From the Department of Biochemistry and
the ¶ Department of Chemistry, University of Nebraska, Lincoln,
Nebraska 68588-0664 and the § Department of Plant Biology,
Arizona State University, Tempe, Arizona 85287-1601
Received for publication, January 2, 2001, and in revised form, February 13, 2001
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ABSTRACT |
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Two classes of cystathionine Cystathionine -synthases have
been identified in eukaryotes, the heme-independent enzyme found in
yeast and the heme-dependent form found in mammals. Both
classes of enzymes catalyze a pyridoxal phosphate
(PLP)-dependent condensation of serine and homocysteine to
produce cystathionine. The role of the heme in the human enzyme
and its location relative to the PLP in the active site are unknown.
31P NMR spectroscopy revealed that spin-lattice
relaxation rates of the phosphorus nucleus in PLP are similar in both
the paramagnetic ferric (T1 = 6.34 ± 0.01 s) and
the diamagnetic ferrous (T1 = 5.04 ± 0.06 s)
enzyme, suggesting that the two cofactors are not proximal to each
other. This is also supported by pulsed EPR studies that do not provide
any evidence for strong or weak coupling between the phosphorus nucleus
and the ferric iron. However, the 31P signal in the
reduced enzyme moved from 5.4 to 2.2 ppm, and the line width decreased
from 73 to 16 Hz, providing the first structural evidence for
transmission to the active site of an oxidation state change in the
heme pocket. These results are consistent with a regulatory role
for the heme as suggested by previous biochemical studies from our
laboratory. The 31P chemical shifts of the resting forms of
the yeast and human enzymes are similar, suggesting that despite the
difference in their heme content, the microenvironment of the PLP is
similar in the two enzymes. The addition of the substrate, serine,
resulted in an upfield shift of the phosphorus resonance in both
enzymes, signaling formation of reaction intermediates. The resting
enzyme spectra were recovered following addition of excess
homocysteine, indicating that both enzymes retained catalytic activity
during the course of the NMR experiment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-synthase catalyzes the condensation of serine
and homocysteine to form cystathionine in a pyridoxal phosphate (PLP1)-dependent
reaction. The mammalian enzyme is novel in its dependence on a second
cofactor, heme, which distinguishes it from all other members of the
PLP family of enzymes in which this combination of cofactors is not
seen (1). In addition, the mammalian enzyme is allosterically activated
by S-adenosylmethionine (2), a regulatory feature
that is lacking in the related yeast enzyme (3). The reaction is
postulated to involve a series of PLP-bound intermediates that are
analogous to other PLP enzymes that catalyze
-replacement
reactions (Fig. 1). Thus, the addition of
serine results in a transaldimination reaction in which the Schiff
base-forming lysine in the internal aldimine is replaced by serine to
form the external aldimine. Binding of the second substrate,
homocysteine, is followed by abstraction of the
-proton of serine
and
-elimination of water to form the
-aminoacrylate
intermediate, which is poised for nucleophilic addition by the thiolate
of homocysteine. A second transaldimination reaction results in product
release and regeneration of the resting enzyme. Fluorescence
spectroscopy provides evidence for this scheme (4, 5). Formation of the
aminoacrylate species in the ternary rather than the binary complex is
supported by a very low level of tritium washout from the
-carbon of
serine in the absence of homocysteine and a significant enhancement of this exchange in the presence of homocysteine (6). In contrast, the
yeast enzyme appears to utilize a ping-pong mechanism, and the
-aminoacrylate intermediate is observed in the presence of serine
(7).
View larger version (17K):
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Fig. 1.
Postulated reaction mechanism of
cystathionine -synthase.
Sequence analysis of the N-terminal domain of cystathionine
-synthase revealed a phylogenetic relationship to a number of other
PLP-dependent enzymes, notably O-acetylserine
sulfhydrylase, tryptophan synthase, and threonine deaminase, and led to
the assignment of the PLP binding pocket to the region extending
between residues 68 and 209 in the human sequence (5). The alignment of
the yeast and human cystathionine
-synthases with conserved active site residues that interact with PLP in O-acetyl serine
sulfhydrylase indicates that the region of homology is in fact more
extensive and extends between residues 85 and 350 in the human sequence (Fig. 2). This assignment is confirmed by
the identification of Lys-119 as the residue involved in Schiff base
formation with the PLP in the resting enzyme (8). Weak sequence
similarity between a few hemeproteins and the central region of
cystathionine
-synthase extending between residues 241 and 341 suggested that this region may be important in heme binding (5).
