Detection of reaction intermediates during human cystathionine β-synthase-monitored turnover and H2S production

Article abstract of DOI:10.1074/jbc.M112.414722

Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5′-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H₂O or H ₂S, respectively. The presence of he

Full text of DOI:10.1074/jbc.M112.414722

Enzymology:
Detection of Reaction Intermediates during
Human Cystathionine β
-
Synthase-monitored Turnover and H2S
Production
Pramod Kumar Yadav and Ruma Banerjee
J. Biol. Chem. 2012, 287:43464-43471.
doi: 10.1074/jbc.M112.414722 originally published online November 2, 2012
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 52, pp. 43464–43471, December 21, 2012
2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
©
Detection of Reaction Intermediates during Human
Cystathionine ␤-Synthase-monitored Turnover and H S
2
*
Production
Received for publication, August 29, 2012, and in revised form, November 2, 2012 Published, JBC Papers in Press, November 2, 2012, DOI 10.1074/jbc.M112.414722
1
Pramod Kumar Yadav and Ruma Banerjee
From the Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0600
Background: Human cystathionine ␤-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to
give cystathionine and H O or H S.
2
2
Results: Difference stopped-flow spectroscopy was employed to characterize reaction intermediates.
Conclusion: An aminoacrylate intermediate common to both condensation reactions was detected.
Significance: This is the first pre-steady-state kinetic analysis of H S production by human CBS.
2
Human cystathionine ␤-synthase (CBS), a novel heme-con- other physiological effects (4). The role of CBS in H S produc-
2
taining pyridoxal 5؅-phosphate enzyme, catalyzes the condensa- tion is believed to be particularly important in brain, where H S
2
tion of homocysteine and serine or cysteine to produce cystathi- functions as a neuromodulator (5). The other major source of
onine and H O or H S, respectively. The presence of heme in
2
2
H S, particularly in the periphery, is ␥-cystathionase, the sec-
2
CBS has limited spectrophotometric characterization of reac-
ond enzyme in the transsulfuration pathway (6). A third
enzyme, mercaptopyruvate sulfurtransferase, which is involved
in cysteine catabolism, generates protein-bound persulfide,
tion intermediates by masking the absorption of the pyridoxal
5
؅-phosphate cofactor. In this study, we employed difference
stopped-flow spectroscopy to characterize reaction intermedi-
ates formed under catalytic turnover conditions. The reactions
of L-serine and L-cysteine with CBS resulted in the formation of
which could release H S upon reduction (7, 8). The contribu-
2
tion of the sulfurtransferase at physiological concentrations of
the substrate (mercaptopyruvate) remains to be evaluated (3).
a common aminoacrylate intermediate (k ؍ 0.96 ؎ 0.02 and
obs
CBS generates H S via multiple reactions (Fig. 1, Reactions
2
؊
1 ؊1
0
.38 ؎ 0.01 mM
s
, respectively, at 24 °C) with concomitant
2
–4) (9). Each of these represents a ␤-replacement reaction,
loss of H O and H S and without detectable accumulation of the
2
2
and under maximum velocity (V ) conditions, the turnover
max
external aldimine or other intermediates. Homocysteine
number for the condensation of cysteine and homocysteine (i.e.
Reaction 2) is ϳ20- and 40-fold faster than for Reactions 3 and
reacted with the aminoacrylate intermediate with k ؍ 40.6 ؎
obs
؊
1
3
.8 s and re-formed the internal aldimine. In the reverse
4
, respectively. In fact, the canonical ␤-replacement reaction of
direction, CBS reacted with cystathionine, forming the amino-
serine by homocysteine (Reaction 1) proceeds ϳ4-fold slower
؊
1 ؊1
acrylate intermediate with k ؍ 0.38 ؎ 0.01 mM
s
. This
obs
than Reaction 2 under V
conditions. At physiologically rel-
max
study provides the first insights into the pre-steady-state kinetic
mechanism of human CBS and indicates that the reaction is
likely to be limited by a conformational change leading to prod-
uct release.
evant substrate concentrations, the condensation of 2 mol of
cysteine is predicted to be the least efficient route for H S gen-
2
eration, accounting for 1.6% of the H S produced, whereas the
2
condensation of homocysteine and cysteine is by far the most
efficient, accounting for 96% of the H S (9).
2
Human CBS is unique in being a pyridoxal 5Ј-phosphate
2
Cystathionine ␤-synthase (CBS) serves two major functions
(
PLP)-dependent enzyme that is also a heme protein. The heme
in the mammalian sulfur network. First, it plays a pivotal role in
removing excess sulfur and maintaining low homocysteine lev-
els by catalyzing its conversion to cystathionine (Fig. 1, Reac-
tion 1) (1). Genetic defects in CBS, which catalyzes the first step
in the transsulfuration pathway, are the single most common
cause of homocystinuria, characterized by aggressive occlusive
arterial disease and other severe pathologies (2). Second, CBS is
cofactor resides ϳ20 Å away from the active site (10, 11) and
plays a regulatory role (12), switching off enzyme activity in
response to CO (13–15) and NO (16). The heme in CBS is
6
-coordinate in the ferric and ferrous states, and the redox
potential for the ferrous/ferric couple for the full-length
enzyme is estimated to be approximately ϳ350 Ϯ 4 mV (17).
