From the Institute of Biochemical Sciences and the
National Institute for the Physics of Matter, University of
Parma, 43100 Parma, Italy; § M. E. Müller Institute
for Structural Biology, Biozentrum University of Basel, CH-4056, Basel,
Switzerland; and the ¶ Department of Pediatrics, University of
Colorado School of Medicine, Denver, Colorado 80262
Received for publication, August 29, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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Human cystathionine High plasmatic levels of homocysteine have recently been
associated with an increased risk of cardiovascular disease (1). Homocysteine is formed from S-adenosylhomocysteine and is
either removed by cystathionine Structural studies of catalytic intermediates of tryptophan synthase
and O-acetylserine sulfhydrylase (11-18) have unveiled the
nature of enzyme action. A common feature is an open to closed conformational transition of the active site taking place along the
catalytic pathway. To fully exploit the structural information as well
as to determine the structure of as many as possible catalytic intermediates, it is of paramount relevance to investigate the functional properties of the enzyme in the crystalline state by polarized absorption microspectrophotometry (19). This approach was
pioneered in the late sixties by Rossi and Bernhard (20). In the
case of tryptophan synthase (17, 21-23) and O-acetylserine sulfhydrylase (24), several catalytic intermediates were isolated and
characterized in the crystalline state, opening the way to their
structural determination. We have recently expressed and purified to
near homogeneity recombinant human CBS comprising amino acid residues
2-413. This enzyme, missing 138 C-terminal residues, forms dimers, is
not activated by S-adenosyl-L-methionine, and
does not exhibit the aggregating properties of the full-length enzyme.
In addition, the recombinant CBS polypeptide contains a 23-amino acid
spacer at its N terminus. The truncated enzyme still binds PLP and heme
and is about 2 times more active than the full-length CBS (25).
Crystals of the recombinant active core of CBS have been obtained, and
the three-dimensional structure is being determined (26). Here, we have
studied the reactivity of these crystals by polarized absorption
microspectrophotometry as an essential prerequisite to the
crystallographic analysis of the enzyme and the structure to function correlation.
Crystallization--
The recombinant truncated form of CBS was
purified as described previously (25) and concentrated to 26 mg/ml in
20 mM HEPES, pH 7.4. Crystals of CBS were obtained using
the vapor diffusion hanging drop method (26). 2 µl of mother liquor
containing 30% w/v PEG Mr 1000, 80 mM HEPES, pH 7.5, and 0.4 mM FeCl3
were mixed with 2 µl of protein solution and equilibrated against 1 ml of reservoir solution at room temperature. Given the dimeric nature of the truncated CBS enzyme, the crystals contained either two or three
dimers per asymmetric unit corresponding to a solvent content of 64 or
46% and a calculated Matthews volume VM of 3.4 or 2.3 Å3/Da, respectively. The crystals belong to the trigonal
space group P31 or P32 with unit cell
dimensions a = b = 144.46 Å and c = 108.21 Å (25).
Crystals were stored in 25% PEG Mr 1000, 80 mM HEPES, 2 mM ferric chloride, pH 8.0.
Chemicals--
All chemicals were of the best commercial quality
and were used without further purification. L-Homocysteine
was prepared from L-homocysteine thiolactone and titrated
with 5,5'-dithiobis-2-nitrobenzoic acid (27).
Microspectrophotometric Measurements--
Single crystals of CBS
were resuspended at least six times in a solution containing 35% PEG
Mr 1000, 80 mM HEPES, pH 8.0 and mounted in a quartz flow cell. Replacement of the suspending medium was
carried out by passing solutions through the cell. The cell was placed
on the thermostatted stage of a Zeiss MPM03 microspectrophotometer, equipped with a × 10 Zeiss UV-visible ultrafluar objective (19, 22). Polarized absorption spectra were collected between 300 and 700 nm
with the electric vector of the linearly polarized light parallel and
perpendicular to the c axis of the trigonal crystals. All
the experiments were carried out at 15 °C.