However, the recent recognition that the closely related yeast enzyme
lacks heme as well as a 66-residue N-terminal extension suggests that the N terminus of the human enzyme is involved in heme binding. Resolution of the question of whether or not the central region is also
involved in the architecture of the heme pocket in the tertiary
structure awaits solution of the crystal structure of the
protein.
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Regardless of the location of the heme binding domain along the primary
sequence, the function of the heme in the reaction catalyzed by human
cystathionine -synthase is unknown. A catalytic function for heme
had been proposed in which the thiolate of homocysteine is activated
for nucleophilic attack by direct coordination with the heme replacing
the endogenous cysteinate ligand (9). However, resonance Raman
spectroscopy failed to provide evidence for this hypothesis (10). More
recently, the generation of a heme-free enzyme in the crystalline state
of the protein has permitted detection of PLP-bound intermediates that
are formed in the presence of the two substrates (11). The heme-free
enzyme is able to form the
-aminoacrylate species in the
presence of serine, and the internal aldimine was reformed when
homocysteine was added, providing evidence for the ability of the
enzyme to support catalysis in the absence of heme. These studies,
along with the observation that the related heme-independent yeast
enzyme catalyzes the same overall reaction with similar kinetic
parameters, argue against an essential catalytic role for this cofactor
in the human enzyme.
Studies in our laboratory have revealed that perturbations in the heme
environment influence the reaction catalyzed by human cystathionine
-synthase (4). Thus, reduction of the heme from the ferric to
ferrous state is correlated with an ~2-fold diminution in enzyme
activity, whereas binding of CO to the ferrous enzyme results in
complete inhibition of enzyme activity (4). These results indicate that
the heme in cystathionine
-synthase is likely to play a regulatory
role and that changes in the heme pocket are transmitted to the active
site where the PLP-dependent transformation of substrate to
product occurs.
In principle, NMR and pulsed EPR methods can be employed to probe
interactions between the magnetic nucleus, 31P in PLP, and
the paramagnetic ferric iron to map the distance between the two
cofactors, PLP and heme. Relaxation of magnetic nuclei occurs because
of the exchange of magnetic energy with the environment. Paramagnetic
ions with unpaired electrons having a 658-fold greater magnetic moment
than protons are effective in enhancing the relaxation process (12).
Paramagnetic effects on the spin-lattice (T1) and spin-spin
(T2) relaxation rates can be evaluated by subtracting
the relaxation rate measured in the presence of a diamagnetic center
(e.g. ferrous heme in cystathionine -synthase) and the
corresponding paramagnetic state (ferric heme). This approach has been
applied to map distances between magnetic nuclei and paramagnetic
centers in a number of enzymatic systems (13, 14).
A second approach to determining distances between electronic and nuclear spins involves measurement of the hyperfine coupling between the two magnetic moments. Specifically the dipolar component of the coupling, which is anisotropic, varies in inverse proportion to the third power of the interspin distance. In cases where the overall hyperfine coupling is relatively large, it may be detected as a resolved splitting in the continuous wave EPR spectrum. More often, however, these splittings are obscured by inhomogeneous line-broadening effects, and one must resort to high resolution techniques such as ESEEM or ENDOR to obtain coupling constants. In both techniques a spectrum of transition energies of hyperfine-coupled nuclear spins is produced, and the orientation dependence of the hyperfine coupling yields the desired distance. Because the g-factor for low spin Fe3+ is anisotropic, determination of hyperfine couplings to magnetic nuclei of ligand atoms usually requires the use of high resolution EPR methods.
In this study we employed a combination of NMR and pulsed EPR
spectroscopic methods to evaluate the interaction, if any, between the
heme and PLP cofactors in the human enzyme. Interestingly, whereas
these studies do not provide evidence for coupling between the
paramagnetic ferric ion and the phosphorus nucleus, they reveal that the heme oxidation state influences the microenvironment of the
PLP and provides the first structural evidence for communication between the two cofactor binding sites. We have also compared the
31P NMR spectra of the heme-independent yeast and
heme-dependent human enzymes that lead us to conclude that
the PLP environment is similar in the two proteins.