Kinetic coupling of reduction of the heme, which has a fairly
low redox potential, to carbonylation allows formation of the
ferrous CO species with physiological reductants (13). Changes
in the heme ligation state are communicated to the active site
and lead to a shift in the tautomeric equilibrium of the Schiff
one of two significant contributors to the biogenesis of H S (3),
2
a gaseous signaling molecule that has cardioprotective and
*
Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant HL58984.
1
To whom correspondence should be addressed: University of Michigan, base of PLP from the predominantly active keto enamine to the
3
7
320B MSRB III, 1150W. MedicalCenterDr., AnnArbor, MI48109-0600. Tel.:
34-615-5238; E-mail: rbanerje@umich.edu.
inactive enol imine (14). The presence of the heme with a high
extinction coefficient obscures the UV-visible spectrum of the
PLP cofactor and renders challenging the direct observation of
2
The abbreviations used are: CBS, cystathionine ␤-synthase; PLP, pyridoxal
Ј-phosphate; dCBS, Drosophila CBS; yCBS, yeast CBS.
5
4
3464 JOURNAL OF BIOLOGICAL CHEMISTRY
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Rapid Reaction Kinetic Analysis of Human CBS
FIGURE 1. Reactions catalyzed by CBS. Reaction 1 represents the “canonical” reaction in the transsulfuration pathway, whereas Reactions 2–4 generate
H2S.
nation of the ␣-proton followed by ␤-elimination of H O from
2
the external aldimine of serine (or H S from the external aldi-
2
mine of cysteine) yields the aminoacrylate intermediate. In the
second half-reaction, the aminoacrylate intermediate under-
goes nucleophilic attack by the thiolate of homocysteine to
yield the external aldimine of the product, cystathionine. Alter-
natively, addition of cysteine or water yields lanthionine or ser-
ine, respectively, bound as external aldimines. Release of cysta-
thionine, lanthionine, or serine by a second transchiffization
reaction restores the resting internal aldimine state of the
enzyme.
Spectroscopic characterization of a hemeless variant of
human CBS lacking the N-terminal heme-binding domain has
been reported (21). This variant retains 40% of the activity of
wild-type human CBS, and its characterization was limited by
its instability and proteolytic susceptibility, leading to poor
yields (21). Insights from studies on the hemeless variant of
human CBS were also limited, as deletion of the heme domain
FIGURE 2. Proposed catalytic cycle of CBS. The resting enzyme exists as the
internal aldimine with Lys-119 in human CBS involved in Schiff base linkage. unexpectedly resulted in the enzyme being inhibited rather
Addition of serine (or cysteine) gives a gem-diamine intermediate, which col-
lapses to the external aldimine of serine (or cysteine). Elimination of water (or
H2S) leads to the aminoacrylate intermediate to which homocysteine (cys-
teine or water) adds to form the external aldimine of the product cystathio-
nine (lanthionine or serine). The latter is released via the transient formation
of a gem-diamine intermediate in a transchiffization reaction involving the
than activated by S-adenosylmethionine. CBS contains a C-ter-
minal regulatory domain to which S-adenosylmethionine
binds, and the juxtaposition of the regulatory, catalytic, and
heme domains was recently revealed in the structure of full-
active site lysine. Elimination of cystathionine from the active site completes length Drosophila CBS (dCBS) (22). In contrast to human
the catalytic cycle and regenerates the resting enzyme. The numbers in paren-
and fly CBS, the absence of heme in yeast CBS (yCBS) has per-
theses represent the absorption maxima of the intermediates.
mitted ready characterization of PLP-based intermediates.
Detailed rapid reaction kinetics of the yCBS-catalyzed canoni-
tautomeric states and reaction intermediates during the cata-
lytic cycle. Indeed, the effect of ferrous CO on the PLP cofactor
could be observed by fluorescence and resonance Raman spec-
troscopies rather than absorption spectroscopy (14).
cal and H S-generating reactions have been reported (23–25).
2
In this study, we used difference electronic absorption spec-
troscopy to monitor the reaction kinetics of full-length human
CBS under pre-steady-state conditions. We recently estab-
lished the utility of this approach by spectroscopic detection of
CBS belongs to the fold II family of PLP enzymes, which
includes well studied members, e.g. O-acetylserine sulfhydry-
lase (18), threonine deaminase (19), and tryptophan synthase the aminoacrylate intermediate in dCBS, which was also cap-
(
20). The PLP is tethered in the active site of human CBS via a tured in the crystal structure (22). The reactions catalyzed in
Schiff base linkage with Lys-119 (Fig. 2). Binding of serine or the presence of serine or cysteine provide, for the first time,
cysteine leads to formation of the external aldimine in a multi- kinetic insights into the canonical transsulfuration reaction and
step reaction involving a gem-diamine intermediate. Deproto- the H S-producing reactions catalyzed by human CBS.
