apo- or holo-eSHMT, 1.5 mg/ml, was diluted 10-fold into a series
of urea solutions at the indicated concentrations and temperature, with
20 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 7.5. After
2 h at 30 °C or 20 h at 4 °C, the CD spectrum, fluorescence
emission spectrum, and enzymatic activity of each solution were
determined. A, CD signal and activity changes observed at 30
°C: , CD
of apo-eSHMT;
, CD
of holo-eSHMT; ▴, CD
of holo-eSHMT;
, enzymatic activity. F
is the apparent
fraction of native protein as defined by F
= (Y
- Y
)/(Y
- Y
). Y
is the observed value of the
parameter, and Y
and Y
are the values of the
parameter for the unfolded and native protein, respectively. B, fluorescence changes at different temperatures:
,
apo-eSHMT at 30 °C;
, apo-eSHMT at 4 °C;
,
holo-eSHMT at 30 °C; ▴, holo-eSHMT at 4 °C. Fluorescence
excitation was at 290 nm and fluorescence emission monitored at 335
nm.
Trp fluorescence properties of the samples described in Fig. 2A are recorded in Fig. 2B for holo- and apoenzyme at both 30 and 4 °C. There is little difference in the fluorescence properties of apo-eSHMT at 4 and 30 °C as a function of urea concentration. The curve is not smooth showing a small but reproducible shoulder at about 1.8 M urea. The studies at 4 °C were allowed to equilibrate for 20 h.
The fluorescence properties of the holoenzyme in the presence of a 3-fold excess of PLP are different with respect to temperature. At 30 °C the fluorescence signal increases from a value that is about one-third that exhibited by native apoenzyme at 0.2 M urea to a value where they are equal in fluorescence at 2 M urea. As noted above, bound PLP quenches the Trp fluorescence of eSHMT, therefore at 1-2 M urea a conformational change takes place in holo-eSHMT, which results in loss of bound PLP accounting for the increase in fluorescence to the value observed for apoenzyme. At 4 °C the transition for holoenzyme, which corresponds to the loss of PLP, is shifted to lower urea concentration. The holoenzyme attains the fluorescence properties of the apoenzyme at 1.7 M urea. This loss of bound PLP at lower temperature was confirmed by the disappearance of the CD signal at 420 nm.
The shape of the equilibrium unfolding profile, shown in Fig. 2, varies with both enzyme concentration and the addition of amino acid substrates. Lower enzyme concentrations result in a decrease for fluorescence changes at lower urea concentrations. However, the urea concentration for reaching the final unfolded state is not significantly changed at lower enzyme concentrations. Addition of L-serine shifts the unfolding curve to slightly higher urea concentrations, suggesting that the PLP external aldimine at the active site helps stabilize the holoenzyme from unfolding in urea.
To investigate the oligomeric state of the protein, 0.15 mg/ml holoenzyme was equilibrated with 0-3 M urea at 30 °C for 2 h and aliquots injected into a HPLC sizing column equilibrated with the corresponding urea concentration. The elution volume of the enzyme did not change up to 2 M urea and was characteristic of a protein with a size of 95 kDa. This indicates that eSHMT remains a dimer up to about 2 M urea. Therefore, the conformational change that results in loss of PLP takes place at lower urea concentrations then dimer dissociation. Above 3 M urea the HPLC elution profile becomes broadened with no defined absorption peaks at 280 nm. This suggests that the equilibration between dimers, monomers, and unfolded species occurs faster than their separation on the column.
When PLP is added to the refolding buffer, there is an additional change in the fluorescence of Trp residues (Fig. 3, curve 2). After nearly reaching the maximum fluorescence of apoenzyme, there is a slower decrease in fluorescence until the value of native holo-eSHMT is reached. This decrease in fluorescence is the result of the quenching of Trp fluorescence upon formation of holoenzyme (Fig. 1C, curve 2). The rate of this slower phase increased as the PLP concentration increased in the refolding solution, further verifying this phase results from PLP binding (data not shown). The rapid increase in fluorescence to the value of native apo-eSHMT, followed by the slow decrease in fluorescence to the level of native holo-eSHMT observed in these refolding studies, suggests that PLP binding occurs very late in the folding process.