Oxidized and Reduced CBS Crystals--
The crystals of oxidized
CBS were suspended in a deoxygenated solution containing 35% PEG
Mr 1000, 80 mM HEPES, pH 8.0 and then in a solution of the same composition plus 5 mM sodium
dithionite. When sodium dithionite was removed, the crystals
re-oxidized completely within the time required for the solution
exchange. The spectrum of the oxidized form did not change when the
crystals were treated with 5 mM potassium ferricyanide.
Removal of Heme from CBS Crystals--
Crystals of CBS were
anaerobically suspended in 35% PEG, 80 mM HEPES, pH 8.0, 5 mM sodium dithionite, saturated with CO at 1 atm. Polarized
absorption spectra were recorded as a function of time on crystals
stored at 4 °C.
Re-binding of Heme to Heme-free Crystals--
Heme-free crystals
were anaerobically suspended in a deoxygenated solution containing 35%
PEG Mr 1000, 80 mM HEPES, pH 8.0, 5 mM sodium dithionite, and increasing concentrations of
hemin. Polarized absorption spectra were recorded as a function of time on crystals stored at 4 °C.
Measurements on Heme-free CBS Crystals--
Heme-free CBS
crystals were washed and stored in a CO-free solution of 35% PEG
Mr 1000, 80 mM HEPES, pH 8.0. Individual crystals were mounted in the flow cell and resuspended in
reagent before collecting spectra.
Redox States of CBS Crystals--
Polarized absorption spectra of
CBS crystals under nonreducing conditions (Fig.
1a) exhibited peaks at 428 and
550 nm, similar to solutions for the oxidized form of the enzyme. A
shoulder at 590 nm was more evident when spectra were collected with
light polarized parallel to the c crystallographic axis,
indicating an unequally polarized x, y electronic transition of the
heme (28). This was also reflected in the variation of the polarization ratio, i.e. the ratio of the absorption intensity parallel
and normal to the c axis of the crystal (Fig.
1a). The isotropic spectrum, calculated from the equation
When CBS crystals were suspended in a solution containing sodium
dithionite, polarized absorption spectra exhibited bands at 450, 538, and 576 nm (Fig. 1b). In solution these peaks are indicative
of the ferrous state of the heme iron. The shoulder at about 430 nm was
more prominent in the isotropic spectrum calculated from crystal
polarized absorption spectra (Fig. 1b, inset)
with respect to solution (5, 6). This finding might suggest an incomplete reduction. However, in the isotropic spectrum (Fig. 1b, inset), the ratio of the peaks at 450 and 576 nm was the same as in solution (5, 6), indicating that the crystalline
enzyme was fully reduced. Heme reduction takes place without any
apparent crystal damage. Furthermore, when reduced enzyme crystals were resuspended in a dithionite-free solution, the fully oxidized form
of the enzyme was readily obtained (data not shown). Therefore, it is
possible to prepare oxidized and reduced forms of CBS crystals suitable
for crystallographic analysis. This study may allow us to determine the
conformational changes associated with different heme redox states
controlling catalytic properties of PLP catalysis (6).
Formation of Heme-free CBS Crystals--
Crystals of reduced CBS
were suspended in a solution containing 5 mM sodium
dithionite, saturated with carbon monoxide at 1 atm. The spectra
collected immediately after exposure to CO showed a progressive
decrease of absorbance intensity without any change of the spectral
shape (Fig. 2). Prolonged incubation of
CBS crystals under these experimental conditions resulted in the
complete loss of the absorption intensity of the heme (Fig. 2). This
process takes place in 2-3 days depending on the crystal size. The
resulting polarized absorption spectra (Fig. 2) were characterized by
an absorption peak at 412 nm, typical of a PLP Schiff base, as observed
in other PLP-dependent enzyme crystals (22, 24). Some
spectral differences among CBS crystals were observed in the 300-340
nm region likely because of crystal aging, as previously observed in
other PLP-enzymes.2 These
findings indicate that under these conditions the heme groups that were
bound to CBS had been completely released. When the oxidized form of
the heme-containing enzyme was exposed to carbon monoxide, no release
of heme was observed (data not shown). Furthermore, reduction of the
iron or other protein groups, i.e. disulfide bridges, by
dithionite did not trigger heme release (data not shown). The presence
of either dithiothreitol or nitric oxide was also ineffective in heme
release. These results indicate that carbon monoxide binds only to the
reduced form of CBS, as observed in solution (30), and the weakening of
heme affinity to the enzyme is very specific. Carbon monoxide likely
replaces a protein residue bound as an axial ligand to the iron,
decreasing significantly the heme affinity to the protein.