2
DECEMBER 21, 2012•VOLUME 287•NUMBER 52
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Rapid Reaction Kinetic Analysis of Human CBS
EXPERIMENTAL PROCEDURES
For experiments in which cystathionine was employed, a
stock solution was made in 100 mM HEPES (pH 7.4), followed
by the gradual addition of 5 M NaOH until the solution was
clear. Further dilutions of cystathionine were made in 100 mM
HEPES (pH 7.4). Data from the stopped-flow experiments were
Materials—L-Cysteine, DL-homocysteine, L-cystathionine,
14
L-serine, and PLP were purchased from Sigma. [ C]Serine (164
mCi/mmol) was purchased from PerkinElmer Life Sciences.
GST-Sepharose 4B was purchased from GE Healthcare. All
other chemicals were purchased from Fisher.
fitted using SigmaPlot. The observed rate constants (k ) for
obs
the reactions with serine, cysteine, homocysteine, and cysta-
Expression and Purification of CBS—Unless specified other-
wise, CBS refers to the recombinant human enzyme used in this
study. Wild-type CBS was expressed and purified as described
previously (26). Briefly, the cell pellet obtained from a 6ϫ 1 liter
culture was resuspended in 300 ml of 50 mM Tris buffer (pH 8.0)
containing a single protease inhibitor mixture tablet (Roche
Applied Science) and 100 mg of lysozyme and stirred for 1 h at
thionine were determined from fits using a single-exponential
equation. The k values obtained at various concentrations of
obs
substrates were fitted to a linear equation to obtain the appar-
ent second-order rate constants.
The observed rate constant for the disappearance of the ami-
noacrylate intermediate in the presence of homocysteine (Hyc)
showed a hyperbolic dependence on substrate concentration,
and the data were fitted using Equation 1, in which K ϭ k /k .
4
°C. The cells were lysed by sonication, and the supernatant
2
1
2
1
was obtained by centrifugation at 12,000 ϫ g for 25 min. The
supernatant was loaded onto a glutathione-Sepharose column
pre-equilibrated with 1ϫ PBS. The column was washed with
kobs ϭ ͑k ͓Hcy]͒/͑K ϩ [Hcy]͒ ϩ k
4
(Eq. 1)
3
21
1
ϫ PBS, and the GST-fused CBS was eluted with 50 mM Tris RESULTS
(
pH 8.0) containing 10 mM glutathione. This step was followed
Reaction of CBS with Serine—Rapid scanning stopped-flow
by thrombin digestion at 4 °C to remove the GST tag. The pro-
spectra recorded after mixing CBS with serine (Fig. 3A) showed
tein was loaded onto a Q-Sepharose column equilibrated with that although the absorption maxima associated with ferric
5
0 mM Tris buffer (pH 8.0), and the column was then washed heme (Soret peak at 428 nm and the ␣/␤-bands centered at 550
with the same buffer. The protein was eluted with a 500-ml nm) did not change, there was an increase in absorption at 465
gradient ranging from 0 to 500 mM NaCl in 50 mM Tris-HCl nm (assigned to a PLP intermediate). To better characterize the
(
pH 8.0). The fractions of interest were concentrated and stored PLP-dependent absorption changes elicited by serine, differ-
at Ϫ80 °C.
ence spectra were analyzed (Fig. 3B). A disappearance in the
Steady-state Enzyme Assay—Enzyme activity was measured absorbance at 406 nm (assigned to the internal aldimine) and
as described previously (26) in the presence of varying concen- the appearance of absorbance at 465 nm (assigned to the ami-
14
noacrylate intermediate) were observed. The difference spectra
exhibit two apparent isosbestic points at 357 and 422 nm,
respectively, indicating that the external aldimine did not accu-
mulate at measurable concentrations. For comparison, addi-
tion of serine led to the formation of a broad absorption peak
between 460 and 510 nm with the hemeless variant of CBS (21)
and at 465 nm with yCBS (23, 24). Rates for the disappearance
of the internal aldimine (Fig. 3C) and the appearance of the
aminoacrylate species (Fig. 3D) showed a linear dependence on
the concentration of serine (1–55 mM). The absence of observ-
able intermediates between the internal aldimine and the ami-
trations of homocysteine and 30 mM [ C]serine (164 mCi/
mmol) in 100 mM HEPES (pH 7.4) at 24 °C. The turnover num-
ber for the enzyme obtained under these conditions was used as
a reference point for the observed reaction rates measured
under pre-steady-state conditions.