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EXPERIMENTAL PROCEDURES |
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Materials--
Serine, ampicillin, TLCK, TPCK, leupeptin,
pepstatin, aprotinin, benzamidine and
isopropyl-1-thio--D-galactopyranoside were purchased from Sigma. D2O (99%) was from Cambridge Isotope
Laboratories. Homocysteine was generated from homocysteine thiolactone
(Sigma) as described (15), and the concentration was determined
spectrophotometrically using Ellman's reagent (16).
Purification of Human Cystathionine -Synthase--
Truncated
human cystathionine
-synthase representing the active catalytic core
of the enzyme was purified as described previously (5) using an
expression vector provided by Warren Kruger (Fox Chase Cancer Center, Philadelphia).
Purification of Yeast Cystathionine
-Synthase--
Full-length yeast cystathionine
-synthase was
purified using a recombinant expression vector, pSEC, provided by Edith
Miles (National Institutes of Health). Escherichia coli
containing the pSEC vector were cultured at 37 °C in 1 liter of
super broth supplemented with 1 × Vogel and Bonner medium
containing ampicillin (100 mg/liter) and induced with 0.1 mM isopropyl-1-thio-
-D-galactopyranoside at
an OD650 of 2.1 as described previously (3, 17). Cells were
harvested after 20 h and collected by centrifugation. The cell
paste was suspended in 80 ml of buffer containing 50 mM
Tris (pH 8.0), 10 mM EDTA, 1 mM
benzamidine-HCl, 10 mM
-mercaptoethanol, 0.1 mM TLCK, 0.1 mM TPCK, 1 mg/liter leupeptin, 1 ml/liter aprotinin, and 1 mg/liter pepstatin. The cells (~12 g
wet weight) were disrupted with a Heat Systems ultrasonic processor XL,
operated at an output setting of 7 for 7 × 0.5 min with 3-min
breaks between cycles to prevent overheating. The suspension was
stirred for 1 h at 4 °C in the presence of 6 mg of lysozyme, 2 mg of DNase I, and 10 mM MgCl2 and then
centrifuged at ~12,000 × g for 30 min to remove cell
wall debris and unbroken cells. The enzyme was purified by a
modification of the reported procedure as described below. The
supernatant was diluted 3-fold with distilled water and loaded onto a
5 × 7 cm fast flow hydroxylapatite column (from Calbiochem) equilibrated with buffer A containing 50 mM potassium
phosphate (pH 7.8), 10 mM EDTA, 10 mM
-mercaptoethanol, 0.1 mM PLP, 1 mM benzamidine, 0.1 mM TLCK, 0.1 mM TPCK, 1 ml/liter aprotinin, 2 mg/liter leupeptin, and 2 mg/liter
pepstatin A. The column was washed with ~300 ml of buffer A. Proteins were eluted with a 2-liter linear gradient
ranging from 0-0.4 M KCl at a flow rate of 4 ml/min. The
enzyme was collected in two pools eluting between 0.05 and 0.12 M KCl and 0.12 and 0.2 M KCl. The second
fraction was ~95% pure and was concentrated and stored at
80 °C. The first pool of enzyme was concentrated and loaded onto a
second hydroxylapatite column (5 × 7 cm) equilibrated with buffer
B, which differed from buffer A by containing 12.5 mM
(instead of 50 mM) potassium phosphate, pH 7.8. The column
was washed with 300 ml of buffer B and eluted with a 2-liter
linear gradient of potassium phosphate ranging from 12.5 mM
to 250 mM at a flow rate of 4 ml/min. Highly pure enzyme
eluted between 150 and 250 mM potassium phosphate and was concentrated and frozen at
80 °C. A total of 286 mg of yeast cystathionine
-synthase was obtained from a 1-liter culture and had
a specific activity of 495 µmol/mg/h at 37 °C, which is comparable with the reported value (17).