Alternatively, carbon monoxide-bound heme triggers displacement of iron
into the heme plane, which in turn may be transmitted to the protein matrix by movement of the second heme axial ligand, similar to that
observed in hemoglobin (31).
The heme release is compatible with the crystal integrity, as evidenced
by the significant difference of absorption intensity of heme-free CBS
crystals along the two optical directions (compare Fig. 2, a
and b), a property of well ordered chromophoric crystals. The heme release is reversible, because incubation of heme-free CBS
crystals with 0.3 mM reduced hemin led to the re-binding of hemes with the retention of the original polarization ratios (data not
shown). On the basis of the spectra of the native and reconstituted enzyme, it was estimated that about 50% of heme content was recovered. Higher concentrations of hemin might be required to attain a
stoichiometric recovery. These findings enable structural determination
of the enzyme in the presence and absence of heme, and, thus,
characterization of the conformational changes associated with heme
binding and its regulation of PLP catalysis.
Reactivity of Heme-free CBS Crystals--
In solution, CBS reacts
with L-serine to form the
Finally, when
Overall, these findings indicate that the heme-free enzyme is
catalytically competent not only in the Cryo-crystallography (33) and kinetic crystallography (34), making
use of either slowly reacting substrates and substrate analogues (20)
or slowly reacting mutant enzymes (35), have considerably expanded the
capability to detect and characterize not only the native form of
enzymes and proteins but also transiently accumulating species.
Detailed functional studies of protein crystals by spectroscopic
techniques have allowed us to define the experimental conditions for
the accumulation of catalytic intermediates, thus directing the
crystallographic measurements. These conditions are not always similar
to those derived by experiments in solution, because crystal lattice
forces affect the relative stability of catalytic intermediates in
unpredictable ways. Examples are the different effect of cations on the
accumulation of the quinonoid species of tryptophan synthase in the
crystal and in solution (22) and the different affinity of the natural
substrate O-acetylserine to several crystal forms of
O-acetylserine sulfhydrylase, where one form is 500-fold
less active than the enzyme in solution and another is completely
inactive (24).
The present investigation of the active core of human CBS crystals has
allowed to prepare the oxidized and reduced forms of the enzyme, the
heme-free protein, and the key catalytic intermediate -synthase is a
pyridoxal 5'-phosphate enzyme containing a heme binding domain and an
S-adenosyl-L-methionine regulatory site. We
have investigated by single crystal microspectrophotometry the
functional properties of a mutant lacking the
S-adenosylmethionine binding domain. Polarized absorption
spectra indicate that oxidized and reduced hemes are reversibly formed.