Stopped-flow Spectroscopy—Pre-steady-state experiments
were performed using an Applied Photophysics stopped-flow
spectrophotometer (SX.MV18) in the photodiode array mode
as described (22). In brief, a solution of CBS (60 ␮M calculated
per 63-kDa monomer) in 100 mM HEPES (pH 7.4) was rapidly
mixed with various concentrations of substrates (cysteine, ser-
ine, or cystathionine) in the same buffer. The time-dependent
formation of reaction intermediates was followed by difference
spectroscopy ((enzyme ϩ substrate) Ϫ enzyme). The tempera-
ture was maintained at 24 °C using a circulating water bath. To
ensure that changes in the heme spectrum did not occur during
enzyme-monitored turnover, the spectrum of CBS (20 ␮M) was
recorded following mixing with serine (60 mM) in a stopped-
flow spectrophotometer. To verify that the kinetic analysis of
difference spectra did not introduce artifacts, we also moni-
tored the kinetics of formation of reaction intermediates at a
single wavelength (465 or 406 nm) following mixing of 60 ␮M
CBS with varying concentrations (2, 20, and 60 mM) of serine.
When the reaction of preformed aminoacrylate with homocys-
noacrylate species accounts for the linear dependence of k on
obs
serine concentration. From the analysis in Fig. 3 (C and D),
bimolecular rate constants of comparable value were obtained:
Ϫ1 Ϫ1
1
.08 Ϯ 0.04 mM
s
for the disappearance of the internal
Ϫ1 Ϫ1
aldimine and 0.96 Ϯ 0.02 mM
s
for the formation of the
aminoacrylate intermediate. To ensure that the kinetic analysis
of difference spectrum data did not skew the estimates, the
experiments were repeated under single-wavelength monitor-
ing conditions at 406 and 465 nm, respectively, at three concen-
trations of serine (Fig. 3E). The bimolecular rate constant
Ϫ1
obtained from this analysis was identical (1.14 Ϯ 0.13 mM
Ϫ1
s
) within experimental error to that estimated from multiple-
wavelength monitoring.
Ϫ1 Ϫ1
From the ratio of the slope (1.05 Ϯ 0.02 mM
s
) to inter-
Ϫ1
teine was monitored, 60 ␮M enzyme was premixed with 25 mM cept (2.15 Ϯ 0.82 s ), the apparent K for serine was estimated
d
serine in 100 mM HEPES (pH 7.4), and the resulting amino- to be 2.04 Ϯ 0.72 mM (Fig. 3C). We also employed the depen-
acrylate intermediate was rapidly mixed with various concen- dence of the change in amplitude at 0.3 and 0.1 s at either 406
trations of homocysteine as described previously (24).
nm (Fig. 3C, inset) or 465 nm (Fig. 3E), respectively, to estimate
4
3466 JOURNAL OF BIOLOGICAL CHEMISTRY
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Rapid Reaction Kinetic Analysis of Human CBS
FIGURE 3. Spectroscopic and kinetic monitoring of the aminoacrylate intermediate formed in the presence of serine. A and B, spectra were recorded
following mixing of 20 ␮M (before mixing) CBS with 60 mM serine (A), whereas difference spectra ((CBS ϩ serine) Ϫ CBS) were acquired every 2 s following rapid
mixing of CBS (60 ␮M in active sites, before mixing) with 50 mM serine in 100 mM HEPES (pH 7.4) at 24 °C (B). The internal aldimine has an absorption maximum
at ϳ406 nm, and upon addition of serine, it converts to the aminoacrylate intermediate with absorption maxima at 465 and 320 nm. C, dependence of the
observed rates for disappearance of the internal aldimine on the concentration of serine. Inset, dependence of the absorbance change at 406 nm at 0.3 s after
mixing on serine concentration. D, dependence of the observed rates for formation of the aminoacrylate on the concentration of serine obtained from the
difference spectra. Inset, representative kinetic traces at 465 nm obtained in the presence of 1–55 mM serine (from bottom to top) are shown. E, dependence of
the absorbance change at 465 nm at 0.1 s after mixing on serine concentration. F, the kinetics of aminoacrylate formation were recorded at single wavelengths
(406 and 465 nm) at the indicated concentrations of serine. Inset, dependence of kobs at 465 nm on serine concentration.
the K for serine. This analysis yielded values of 2.3 Ϯ 0.5 and diate without detectable formation of the gem-diamine or the
d
2
.5 Ϯ 0.6 mM, respectively. We note, however, that interpreta- external aldimine intermediates. Formation of the aminoacry-
tion of the dependence of the observed rate constants on the late intermediate with absorption features at ϳ320 and 460 nm
concentration of serine is complex because k does not simply has also been reported for yCBS (25) and dCBS (22).
obs
correspond to the binding of serine. Formation of the amino-
Reaction of CBS with Cysteine—Addition of cysteine resulted
acrylate from the internal aldimine involves multiple steps (e.g. in the appearance of difference absorption spectra (Fig. 4A) that
the gem-diamine, external aldimine, and carbanion intermedi- were comparable with those seen in the presence of serine (Fig.
ates) that are spectroscopically and kinetically unresolved.