NMR Measurements--
Fourier transform 31P NMR
spectra were collected at 202.4 MHz on a GE-Omega 500-MHz spectrometer
using a 10-mm multinuclear probe head with broadband 1H
decoupling using a standard WALTZ-16 decoupler phase modulation scheme.
The NMR tube contained the sample (1.8 ml) and D2O (0.2 ml)
as field frequency lock and was maintained at 10 °C using a
thermostated liquid nitrogen flow. A spectral width of 8298 Hz was
acquired in 4096 complex data points with a pulse angle of 60°.
Positive chemical shifts in ppm were reported versus 85% H3PO4. Chemical shift referencing was achieved
by inserting a capillary containing 85% H3PO4
into the sample and adding 4-8 additional transients to the
accumulated spectrum. Spin-spin relaxation times
(T2) were determined using
Carr-Purcell-Meiboom-Gill spin-echo experiments, and the results were
fitted to a 3-parameter exponential decay using a nonlinear regression
algorithm. Spin-lattice relaxation times (T1)
were determined via progressive saturation using 90° excitation
pulses with progressively shorter pulse-recycle delays until the signal
was eliminated by saturation and the data were fit to the appropriate
exponential decay function. The progressive saturation method was used
rather than the more common inversion-recovery technique because of the
sensitivity limitations of these samples. The spectrum of the reduced
enzyme (Fig. 3A) was acquired
on a GE-Omega 300 instrument operating at 121.65 MHz for
31P observation with 60° pulses. The substrates, serine
and homocysteine, were added at the concentrations described in the
figure legends.
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Pulsed EPR Measurements-- Pulsed ENDOR, ESEEM, and HYSCORE spectra were obtained using a Bruker ELEXSYS E580 spectrometer. A Bruker Flexline variable-Q dielectric resonator with an integral radiofrequency coil for ENDOR was employed for pulsed measurements. Radiofrequency power was delivered by an ENI model A-500 amplifier with a 500-watt nominal full output. An Oxford Instruments CF935 liquid helium cryostat maintained samples at a constant temperature for measurement.
All ESEEM data were obtained by the use of a stimulated echo pulse
sequence (18). ESEEM experimental conditions were: sample temperature,
14 K; microwave frequency, 9.695 GHz; (delay between first and
second microwave pulses), 200 ns; increment of T (delay between second and third microwave pulses), 16 ns; points per spectrum,
512;
/2 pulse duration, 16 ns; each point is the average of ~5000
individual spin echoes. Each time domain ESEEM scan was fitted to a
fourth-order polynomial function that was subtracted from the data set.
Apodization with a trapezoid function, zero-filling to 1024 points, and
calculation of the absolute-value Fourier transform yielded the final
frequency domain spectrum.
HYSCORE is a two-dimensional ESEEM method that helps to deconvolute complex spectra by revealing correlations between nuclear spin transition frequencies. For HYSCORE, the standard four-pulse sequence was used (19). The 128 × 128 point data sets were base line corrected for ESEEM and processed by applying a two-dimensional fast Fourier transform.
A Mims pulse sequence was used for all ENDOR measurements (20). ENDOR
experimental conditions were: sample temperature, 14 K; , 140-240
ns; T, 12 µs; radiofrequency pulse duration, 6.0 µs;
microwave frequency, 9.695 GHz; magnetic field, 278.0 mT; pulse train
repetition rate, 1.0 kHz; each point is the average of ~2000
individual spin echoes.
Spin-echo-detected EPR spectra were obtained by monitoring a two-pulse
electron spin echo ( = 140 ns) during a magnetic field scan.