When unfolded eSHMT in 8 M urea is diluted 10-fold at 4 °C, the CD spectrum at 220 nm returns to the value of native apo-eSHMT in less than 30 s. A full CD spectrum, which requires several minutes, shows that the spectrum of native apo-eSHMT has also returned (data not shown). This is in contrast to the changes in fluorescence during refolding at 4 °C which takes 10 min to reach the values exhibited by native apoenzyme. However, as observed at 30 °C, more than 50% of the fluorescence changes occur within the 5 s it takes to measure a signal. These results suggest that secondary structure and some tertiary structure return rapidly upon dilution of the unfolded enzyme to 0.8 M urea at 4 °C, but that forming all of the tertiary structure characteristic of native apoenzyme is much slower than at 30 °C.
Figure 4:
Rate of folding of eSHMT monitored by
reactivation. Folding was initiated by a 10-fold dilution of enzyme in
8 M urea into 20 mM Tris-HCl containing 0.1 mM PLP, 50 mM glycine, 0.2 mM 5-CHO-HPteGlu, 5 mM 2-mercaptoethanol, pH
7.5, at 30 and 4 °C. The lines are the absorbance at 502 nm, due to
the formation of a correctly folded
eSHMT
Gly
5-CHO-H
PteGlu complex, as a function of
time. Curve 1 is the rate of formation of the complex after
addition of native apo-eSHMT to the refolding assay solution at 30
°C. Curve 2 is for apo-eSHMT that had been unfolded in 8 M urea for 2 min at 4 °C before dilution into refolding
buffer at 30 °C. Curve 3 is for apo-eSHMT that had been
unfolded in 8 M urea for 20 min at 4 °C before dilution
into refolding buffer at 30 °C. Curve 4 is the same as the
conditions used to obtain curve 3, except the temperature of
refolding was at 4 °C.
If PLP was omitted from the refolding solution and added only to the assay solution in determining catalytic activity, we found essentially the same rate of return to native enzyme, including the lag time of about 20 s. Attempts were made to verify the effect of PLP on forming active holoenzyme by including trypsin (20 µg/ml) in the assay buffer to rapidly destroy enzyme that was still not completely folded, thus stopping the refolding process during the assay for active enzyme. Under these conditions the addition of PLP to the refolding buffer increased the rate of formation of active enzyme by about 30%, compared with refolding buffer not containing PLP. This could be the result of some of the more trypsin labile apoenzyme being digested in the assay solution before PLP converted it to holoenzyme. We conclude that the presence of PLP in the renaturation buffer does not significantly accelerate the rate of formation of active holo-eSHMT.
Dependence of the refolding rate on enzyme concentration was examined by the same method used to obtain curve 3 in Fig. 4. Neither a decrease of the lag period nor an increase in rate of appearance of absorbance at 502 nm was observed over a concentration range of 128-fold (0.014-1.8 mg/ml). These results show that dimerization is not rate controlling for either the reaction causing the 20-s lag phase or the formation of native holoenzyme. Above 0.8 mg/ml, the yield of holo-eSHMT decreased to a value of 70% at 1.8 mg/ml eSHMT. This suggests that at high unfolded enzyme concentrations used in this study, some aggregation occurs which blocks forming native enzyme.
Attempts were made to fit the rate of formation of native enzyme (as shown by curve 3 in Fig. 4) to a simple kinetic model. The results could not be fit by a model of two first-order reactions or a first-order reaction followed by a second-order reaction. The results suggest that the 20-s lag is not the result of a simple accumulation of an intermediate. Since proline isomerization can occur in the unfolded state an attempt was made to determine if this was the cause of the 20-s lag(20) . At 0 °C the rate of proline isomerization has been determined to have a half-life of 1000 s(21) . This suggests that at about 4 °C, greater than 90% of all prolines in the unfolded state will reach cis-trans equilibrium in 10 min, whereas at 100 s less than one-half-life will have occurred.
We investigated the effect of unfolding time in 8 M urea versus the lag time observed in the refolding studies. Curves 2 and 3 in Fig. 4compare enzyme that has been in 8 M urea at 4 °C for 100 s and 20 min, respectively, before initiating refolding at 30 °C. As noted in the unfolding studies, in 8 M urea at 4 °C the changes in the far-UV CD spectrum and Trp fluorescence are complete in 100 s. The lag period in forming holo-eSHMT at 30 °C is greatly decreased, but not eliminated, when enzyme has been in unfolding buffer at 4 °C for only 100 s (Fig. 3, curve 2). Increasing the time in unfolding buffer at 4 °C beyond 20-min results in no further increase in the lag time in refolding. These results suggest that the 20-s lag period in refolding is at least partly the result of proline isomerization. To further test this hypothesis, we added proline isomerase to the refolding buffer. At an optimum concentration of proline isomerase the lag period was reduced about 30%, but we could show no correlation between lag period and the concentration of this enzyme. This suggests that the 20-s lag seen in the refolding studies is the result of complex factors, which include proline isomerization and the build-up of an intermediate(s) on the refolding pathway.