3B). The spectral changes were consistent with the conversion
Addition of serine to CBS also gave rise to a second absorp- of the internal aldimine (406 nm) to the aminoacrylate interme-
tion peak centered at 320 nm, which we assigned as the enol diate (465 and 320 nm) with apparent isosbestic points at 357
imine tautomer of the aminoacrylate species (Fig. 3B). The rate and 422 nm. The observed rates for the disappearance of the
of increase in absorbance at 465 and 320 nm was identical (not internal aldimine and the formation of the aminoacrylate inter-
shown), consistent with our assignment that the internal aldi- mediate showed linear dependences on the concentration of
mine converts to the tautomers of the aminoacrylate interme- cysteine (1–50 mM) (Fig. 4, B and C). The 320 nm peak formed
DECEMBER 21, 2012•VOLUME 287•NUMBER 52
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Rapid Reaction Kinetic Analysis of Human CBS
Ϫ1 Ϫ1
and 0.38 Ϯ 0.01 mM
s
for the formation of the aminoacry-
late, respectively. These rate constants were ϳ2-fold lower than
the corresponding biomolecular rate constants obtained with
serine (Fig. 3). We note, however, that the reaction kinetics of
cysteine with CBS are complicated by the simultaneous occur-
rence of two reactions, i.e. the ␤-replacement of cysteine with
H O and the ␤-replacement of cysteine with a second mole of
2
cysteine (Fig. 1, Reactions 3 and 4). Hence, a simple comparison
with the bimolecular rate constants obtained with serine and
cysteine is not useful.
Ϫ1 Ϫ1
From the ratio of the slope (0.4 Ϯ 0.03 mM
s
) to intercept
Ϫ1
(
2.95 Ϯ 0.58 s ), the apparent K for cysteine was estimated to
d
be 7.4 Ϯ 1.5 mM (Fig. 3C). The dependence of the change in
amplitude at 0.3 s at either 406 nm (Fig. 4B, inset) or 465 nm
(
Fig. 4C, inset) on cysteine concentration yielded K estimates
d
of 7.4 Ϯ 1.4 and 6.1 Ϯ 1.8 mM, respectively. These values are
similar to the K value of 6.8 Ϯ 1.7 mM for cysteine binding to
m1
the PLP site in CBS and distinct from the K value of 27.3 Ϯ 3.7
m2
mM for cysteine binding to the second site in CBS (9).
Reaction of the Aminoacrylate Intermediate with Homo-
cysteine—In the second half-reaction catalyzed by CBS, the
aminoacrylate intermediate (generated by elimination of H O
2
from serine or H S from cysteine) reacts with homocysteine to
2
form cystathionine (Fig. 2). To determine the kinetics of cysta-
thionine formation, the aminoacrylate intermediate was pre-
formed by mixing 60 ␮M CBS and 25 mM serine, which were
then rapidly mixed with varying concentrations of homocys-
teine. The difference spectra obtained following mixing of the
aminoacrylate intermediate with 30 mM L-homocysteine show
the disappearance of the 320 and 465 nm-absorbing aminoac-
rylate intermediate and the appearance of a 406 nm-absorbing
aldimine species (Fig. 5A). These spectral changes occur with
two apparent isosbestic points at 357 and 422 nm. The rate of
disappearance of the aminoacrylate intermediate exhibits a
hyperbolic dependence on homocysteine concentration and
Ϫ1
yields a maximum rate of 40.6 Ϯ 3.8 s for formation of the
aldimine and a K
m
conditions identical to those used in the stopped-flow experi-
ments is 2.8 Ϯ 0.6 mM.
for homocysteine of 2.1 Ϯ 0.5 mM. The
m(app)
K
for homocysteine obtained in the steady-state assay using
Reaction of CBS with L-Cystathionine—When CBS was rap-
idly mixed with L-cystathionine, the disappearance of the
absorbance feature centered at 406 nm and the appearance of
the 320 and 465 nm-absorbing aminoacrylate species were
observed (Fig. 6A). Formation of the aminoacrylate intermedi-
FIGURE 4. Spectroscopic and kinetic monitoring of the aminoacrylate
intermediate formed in the presence of cysteine. A, difference spectra
(
(CBS ϩ cysteine) Ϫ CBS) obtained following rapid mixing of CBS with cys- ate from cystathionine indicates that the reaction is partially
teine in 100 mM HEPES (pH 7.4) at 24 °C. The spectra were acquired every 2 s
following mixing of 60 ␮M CBS with 50 mM cysteine. The internal aldimine has
an absorption maximum at ϳ406 nm, and upon addition of serine, it rapidly
converts to the aminoacrylate intermediate with an absorption maximum at
reversible. The observed rate of formation of the aminoacrylate
intermediate was linearly dependent on the concentration of
cystathionine (1–10 mM) (Fig. 6B). From these data, the appar-
4
65 nm. B and C, dependence of the apparent rates for the disappearance of
ent bimolecular rate constant for formation of the aminoacry-
the internal aldimine (B) andthe formationofthe aminoacrylateintermediate
Ϫ1
(
C) on the concentration of cysteine. Insets, dependence of the absorbance late intermediate from cystathionine was 0.38 Ϯ 0.01 mM
changes at 406 and 465 nm, respectively, on cysteine concentration at 0.3 s
after mixing.