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RESULTS AND DISCUSSION |
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The PLP Binding Sites in Human and Yeast Cystathionine
-Synthases Are Similar--
31P NMR spectroscopy has
been a useful tool for studying the environment of PLP bound in the
active sites of enzymes. The chemical shift for PLP free in solution
ranges from 0.75 to 4.3 ppm as the pH is increased from a low to high
value (21). The low field resonances observed in the 31P
NMR spectra of human (5.4 ppm) and yeast (5.17 ppm) cystathionine
-synthase suggests that the phosphate group of PLP is bound as a
dianion in the active sites of both enzymes (Fig. 3). A similar chemical shift has been reported in the PLP enzyme,
O-acetylserine sulfhydrylase (5.2 ppm) (22). A correlation
between the 31P chemical shift and the smallest
O-P-O bond angle has been reported by Gorenstein (23). For
free PLP in solution, the observed 31P chemical shift is
presumed to arise from the minimal energy conformation of the
phosphate. In human and yeast cystathionine
-synthases, the
magnitude of the downfield chemical shifts for the bound PLP is greater
than that for the free cofactor (4.3 ppm for the dianion) and suggests
that the conformation of the phosphate moiety is strained. A resonance
at an even lower field has been observed for PLP bound to ornithine
decarboxylase (6.2 ppm) (24). The broadness of the 31P
signals in both yeast and human enzymes indicates that the PLP is bound
tightly. The signal associated with the yeast enzyme is significantly
broader than that of the human enzyme (73 Hz line width) because of the
larger size of the protein (224 kDa versus 96 kDa for the
truncated human protein), and its line width could not be estimated accurately.
The similarity in the 31P NMR spectra of the yeast and
oxidized human enzyme is consistent with the high degree of
conservation in the PLP binding domains between the two enzymes at the
primary sequence level (Fig. 2). To guide interpretation of our data, we have used the crystal structure of the Salmonella typhimurium O-acetylserine sulfhydrylase to develop a model for human
cystathionine -synthase by employing a molecular replacement
strategy "in silico." All the active site residues that
interact with the PLP in the O-acetylserine sulfhydrylase
(25) are completely conserved in both yeast and human cystathionine
-synthase (Fig. 2). Thus, the glycine-rich loop that is involved in
electrostatic stabilization of the phosphate oxygen atoms, the serine
that hydrogen-bonds with N1 of the PLP ring, and the asparagine that
hydrogen-bonds to the 3'-hydroxyl group are present in all three
enzymes (Fig. 4). The 5'-phosphate is
likely to be dianionic in all three enzymes based on their respective
chemical shifts. In O-acetylserine sulfhydrylase, His-152 is
within hydrogen-bonding distance to a water molecule that in turn
hydrogen-bonds to the 5'-phosphate. It has been postulated that the
histidine may be positively charged, resulting in near neutrality
around the dianionic phosphate moiety (25). A similarly positioned
histidine residue is observed in the modeled structure of cystathionine
-synthase (His-232 in the human sequence). Thus, the sequence
alignments of the human and yeast cystathionine
-synthase and the
O-acetylserine sulfhydrylase provide strong evidence for the
conservation of the PLP binding site in the three enzymes and explain
the similar 31P NMR chemical shifts observed in the
resting forms of these enzymes. More importantly, these results
indicate that the phosphorus group in PLP is shielded from the
paramagnetic ferric heme in the human enzyme and that the two cofactors
are not proximal to each other.
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Pulsed EPR Studies Indicate That the Heme and PLP Binding Sites Are Distant from Each Other-- We have employed ESEEM and pulsed ENDOR spectroscopy to determine whether or not the 31P nucleus of PLP is in the immediate vicinity of the ferric heme iron. Measurements were performed on three states of the enzyme, (i) the resting enzyme, (ii) the enzyme and homocysteine, and (iii) the enzyme, homocysteine, and serine.
The ENDOR spectrum of the resting enzyme, obtained using the pulse
sequence of Mims, exhibits a four-line pattern that is characteristic
of heme nitrogen hyperfine couplings (Fig.
5a). None of the four lines
tracked by magnetic field scan changed at the rate expected for
31P, and none was altered upon addition of one or both
substrates (Fig. 5b). A weak 31P coupling should
produce a pair of transitions positioned symmetrically about the Larmor
frequency for that nucleus and positioned close to that frequency. In
no case was a transition observed at or above the Larmor frequency of
31P, except for 1H features well above 10 MHz.
Thus, the ENDOR spectra show no evidence of the proximity of the
31P nucleus to the heme and render unlikely its presence in
the iron coordination sphere. Furthermore, substrate addition
did not change the spin-echo-detected EPR spectrum (Fig. 5,
inset showing a representative spectrum), again indicating
that heme coordination by homocysteine is very unlikely.