To investigate the oligomeric state of the trapped intermediate,
unfolded enzyme was put in renaturation buffer at 4 °C and at
varying time intervals passed through a HPLC BIOSEP SEC 3000 molecular
sieve column, also at 4 °C. The earliest refolding time points show
that the enzyme is a mixture of two sizes (Fig. 5A).
Comparing these sizes with a standard curve, developed with known
molecular weight markers (Fig. 5B), shows that the
earliest eluting peak has an estimated size of 95 kDa, and the latter
peak corresponds to a protein with a size of about 45 kDa. This
suggests that the refolding solution contains a mixture of dimer and
monomer species. Increased length of time of refolding at 4 °C
results in a decrease in the amount of monomer with a concomitant
increase in the amount of dimer (Fig. 5A). After 60 min
of refolding at 4 °C, the enzyme is essentially in the dimeric
state. However, at this time point less than 10% of the absorbance at
502 nm returns when glycine and 5-CHO-HPteGlu are added.
These results suggest that both monomeric and dimeric forms of the
enzyme accumulate at 4 °C that are not yet fully formed native
enzyme.
Figure 5: Determination of the size of refolding intermediates of eSHMT at 4 °C. Unfolded eSHMT in 8 M urea was diluted in renaturation buffer at 4 °C. After initiation of refolding aliquots were passed through a HPLC BIOSEP SEC 3000 column equilibrated with 100 mM NaCl in the buffer. A, elution profiles of eSHMT after the indicated times of refolding at 4 °C. M and D` refer to the structures shown in Fig. SI. B, elution time of the two absorption peaks observed in A with respect to the elution time of a set of standard proteins.
Figure SI:
Scheme IMinimal mechanism showing
intermediates on the folding pathway of eSHMT. U and U
are the fast folding
species of unfolded enzyme with either all prolines in the correct
cis-trans isomer or a slower folding species with some isomerized
proline residues, respectively. MG is a rapidly formed
collapsed structure which contains considerable secondary and some
tertiary structure. M is a monomer which exhibits CD and
fluorescence properties similar to the native apoenzyme. D` is
a dimer which exhibits the CD and fluorescence properties of the native
apoenzyme, but does not bind PLP. NAD is the native apodimer. NHD is the native holodimer.
Figure 6:
Formation of holo-eSHMT as determined by
CD. Curve 1 is the addition of PLP to native
apo-eSHMT (0.6 mg/ml) at 30 °C. Curve 2 is the addition of
PLP to native apo-eSHMT at 4 °C. Curve 3 is the addition
of PLP to unfolded apo-eSHMT at 4 °C. At the arrow in curve 3, the temperature was increased rapidly to 30
°C.
When unfolded enzyme was added to renaturation buffer at 4 °C in the presence of PLP, essentially no increase in optical activity was observed for 60 min at 420 nm (curve 3). After 60 min of incubation at 4 °C, the temperature was increased to 30 °C and the optical activity appeared in only a few minutes.
Addition of PLP to the trapped intermediate was also investigated using the quenching of Trp fluorescence observed when PLP binds (Fig. 1A). When unfolded enzyme is added to renaturation buffer containing a 100-fold excess concentration of PLP at 4 °C, there is a rapid increase in Trp fluorescence to values characteristic of native apo-eSHMT, which remain unchanged for 100 min (data not shown). However, if refolding is done at 30 °C, there is a rapid increase in Trp fluorescence followed by a decrease to a fluorescence level characteristic of holoenzyme (Fig. 2, curve 2). These results again suggest that PLP does not bind to either the trapped monomeric or dimeric intermediate at 4 °C, but does bind to a dimeric species which is formed from the trapped intermediate at 30 °C.
Free PLP has a very reactive aldehyde group at the 4`-position which forms aldimines with primary amino groups on many proteins and amino acids. As one example, it was shown nearly 40 years ago that free PLP binds tightly to serum albumin(22) . Since that time many have used PLP as a probe for the presence of critical lysyl residues in a variety of enzymes. The vitamin form of PLP is pyridoxine, which contains the nonreactive alcohol at the 4` position. The enzymes pyridoxol kinase and pyridoxol phosphate oxidase convert this alcohol to the phosphorylated aldehyde form in the cell(23, 24) . A question that has not been adequately addressed is how the newly formed PLP from the pyridoxol phosphate oxidase reaction in the cell is targeted to newly synthesized PLP requiring apoenzymes. How does the cell target this reactive coenzyme rather than letting the PLP react with free amino acids, thiols, and other non-PLP proteins in the cell? As a first step in addressing this question we have studied the mechanism of folding of eSHMT and determined at which step free PLP reacts to form holo-eSHMT.