Ϫ1
s
, and the K
for cystathionine was estimated to be 2.5 Ϯ
d(app)
Ϫ1 Ϫ1
0
.7 mM from the ratio of the slope (0.38 Ϯ 0.03 mM
s
) to
Ϫ1
at the same rate as the 465 nm peak (data not shown) but was of intercept (0.94 Ϯ 0.26 s ) (Fig. 6B). A K for cystathionine of
d
lower intensity. Data analysis at 406 and 465 nm yielded bimo- 2.1 Ϯ 0.7 mM was also determined from the dependence of the
lecular rate constants that were of similar magnitude: 0.40 Ϯ amplitude change at 465 nm at 0.5 s on the concentration of
Ϫ1 Ϫ1
0
.03 mM
s
for the disappearance of the internal aldimine cystathionine (Fig. 6B, inset).
4
3468 JOURNAL OF BIOLOGICAL CHEMISTRY
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Rapid Reaction Kinetic Analysis of Human CBS
FIGURE 5. Spectroscopic and kinetic monitoring of the conversion of the FIGURE 6. Spectroscopic and kinetic monitoring of the aminoacrylate
aminoacrylate intermediate in the presence of homocysteine. A, differ- intermediate formed in the presence of cystathionine. A, difference spec-
ence spectra ((CBS-aminoacrylate ϩ homocysteine) Ϫ CBS-aminoacrylate) tra ((CBS ϩ cystathionine) Ϫ CBS) obtained following rapid mixing of CBS
obtained following rapid mixing of preformed CBS-aminoacrylate with with cystathionine in 100 mM HEPES (pH 7.4) at 24 °C. The experiment was
homocysteine in 100 mM HEPES (pH 7.4) at 24 °C. To obtain the spectra, 60 ␮M initiated by mixing 60 ␮M CBS with 20 mM L-cystathionine, and the spectra
CBS was premixed with 25 mM serine and then mixed with 30 mM homocys- wererecordedevery2s. Theinternalaldiminehasanabsorptionmaximumat
teine The spectra were recorded every 2 s. Addition of homocysteine resulted ϳ406 nm, and upon addition of serine, it converts to the aminoacrylate inter-
in the disappearance of the aminoacrylate intermediate (320 and 465 nm) mediatewithanabsorptionmaximumat320or465nm. B, dependenceofthe
and the re-formation of the internal aldimine with an absorption maximum at apparent rate of formation of the aminoacrylate on the concentration of cys-
ϳ406 nm. B, dependence of the apparent rates for disappearance of the tathionine. Inset, dependence of the absorbance changes at 465 nm on cys-
aminoacrylate intermediate on the concentration of homocysteine.
tathionine concentration at 0.5 s after mixing.
Our study provides the first insights into the reaction cata-
lyzed by full-length human CBS. The aminoacrylate is the only
distinct intermediate detected in either direction by rapid scan-
ning stopped-flow spectroscopy. The lack of detectable accu-
mulation of the preceding external aldimine intermediate sug-
gests that removal of the ␣-proton, needed to initiate formation
of the aminoacrylate intermediate, might not be rate-limiting.
This is consistent with the lack of a primary kinetic isotope
effect on aminoacrylate formation in yCBS (25). In contrast, a
primary kinetic isotope effect on formation of an aminoacrylate
intermediate is seen in both O-acetylserine sulfhydrylase and
tryptophan synthase, which are fold II PLP enzymes that cata-
lyze ␤-replacement reactions (20, 29, 30).
DISCUSSION
The critical role of CBS in mammalian sulfur metabolism is
exemplified by the pleiotropic clinical consequences of its
impairment, inherited as an autosomal recessive disorder (27).
Studies on the effects of CBS deficiency have been focused on
the homocysteine management aspect of the reaction because
homocystinuria is a hallmark of this condition. However, CBS
also plays a role in H S biogenesis, particularly in brain. Study-
2
ing the detailed kinetic mechanism of human CBS is therefore
of significant interest but has been limited by the presence of
the heme cofactor, whose absorption spectrum masks that of
the PLP cofactor. In contrast, yCBS, devoid of the heme cofac-
tor, has been the subject of detailed kinetic analyses (23–25).
In general, the rates of formation and disappearance of the
However, significant differences exist in the catalytic and regu- aminoacrylate intermediate are slower for human versus yeast
latory properties of yeast and human CBS (28), warranting CBS (Table 1). This is consistent with the ϳ5-fold lower k for
cat
direct characterization of the human enzyme.