Exposure of the reduced form of enzyme crystals to carbon monoxide led
to the complete release of the heme moiety. This process, which takes
place reversibly and without apparent crystal damage, facilitates the
preparation of a heme-free human enzyme. The heme-free enzyme crystals
exhibited polarized absorption spectra typical of a pyridoxal
5'-phosphate-dependent protein. The exposure of these
crystals to increasing concentrations of the natural substrate
L-serine readily led to the formation of the key catalytic
intermediate
-aminoacrylate. The dissociation constant of
L-serine was found to be 6 mM, close to that
determined in solution. The amount of the
-aminoacrylate Schiff base
formed in the presence of L-serine was pH independent
between 6 and 9. However, the rate of the disappearance of the
-aminoacrylate, likely forming pyruvate and ammonia, was found to
increase at pH values higher than 8. Finally, in the presence of
homocysteine the
-aminoacrylate-enzyme absorption band readily
disappears with the concomitant formation of the absorption band of the
internal aldimine, indicating that cystathionine
-synthase crystals
catalyze both
-elimination and
-replacement reactions. Taken
together, these findings demonstrate that the heme moiety is not
directly involved in the condensation reaction catalyzed by
cystathionine
-synthase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
-synthase (EC 4.2.1.22,
CBS)1 in the
trans-sulfuration pathway or remethylated to methionine in the
methionine cycle. Deficiency of CBS is the major cause of inherited
homocystinuria. CBS is a pyridoxal 5'-phosphate
(PLP)-dependent enzyme catalyzing the synthesis of
cystathionine from homocysteine and L-serine. The reaction
proceeds via a
-replacement mechanism, similar to that of tryptophan
synthase and O-acetylserine sulfhydrylase (2). These enzymes
belong to the
-family and fold II type within the
PLP-dependent enzymes classification (3, 4). Other members
of the
-family are serine and threonine dehydratases. The human CBS
is a 63-kDa homotetramer containing one PLP and one heme per subunit
(5). Whereas the functional role of PLP is known, the role of the heme
is less clear. It was demonstrated that the heme redox state affects
the affinity of the enzyme for the substrates (6). Moreover, the
catalytic activity of CBS is controlled by
S-adenosylmethionine, which specifically binds to a
C-terminal site. The trypsinolysis of a 18 kDa C-terminal fragment
leads to a dimeric form, 2-fold more active than the native species and
no longer regulated by S-adenosylmethionine (7). Similar
results have been obtained on other truncated forms of CBS, obtained by
insertion of nonsense mutations (8). Investigation of the catalytic
reaction brought about by PLP is complicated by the overlapping
chromophoric properties of the heme. The recent investigation of the
yeast enzyme, which does not contain heme groups, permitted a better
characterization of the PLP role in the catalytic steps (9, 10). An
-aminoacrylate species, absorbing at 470 and 320 nm, was observed
upon reaction with L-serine. The nucleophilic attack of
homocysteine on the
-aminoacrylate led to the formation of the
product cystathionine. However, it is not yet known how closely the
functional properties of the yeast enzyme resemble those of the human source.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
= 1/3(
c+2
c)
exhibited the same ratio of absorbance intensity at 428 and 550 nm as
in solution (Fig. 1a, inset). Oxidized CBS
crystals, titrated between pH 6.0 and 8.0 (data not shown), did not
exhibit any significant spectral changes, suggesting that the heme iron
is not coordinated to a water molecule, which is different from what
was observed for metmyoglobin and methemoglobin (29).
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Fig. 1.
Polarized absorption spectra of 45-kDa CBS
crystals in the ferric (a) and ferrous
(b) state. A single crystal of CBS was suspended
in a solution containing 35% (w/v) PEG Mr 1000, 80 mM HEPES, pH 8.0. The ferrous state was obtained by
resuspending the crystal in a deoxygenated solution containing 2 mM sodium dithionite. Spectra were recorded with light
linearly polarized parallel (solid line) and normal
(dashed line) to the c crystal axis at 15 °C.
The polarization ratio (P.R.) is reported in the
top of each panel. Inset, calculated isotropic
spectrum (see "Results and Discussion").
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Fig. 2.
Formation of heme-free crystals in the
presence of carbon monoxide under reducing conditions. Crystals of
CBS were suspended in a solution containing 35% (w/v) PEG
Mr 1000, 80 mM HEPES, 2 mM sodium dithionite, pH 8.0. Crystal size does not permit
collecting reliable spectra of the Soret peak parallel (a,
solid line) and normal (b, solid line)
to the c crystal axis. The crystal was then anaerobically
suspended and stored at 4 °C in a solution of the same composition,
previously equilibrated with carbon monoxide at 1 atm. The spectra of
the heme-free CBS crystal were collected after 48 h (a
and b, dashed lines).
