©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Affinity of Pyridoxal 5`-Phosphate for Folding Intermediates of Escherichia coli Serine Hydroxymethyltransferase (*)

(Received for publication, January 9, 1995; and in revised form, June 13, 1995)

Kang Cai Douglas Schirch Verne Schirch (§)

From the Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Ultrapure urea was purchased from Boehringer Mannheim. All coenzymes, amino acids, and buffers were obtained from Sigma and were of the highest grade available.

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.

Unfolding and Refolding of eSHMT

Unfolding experiments were carried out by a 20-fold dilution of concentrated solutions (20 to 30 mg/ml) of either apo- or holo-eSHMT into a solution of freshly prepared 8.3 M urea containing 20 mM Tris-HCl, pH 7.5, 5 mM 2-mercaptoethanol and 1 mM EDTA. The refolding rate and yield were examined with final urea concentrations of 0.2, 0.4, and 0.8 M. Although the rates of refolding were the same, the 0.8 M urea gave the highest yield of native enzyme. Therefore, a 1:10 dilution of unfolded protein into 20 mM Tris-HCl, pH 7.5, containing 5 mM 2-mercaptoethanol was used throughout the study to initiate refolding.

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.

Fluorescence and CD Measurements

Fluorescence spectra were taken on a Shimadzu 5000 fluorimeter with 5-nm slits for both excitation and emission spectra. The concentration of eSHMT was 0.15 mg/ml. CD spectra were taken on a Jasco J-500A spectropolarimeter. The path length of the optical cuvette was 1 cm for measurements at 420 nm and 0.1 cm for far UV measurements. Protein concentration for the 420 nm studies was 0.6 mg/ml, and the scan rate was 20 nm/min. For the far-UV measurements the protein concentration was 0.15 mg/ml, and the scan rate was 2 nm/min.

High Performance Liquid Chromatography

Analysis of the size of eSHMT during refolding experiments was performed on a HP 1090 HPLC with a BIOSEP SEC 3000 column at 4 °C. The column was equilibrated with the refolding buffer containing 0.8 M urea and 100 mM NaCl and calibrated with proteins with known molecular weights. Refolding was initiated by the same method as above, and 50-µl (7.5 µg of protein) aliquots were removed at several time intervals and injected onto the column. The flow rate was 0.5 ml/min, and the detection was determined from absorbance at 220 nm. As a control, the column was calibrated with the following proteins: cytochrome c (12 kDa), chymotrypsinogen (25 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), phosphorylase b (94 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa). Blue dextran (2000 kDa) was used to approximate the exclusion volume.

Enzyme Activity

Catalytic activity was determined using L-serine and tetrahydrofolate as substrates. The rate of product formation, methylene tetrahydrofolate, was continuously monitored at 340 nm by coupling with an excess of methylenetetrahydrofolate dehydrogenase and NADP(18) . The activity was measured as the initial rate of increase in absorbance at 340 nm. A second method used to determine the presence of active enzyme was to determine the formation of an abortive complex formed between holo-eSHMT, glycine (50 mM), and 5-formyltetrahydrofolate (0.2 mM). This complex exhibits a sharp absorption peak with a maximum at 502 nm and a molar absorption coefficient of 40 mM cm(18) . The absorption peak is the result of the loss of the alpha-proton of glycine bound as an imine to the active site PLP. The loss of the proton results in a resonance stabilized structure referred to as the quinonoid complex(18) .


RESULTS

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.

Unfolding of eSHMT

Both apo- and holo-eSHMT show similar far UV CD spectra, characteristic of a protein with both alpha-helix and beta-sheet secondary structure. This signal disappears when the enzyme is unfolded in 8 M urea (Fig. 1A). Unlike the far-UV CD spectra, the visible CD signal is different between apo- and holoenzyme, the latter has a unique peak at 420 nm due to the bound PLP. This peak also disappears upon unfolding of the enzyme (Fig. 1B).


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.



Equilibrium Studies

Fig. 2A shows the relative changes in the CD signal at 220 nm for both holo- and apoenzyme (0.15 mg/ml) incubated in increasing urea concentrations for 2 h at 30 °C. The transition midpoint is at 2.5 M urea. However, the transition of the CD band at 420 nm and enzyme activity have a midpoint of 1.6 M urea (Fig. 2A). This indicates that the equilibrium unfolding of this enzyme is not a simple two-state transition. Instead, a hidden transition, which is not obvious by the far-UV CD spectrum, occurs at a lower urea concentration.


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; bullet, CD of holo-eSHMT; ▴, CD of holo-eSHMT; , enzymatic activity. F is the apparent fraction of native protein as defined by F = (Y(u) - Y)/(Y(u) - Y(n)). Y is the observed value of the parameter, and Y(u) and Y(n) 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; bullet, 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.