Reaction 1 between serine and homocysteine and the ϳ2.8-fold
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Rapid Reaction Kinetic Analysis of Human CBS
TABLE 1
Comparison of parameters obtained from pre-steady-state analyses of yeast and human CBS
Parameter
Human CBS
yCBSa
b
K
k
for Ser
2.04 Ϯ 0.72 mM
14.4 Ϯ 1.7 mM
Ϫ1
d(app)
obs
d(app)
Ϫ1
38.0 Ϯ 3.0 mM sϪ1
(AA from Ser)
0.96 Ϯ 0.02 mM sϪ1
b
K
for Cys
7.4 Ϯ 1.5 mM
ND
0.38 Ϯ 0.01 mM sϪ1
Ϫ1
1.61 Ϯ 0.3 mM
1.6 Ϯ 0.3 mM
Ϫ1
Ϫ1
s
Ϫ1,c 4.1. Ϯ 0.2 mMϪ1 sϪ1d
k
(AA from Cys)
obs
K
b
for cystathionine
2.5 Ϯ 0.7 mM
d(app)
Ϫ1
4.1 Ϯ 0.2 mM sϪ1
0.37 Ϯ 0.02 mM
183 Ϯ 4 mM sϪ1
ND
k
(AA from cystathionine)
0.38 Ϯ 0.01 mM sϪ1
obs
K
e
f
for Hcy, K
for Hcy,
d(app)
g
2.1 Ϯ 0.5 mM
m(app)
Ϫ1
k
(aldimine from AA)
ND
obs
Ϫ1
k
(aldimine from AA)
40.6 Ϯ 3.8 s
max
a
b
c
d
e
f
The values for yCBS are taken from Ref. 23.
The Kd(app) values listed for human CBS were derived from the dependence of kobs on the respective substrate concentrations as discussed under “Results.”
The values represent the rates for Reaction 3 in Fig. 1.
The values represent the rates for Reaction 4 in Fig. 1.
This parameter was determined for human CBS. Hyc, homocysteine.
This parameter was determined for yCBS.
g
AA, aminoacrylate; ND, determined.
tion of Lys-119 to alanine results in a 1000-fold diminution in
enzyme activity compared with wild-type CBS, which is allevi-
ated by ϳ2-fold by provision of an exogenous base, e.g. ethyla-
mine, consistent with the additional role of Lys-119 as a general
base during the catalytic cycle (21). The interactions between
the carbanion intermediate and active site residues are consis-
tent with a greater role for the Schiff base in providing ylide-
type electrostatic stabilization versus delocalization of the neg-
ative charge through the extended conjugated ring system of
the PLP to generate a quinonoid intermediate, which is not
observed under pre-steady-state conditions with either human
or yeast CBS (23–25). Furthermore, the presence of a serine
residue (Ser-349 in the human sequence) proximal to N1 in the
FIGURE7.StructuresofreactionintermediatesinCBS.Thestructuresofthe
carbanion (A) and aminoacrylate (B) intermediates in dCBS show few key PLP ring (versus an aspartate, for example) is expected not to
interactions. The numbering refers to the dCBS sequence, whereas the num-
stabilize a quinonoid intermediate.
bers in parentheses refer to the human CBS sequence. The figures were gen-
erated from Protein Data Bank code 3PC4 (A) and 3PC3 (B).
Although the carbanion intermediate was fortuitously
trapped in the structure of dCBS, it is a transient and reactive
lower k for Reaction 2 between cysteine and homocysteine intermediate that is expected to rapidly undergo ␤-elimination
cat
for the human versus yeast enzymes (23). Other differences in to yield the aminoacrylate intermediate. In the 1.55 Å structure
the pre-steady-state kinetics are also evident between yeast and of the aminoacrylate intermediate, the ⑀-amino group of the
human CBS. Although formation of the aminoacrylate inter- active site lysine has swung away from the C␣ and interacts
mediate by human CBS exhibited a linear dependence on the instead with the phosphate oxygens of PLP (Fig. 7B). The C␤ of
concentrations of serine (Fig. 3C), yCBS exhibited saturation the aminoacrylate intermediate is positioned for nucleophilic
kinetics with evidence for transient accumulation of an external addition (22), which is expected to occur from the same face
aldimine, whose formation was linearly dependent on serine from which the leaving group departed based on stereochemi-
concentration (23, 24). In the presence of cysteine, a single lin- cal analyses reported by Borcsok and Abeles (31).