-aminoacrylate intermediate
(9, 32). This species absorbs at about 460 and 330 nm (9). Crystals of
heme-free CBS were titrated with increasing concentrations of
L-serine. The spectra at high L-serine
concentration (Fig. 3), recorded parallel
to the c crystal axis, show the appearance of bands centered
at about 450 and 320 nm, indicating the accumulation of the
-aminoacrylate Schiff base. Therefore, the enzyme is catalytically
competent in the
-elimination reaction. The spectral changes
observed for light polarized normal to the c crystal axis
are limited. There is a small red shift of the peak and a decrease of
absorption intensity. The high polarization ratio around 450 nm
reflects the spectral differences along the two extinction directions
(Fig. 3). This behavior might be explained by a reduced reactivity of
one of the coenzymes, as suggested in solution for the dimeric CBS (30) and assuming that most of the inactive PLP is observed along the direction normal to the c crystal axis. This partial
reactivity does not seem to be present in the yeast enzyme (9). The
calculated dissociation constant of L-serine for CBS
crystals is 6.4 mM (Fig. 3, inset). In solution,
at pH 8.6, a Km value of 3.0 mM was
determined for the tetrameric CBS (7) and a Km value
of 2.7 mM for the dimeric CBS (7). The amount of
-aminoacrylate Schiff base that was accumulated in the presence of
500 mM L-serine was found to be pH-independent
between 6 and 9 (data not shown). However, the rate of the
-aminoacrylate disappearance, likely to form pyruvate and ammonia
(11, 12), increases at pH higher than 8. A bell-shape dependence on pH,
centered at pH 7.2, was previously observed for the accumulation of the
-aminoacrylate-O-acetylserine sulfhydrylase crystals
(24).
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Fig. 3.
Polarized absorption spectra of heme-free CBS
crystals in the absence and presence of
L-serine. The crystal was suspended in a
solution containing 35% (w/v) PEG Mr 1000, 80 mM HEPES, pH 8.0. Polarized absorption spectra were
recorded with the electric vector either parallel
(E//c) or normal (E c) to
the c crystal axis, in the absence (
) and
presence of 500 mM (
) L-serine. The
corresponding polarization ratio (P.R.) is reported.
Inset, changes of the ratio of absorbance at 470 and 411 nm
as a function of L-serine concentration, recorded with
light polarized parallel to the c crystal axis. The data
points are the average of the values obtained for two crystals in
different titration experiments. The curve through the points is the
fitting to a binding isotherm with a dissociation constant of 6.4 mM.
-aminoacrylate CBS crystals were suspended in a
solution containing 30 mM L-serine and 34 mM homocysteine, the polarized absorption spectra of the
internal aldimine species were readily recovered (Fig.
4). The exposure of CBS crystals to
homocysteine alone did not cause any spectral changes, as observed in
solution for the yeast enzyme (10), suggesting that also in the human
enzyme homocysteine does not form the external aldimine.
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Fig. 4.
Polarized absorption spectra of heme-free CBS
crystals in the absence and presence of L-serine
and homocysteine. Spectra were recorded for heme-free CBS crystals
suspended in a solution containing 35% (w/v) PEG
Mr 1000, 80 mM HEPES, pH 8.0, in the
absence (solid line), presence of 30 mM serine
(dashed line) and 30 mM L-serine and
34 mM homocysteine (dotted line). Spectra
recorded with the electric vector parallel to the c
direction are shown. Spectra of internal aldimines were scaled with
respect to the absorbance intensity at 412 nm to account for different
crystal thickness.
-elimination reaction but
also in the
-replacement reaction, in agreement with preliminary solution experiments.3 It is,
therefore, very unlikely that the heme plays a catalytic role in the
activation of homocysteine, as recently proposed (6). A quantitative
evaluation of native versus heme-free enzyme activity either
using a microcrystalline suspension (19) or the soluble form is planned.
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
-aminoacrylate. For the first time, it has also been demonstrated that the heme does not participate in PLP-dependent
catalysis of CBS. However, further investigations in solution are
required to assess the fine-tuning of ligand binding and catalysis by
the heme moiety.
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FOOTNOTES |
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* This study was supported by grants from the Italian Ministry of University and Scientific and Technological Research PRIN99 and National Research Council (to A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 39-0521-905138; Fax: 39-0521-905151; E-mail: biochim@unipr.it.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.C000588200
2 A. Mozzarelli, unpublished observations.
3 J. Kraus, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
CBS, cystathionine
-synthase;
PLP, pyridoxal 5'-phosphate;
PEG, polyethylene
glycol.
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