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ESEEM spectra of the three forms of the enzyme are quite similar,
containing modulation frequencies in a range generally associated with
14N couplings (Fig. 6). One
rather broad peak is consistently observed at just over 6.0 MHz, the
31P Larmor frequency at an applied magnetic field of 350 mT. However, spectra obtained using the two-dimensional ESEEM
technique, HYSCORE, revealed clearly that this feature is correlated
with a transition at 2.5 MHz (data not shown). The observed correlation
means that the 6.2- and 2.5-MHz transitions must arise from the same
nucleus. For 31P at the field employed for HYSCORE of the
resting enzyme (345.0 mT), the correlated partner transition should
occur at 5.7 MHz and not at 2.5 MHz. Thus, the 6.2-MHz ESEEM feature is
not due to 31P. As in the ENDOR and ESEEM studies, HYSCORE
spectra exhibited no reproducible changes upon addition of substrate(s)
to the enzyme. These results are consistent with resonance Raman data
that failed to provide evidence for direct coordination of the thiolate
of homocysteine to the heme in cystathionine -synthase (10).
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Oxidation State Changes in the Heme Modulate the 31P
NMR Spectrum of PLP--
A change in the oxidation state of the heme
from ferric to ferrous state diminishes the activity of cystathionine
-synthase by a factor of ~2 (9). Binding of CO to ferrous heme
results in complete inhibition of cystathionine
-synthase activity,
indicating that changes in the heme binding site are communicated to
the active site containing PLP, and that the heme may play a regulatory role (4). To provide structural evidence for this allosteric communication, we examined the 31P NMR spectrum of the
ferrous enzyme. Reduction of cystathionine
-synthase results in
conversion of the heme from a low spin paramagnetic ferric to a low
spin diamagnetic ferrous state and is accompanied by significant
changes in the 31P NMR spectrum of the bound PLP (Fig. 3).
The phosphorus resonance shifts to higher field (from 5.4 to 2.2 ppm),
and the signal is substantially narrowed (from 73 to 16 Hz). To exclude
the possibility that the signal observed upon reduction of the heme is
due to free PLP, the enzyme was subjected to filtration in a Centricon concentrator. Free PLP was not detected in the filtrate. In addition, the chemical shift of free PLP in 50 mM Tris, pH 8.3, is
~4.3 ppm. The upfield shift in the resonance position is
consistent with either a change in the ionization state of the
phosphate group or a substantial change in the smallest O-P-O bond
angle. The narrowing of the line width suggests that the phosphate
group of PLP is more mobile and may be less tightly bound in the
ferrous enzyme.
To evaluate whether or not the longitudinal relaxation of the phosphorus nucleus is influenced by the presence of the paramagnetic ferric heme, the relaxation rates were determined by saturation recovery experiments and found to be 6.34 ± 0.01 s and 5.04 ± 0.06 s for the oxidized and reduced enzymes, respectively. The limiting values for the transverse relaxation rate, T2, estimated from the spectral line widths, are 4.4 ms and 21.1 ms for the oxidized and reduced enzymes, respectively (the measured T2 value using the spin echo technique for the oxidized enzyme is 2.8 ms). Because the presence of the paramagnetic ferric heme in the vicinity of PLP is expected to measurably enhance the spin relaxation rate of the phosphorus nucleus primarily by dipolar interactions between the ferric ion and the 31P nucleus, the absence of a paramagnetic effect on the longitudinal relaxation rate indicates that the two cofactors are not proximal.
Interestingly, these results reveal that whereas the heme is not sufficiently proximal to the PLP to affect the T1 relaxation properties of the phosphorus nucleus, a change in the oxidation state is transmitted to the active site resulting in changes in the microenvironment of the PLP cofactor. This is manifested by significant alterations in the spectral line width (and T2) and resonance frequency of the phosphorus signal. These results provide the first structural evidence for communication between the heme and the PLP binding sites and are consistent with functional studies from our laboratory that reveal that perturbations in the heme pocket affect catalytic activity (4).
Changes in the 31P NMR Spectra Are Induced by the
Addition of Substrates to Human Cystathionine -Synthase--
PLP is
bound as a Schiff base to Lys-119 in the active site of human
cystathionine
-synthase (Fig. 1). The addition of the substrate,
serine, results in fluorescent changes that are consistent with the
formation of an external aldimine and the reported absence of tritium
washout from the
carbon of serine in the absence of homocysteine.