Fluorescence and CD Properties during Refolding of eSHMT

A 10-fold dilution of unfolded eSHMT at 30 °C results in an increase in Trp fluorescence that occurs in several stages. For apoenzyme, there is a rapid burst in the increase in fluorescence intensity as well as a blue shift in maximum emission wavelength which occurs in the time it takes to make the first measurement (5 s). This is followed by a slower increase without a further blue shift, lasting about 2 min, to a value that approaches the fluorescence of the native apoenzyme (Fig. 3, curve 1).

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.

Rate and Extent of Refolding to Form Native eSHMT

The extent of refolding of denatured eSHMT at 30 °C was determined by both catalytic activity measurements and the formation of a complex between holo-eSHMT, glycine, and 5-CHO-H(4)PteGlu. This complex has a unique and intense absorption maximum at 502 nm(18) . 5-CHO-H(4)PteGlu is a competitive dead-end inhibitor of the substrate 5,10-CH(2)-H(4)PteGlu, and the formation of this complex indicates a functional active site. The advantage of using this complex as a measure of refolding is that it permits a continuous monitoring of the formation of active enzyme by observing the absorbance at 502 nm. Fig. 4(curve 3) shows the return of absorbance at 502 nm after a 10-fold dilution of unfolded eSHMT, which has been incubated in 8 M urea for 20 min at 4 °C, in the presence of 0.1 mM PLP, 50 mM glycine, and 0.2 mM 5-CHO-H(4)PteGlu. After a lag period of about 20 s, the native enzyme rapidly reappears to absorbance values which ranged between 100 and 115% of the absorbance at 502 nm obtained with the addition of PLP directly to native apo-eSHMT (Fig. 4, curve 1). Aliquots were removed at several time points and assayed for catalytic activity. Within the time constraints of the measurements, activity returned at the same rate as the absorbance at 502 nm (data not shown).


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-H(4)PteGlu, 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 eSHMTbulletGlybullet5-CHO-H(4)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.

Trapping a Folding Intermediate at 4 °C

Refolding at 4 °C exhibits dramatic differences compared with the refolding studies done at 30 °C as recorded in curves 2 and 3 in Fig. 4. The formation of active holoenzyme at 4 °C, as recorded in Fig. 4(curve 4), results in a much slower rate of formation of holo-eSHMT. Even after 1 h of refolding at 4 °C less than 10% of the absorbance at 502 nm has appeared. After 20 h about 90% of the activity of native holoenzyme has returned. After a few minutes of refolding at 4 °C, the far-UV CD spectrum is the same as native holo-eSHMT. Also, about 90% of the Trp fluorescence has returned. Raising the temperature of the 4 °C renaturation solution to 30 °C after 10 min results in the rapid return of active holo-eSHMT at a rate similar to the rate of refolding at 30 °C (data not shown). These results suggest that at 4 °C an intermediate(s) has been trapped which is on the folding pathway. The overall refolding rate is about 50-fold slower at 4 °C compared with 30 °C, suggesting the energy barrier of a key step was increased by at least 5 kcal/mol. The Trp fluorescence and CD spectra of this intermediate suggest that it contains essentially all of the secondary and most tertiary structure of the native apo-eSHMT.

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-H(4)PteGlu 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.



Determining Which Intermediates Can Bind PLP

The ability to trap both monomeric and dimeric intermediates at 4 °C permits an investigation of which intermediate(s) can bind PLP. As mentioned above, two methods can be used to detect PLP binding. First, PLP bound at the active site of eSHMT elicits optical activity at 420 nm as the result of an asymmetric environment. Neither free PLP, apoenzyme, nor unfolded enzyme elicits this optical activity. Second, bound PLP quenches the fluorescence of Trp residues of the apoenzyme. Fig. 6shows the optical activity at 420 nm after the addition of PLP to either native apo-eSHMT at 30 °C (curve 1) or 4 °C (curve 2). At 30 °C the increase in optical activity at 420 nm is complete in 3 min, whereas at 4 °C optical activity increases more slowly, reaching about 90% of its maximal change in 60 min. This experiment shows that native apo-eSHMT can bind PLP at 4 °C.


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.


DISCUSSION

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 beta 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 alpha and beta subunits(27) . The mechanism proposed for the folding pathway of the beta subunit of tryptophan synthase and the binding of PLP are almost identical to our findings with eSHMT.


FOOTNOTES

*
This work was supported by Grant GM 28143 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 804-828-9482; Fax: 804-828-1473.

(^1)
The abbreviations used are: PLP, pyridoxal 5`-phosphate; 5-CHO-H(4)PteGlu, 5-formyltetrahydropteroylglutamate; HPLC, high pressure liquid chromatography; eSHMT, E. coli serine hydroxymethyltransferase.


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