Fig. SIrepresents the minimal mechanism that can account for the observations recorded in this study. eSHMT is reversibly unfolded by 8 M urea. The rate of unfolding occurs in less than 2 min even at temperatures as low as 4 °C (Fig. 2, curve 3). From fluorescence and CD measurements there is no evidence for any significant remaining secondary or tertiary structure (Fig. 1). Both fluorescence and CD measurements suggest that reducing the urea concentration to 0.8 M results in a rapid return of the secondary and tertiary structure characteristic of the native apoenzyme. Fluorescence measurements suggest that the return of the native environment of the 3 Trp residues is at least biphasic with a burst phase occurring in a few seconds followed by a slower phase which takes up to 2 min at 30 °C. In our proposed mechanism, we suggest that the burst increase in fluorescence is the collapse of the unfolded structure to a monomer which would have the characteristics of a molten globule and is listed in Fig. SIas MG. This form then undergoes further conformational changes to form a monomer which has the CD and fluorescence characteristics of the native apoenzyme, we refer to it as M in Fig. SI. Reactions 1 and 2 both occur rapidly, being complete in less than 2 min at 30 °C.
The refolded monomer M has the CD and fluorescence characteristics similar to those of the native apoenzyme. At 4 °C, M is the first dominant species formed as shown by HPLC size exclusion chromatography (Fig. 5). M forms dimers, shown as D` in Fig. SI, over a period of 60 min at 4 °C and probably several minutes at 30 °C. Reaction 3 is not the rate-determining step at protein concentrations as low as 0.014 mg/ml. D` can be observed in the HPLC experiments to exist for hours at 4 °C. It is clear from fluorescence properties and CD measurements at 420 nm that neither M nor D` binds free PLP (Fig. 6).
The intermediate D` is converted into a form which can bind PLP at 30 °C. The simplest interpretation for this step is that an isomerization takes place which affects the active site of D` to generate the native apoenzyme, listed as NAD in Fig. SI. This isomerization is the rate-determining step in forming holoenzyme. The final step is the addition of PLP to NAD to form the native homodimer, listed as NHD in Fig. SI. We conclude that in the refolding pathway of eSHMT the domain required for forming intersubunit contacts occurs more rapidly than forming the domain for binding PLP. We also conclude that PLP binds only near the very end of the folding pathway and does not play any significant role in the rate determining steps where M and D` are converted to NAD (Fig. SI).
The formation of U` in Fig. SIindicates that proline isomerization of some of the 19 proline residues in the unfolded state has some effect on the rate of refolding(20) .
Considerable evidence suggests that at least two separate classes of PLP enzymes have evolved. The major class includes transaminases, SHMT, and probably most decarboxylases(17) . These are all expected to have related three-dimensional structures. It will be informative to determine if a common folding mechanism has been preserved in this group of enzymes. The reversible folding of several members has been demonstrated(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) , and studies suggest that for several of these proteins the addition of PLP occurs near the end of the folding process. However, in only one case has the binding of PLP been studied in detail with respect to folding intermediates. Several studies have confirmed that on the refolding pathway of aspartate aminotransferase a monomeric form exists which can bind PLP prior to forming the native apodimer(10, 12) . This is very different from what we observe with eSHMT. The aspartate aminotransferase studies conclude that the PLP binding domain forms faster then the domain involved in intersubunit contacts. We conclude that with eSHMT the domain for forming the intersubunit contacts forms more rapidly than the PLP binding domain. These observations may not represent a major difference in folding pathways between aspartate aminotransferase and eSHMT, but only reflect a difference in relative rates of similar steps.
The
most extensive studies on folding of a PLP enzyme have been done with
bacterial tryptophan synthase which is a member of the second class of
PLP
enzymes(5, 6, 9, 10, 25, 26, 27) .
It has been demonstrated that a non-native dimer of the subunit
is an obligatory intermediate on the folding pathway(25) .
Evidence suggests that PLP does not bind to the monomer and binds to
the dimer only after a conformation change occurs to form the native
apoenzyme(5, 25, 26) . It has also been shown
recently that PLP and its substrate complexes play an important role in
stabilizing the contacts between the
and
subunits(27) . The mechanism proposed for the folding pathway
of the
subunit of tryptophan synthase and the binding of PLP are
almost identical to our findings with eSHMT.