In the related enzymes tryptophan synthase (26) and
O-acetylserine sulfhydrylase (22), the corresponding
external aldimines rather than the elimination product,
-aminoacrylate, are also observed in the presence of serine alone.
This is in contrast to the reported formation of the
-aminoacrylate
product in heme-free cystathionine
-synthase in the crystalline
state (11). These observations suggest that the presence of heme in the
human enzyme affects the equilibrium between intermediates along the
reaction coordinate and/or that the properties of the enzyme observed
in the crystalline state are different from those observed for human
cystathionine
-synthase in solution.
The addition of 6 mM serine to 1.3 mM
cystathionine -synthase results in the appearance of a new signal at
4.7 ppm (Fig. 7). The smaller signal at
5.4 ppm corresponds to residual internal aldimine. The addition of 8 mM homocysteine to the sample containing the external
aldimine results in reformation of the internal aldimine (at 5.4 ppm).
This is expected because homocysteine was added in excess, and it
establishes that the enzyme used in the NMR experiment remained
catalytically active.
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Changes in the 31P NMR Spectrum Induced by the Addition
of Substrates to Yeast Cystathionine -Synthase--
The addition of
serine to yeast cystathionine
-synthase results in the disappearance
of the 412-nm absorption band assigned as the internal aldimine and in
the appearance of a 460-nm band that is attributed to the
-aminoacrylate (7, 17). We have examined the 31P NMR
spectra of the enzyme in the presence of the two substrates, serine and
homocysteine (Fig. 8). Addition of 4.8 mM L-serine to the enzyme (1.2 mM
in PLP) resulted in an upfield shift of the 5.15 ppm resonance to 4.07 ppm and was accompanied by a decrease in the line width. The latter
suggests increased mobility, whereas the upfield shift in resonance
frequency is consistent with an increase in the smallest
O-P-O bond angle, resulting in increased shielding of the
phosphorus nucleus (23). The addition of 9.6 mM
DL-homocysteine resulted in recovery of the resting
enzyme spectrum.
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Conclusions--
In this study, we employed a combination of NMR
and pulsed EPR studies to detect possible interactions between the
paramagnetic ferric iron and the phosphorus nucleus in PLP in
human cystathionine -synthase. The spectroscopic studies failed to
show a direct coupling between the two nuclei and a paramagnetic effect
on the relaxation rate of the phosphorus nucleus, thus supporting the conclusion that PLP and heme are not proximal in human cystathionine
-synthase. However, reduction of the heme in cystathionine
-synthase is accompanied by chemical shift and line width changes in
the 31P signal, revealing that an oxidation state change in
the heme pocket is transmitted to the active site, consistent with
previous biochemical results from our laboratory that suggest a
regulatory role for heme. The similarity in the 31P NMR
spectra of the resting forms of yeast and human cystathionine
-synthase indicates that the PLP environment in the two enzymes are
similar notwithstanding the absence of heme in the yeast enzyme. Furthermore, the addition of the substrate, serine, results in chemical
shift changes that are consistent with the formation of the
spectroscopically identified intermediates in the two enzymes.
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ACKNOWLEDGEMENT |
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We gratefully acknowledge the assistance of
Bryan Lepore (Rosenstiel Center, Brandeis University) in generating the
modeled structure of human cystathionine -synthase and in preparing
Fig. 4.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL58984.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.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Dept. of Biochemistry, N133
Beadle Center, University of Nebraska, Lincoln, NE 68588-0664. Tel.:
402-472-2941; Fax: 402-472-7842; E-mail: rbanerjee1@unl.edu.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M100029200
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ABBREVIATIONS |
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The abbreviations used are:
PLP, pyridoxal
phosphate;
ESEEM, electron spin-echo envelope modulation spectroscopy;
ENDOR, electron nuclear double resonance spectroscopy;
TLCK, N-p-tosyl-L-lysine
chloromethyl ketone;
TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone;
HYSCORE, hyperfine sublevel correlation spectroscopy..
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REFERENCES |
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1. |
Kery, V.,
Bukovska, G.,
and Kraus, J. P.
(1994)
J. Biol. Chem.
269,
25283-25288 |
2. | Finkelstein, J. D., Kyle, W. E., Martin, J. J., and Pick, A.-M. (1975) Biochem. Biophys. Res. Commun. 66, 81-87[Medline] [Order article via Infotrieve] |
3. |
Jhee, K. H.,
McPhie, P.,
and Miles, E. W.
(2000)
J. Biol. Chem.
275,
11541-11544 |
4. | Taoka, S., West, M., and Banerjee, R. (1999) Biochemistry 38, 2738-2744[CrossRef][Medline] [Order article via Infotrieve] |
5. | Taoka, S., Widjaja, L., and Banerjee, R. (1999) Biochemistry 38, 13155-13161[CrossRef][Medline] [Order article via Infotrieve] |
6. | Borcsok, E., and Abeles, R. H. (1982) Arch. Biochem. Biophys. 213, 695-707[Medline] [Order article via Infotrieve] |
7. | Jhee, K. H., McPhie, P., and Miles, E. W. (2000) Biochemistry 39, 10548-10556[CrossRef][Medline] [Order article via Infotrieve] |
8. | Kery, V., Poneleit, L., Meyer, J. D., Manning, M. C., and Kraus, J. P. (1999) Biochemistry 38, 2710-2724 |
9. |
Taoka, S.,
Ohja, S.,
Shan, X.,
Kruger, W. D.,
and Banerjee, R.
(1998)
J. Biol. Chem.
273,
25179-25184 |
10. | Green, E. L., Taoka, S., Banerjee, R., and Loehr, T. M. (2000) Biochemistry 40, 459-463[CrossRef] |
11. |
Bruno, S.,
Schiarettti, F.,
Burkhard, P.,
Kraus, J. P.,
and Mozzarelli, A.
(2000)
J. Biol. Chem.
276,
16-19 |
12. | Mildvan, A. S., and Gupta, R. K. (1978) Methods Enzymol. 49, 322-359[Medline] [Order article via Infotrieve] |
13. | Fung, C. H., Feldmann, R. J., and Mildvan, A. S. (1976) Biochemistry 15, 75-84[Medline] [Order article via Infotrieve] |
14. |
Makinen, A. L.,
and Nowak, T.
(1989)
J. Biol. Chem.
264,
12148-12157 |
15. | Drummond, J. T., Jarrett, J., Gonzalez, J. C., Huang, S., and Matthews, R. G. (1995) Anal. Biochem. 228, 323-329[CrossRef][Medline] [Order article via Infotrieve] |
16. | Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77[Medline] [Order article via Infotrieve] |
17. | Jhee, K. H., McPhie, P., and Miles, E. W. (2000) Biochemistry 39, 10548-10556[CrossRef][Medline] [Order article via Infotrieve] |
18. | Mims, W. B. (1972) Physiol. Rev. B 5, 2409-2419 |
19. | Hofer, P., Grupp, A., Nebenführ, H., and Mehring, M. (1986) Chem. Phys. Lett. 132, 279-282[CrossRef] |
20. | Mims, W. B. (1965) Proc. Roy. Soc. Lond. A. 283, 452-457 |
21. | Schnackerz, K. D., Wahler, G., Vincent, M. G., and Jansonius, J. N. (1989) Eur. J. Biochem. 185, 525-531[Abstract] |
22. | Schnackerz, K. D., Tai, C.-H., Simmons, J. W., Jacobson, T. M., Rao, G. S. J., and Cook, P. F. (1995) Biochemistry 34, 12152-12160[Medline] [Order article via Infotrieve] |
23. | Gorenstein, D. G. (1975) J. Am. Chem. Soc. 97, 898-900 |
24. | Osterman, A. L., Brooks, H. B., Rizo, J., and Phillips, M. A. (1997) Biochemistry 36, 4558-4567[CrossRef][Medline] [Order article via Infotrieve] |
25. | Burkhard, P., Rao, G. S., Hohenester, E., Schnackerz, K. D., Cook, P. F., and Jansonius, J. N. (1998) J. Mol. Biol. 283, 121-133[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Schnackerz, K. D.,
and Mozzarelli, A.
(1998)
J. Biol. Chem.
273,
33247-33253 |