ear dependence of the rate of aminoacrylate formation was
The k for the reaction of CBS (with serine and homocys-
cat
observed with human CBS, whereas yCBS showed two phases, teine) determined in the steady-state assay using the same
interpreted as representing Reactions 3 and 4, respectively (23). buffer, pH, and temperature conditions as in the stopped-flow
Ϫ1
Crystal structures of full-length dCBS have revealed details of experiments is 0.60 Ϯ 0.05 s . At 30 mM serine, the concen-
how the carbanion and aminoacrylate intermediates interact tration used in the steady-state assay, the rate of aminoacrylate
Ϫ1
with and are stabilized by neighboring residues (Fig. 7A) (22). intermediate formation is estimated to be ϳ32 s . Hence,
The 1.7 Å structure of the carbanion intermediate generated steps between binding of serine and aminoacrylate formation
upon removal of the C␣ proton shows that the ⑀-amino group are not rate-determining in the first half-reaction. The k for
obs
of the active site lysine (corresponding to Lys-119 in the human formation of the aminoacrylate from cysteine is ϳ2.5-fold
sequence) is positioned within 2.1 Å of the C␣ of serine and 3.0 lower than that from serine. Because H S is in fact a better
2
Å from the C4 of PLP, stabilizing the negative charge (Fig. 7A). leaving group than H O at physiological pH, the higher k for
2
obs
On the opposite side of the lysine, the hydroxyl group of serine the reaction with serine suggests that H O is released faster
2
(
Ser-147 in the human sequence) contacts the C␣ and might than H S from the active site. Similarly, the chemical steps
2
play a role in activating the leaving group (OH in serine and SH between the aminoacrylate intermediate and re-formation of
in cysteine). The structure supports a direct role of the active the internal aldimine are not rate-determining (k
ϭ 40.6 Ϯ
max
Ϫ1
site lysine (Lys-119 in the human sequence) as a general base for 3.8 s ). Hence, a step following re-formation of the internal
C␣ proton abstraction and for carbanion stabilization. Muta- aldimine, e.g. product release or a conformational change,
4
3470 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 52•DECEMBER 21, 2012

Rapid Reaction Kinetic Analysis of Human CBS
probably limits the reaction rate. A conformational change
induced by substrate binding/product release is a common fea-
ture of PLP enzymes. Such a conformational change in CBS is
evident upon comparing the crystal structures of the apo- and
substrate-bound forms of dCBS (22). Binding of substrate trig-
gers the closed conformation and repositioning of the active
site serine in the proximity of the cofactor.
12809–12816
1
1
1
5. Taoka, S., West, M., and Banerjee, R. (1999) Characterization of the heme
and pyridoxal phosphate cofactors of human cystathionine ␤-synthase
reveals nonequivalent active sites. Biochemistry 38, 2738–2744
6. Taoka, S., and Banerjee, R. (2001) Characterization of NO binding to hu-
man cystathionine ␤-synthase: possible implications of the effects of CO
and NO binding to the human enzyme. J. Inorg. Biochem. 87, 245–251
7. Singh, S., Madzelan, P., Stasser, J., Weeks, C. L., Becker, D., Spiro, T. G.,
Penner-Hahn, J., and Banerjee, R. (2009) Modulation of the heme elec-
tronic structure and cystathionine ␤-synthase activity by second coordi-
nation sphere ligands: the role of heme ligand switching in redox regula-
tion. J. Inorg. Biochem. 103, 689–697
REFERENCES
1
. Zou, C.-G., and Banerjee, R. (2005) Homocysteine and redox signaling.
Antioxid. Redox Signal. 7, 547–559
2
. Kraus, J. P., Janosík, M., Kozich, V., Mandell, R., Shih, V., Sperandeo, M. P., 18. Rabeh, W. M., and Cook, P. F. (2004) Structure and mechanism of O-
Sebastio, G., de Franchis, R., Andria, G., Kluijtmans, L. A., Blom, H., Boers,
G. H., Gordon, R. B., Kamoun, P., Tsai, M. Y., Kruger, W. D., Koch, H. G.,
Ohura, T., and Gaustadnes, M. (1999) Cystathionine ␤-synthase muta-
tions in homocystinuria. Hum. Mutat. 13, 362–375
acetylserine sulfhydrylase. J. Biol. Chem. 279, 26803–26806
19. Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chin-
chilla, D., and Eisenstein, E. (1998) Structure and control of pyridoxal
phosphate-dependent allosteric threonine deaminase. Structure 6,
465–475
3
4
5
6
. Singh, S., and Banerjee, R. (2011) PLP-dependent H S biogenesis. Biochim.
2
Biophys. Acta 1814, 1518–1527
20. Drewe, W. F., Jr., and Dunn, M. F. (1985) Detection and identification of
intermediates in the reaction of L-serine with Escherichia coli tryptophan
synthase via rapid-scanning ultraviolet-visible spectroscopy. Biochemistry
24, 3977–3987
. Kabil, O., and Banerjee, R. (2010) The redox biochemistry of hydrogen
sulfide. J. Biol. Chem. 285, 21903–21907
. Abe, K., and Kimura, H. (1996) The possible role of hydrogen sulfide as an
endogenous neuromodulator. J. Neurosci. 16, 1066–1071
. Kabil, O., Vitvitsky, V., Xie, P., and Banerjee, R. (2011) The quantitative
21. Evande, R., Ojha, S., and Banerjee, R. (2004) Visualization of PLP-bound
intermediates in hemeless variants of human cystathionine ␤-synthase:
evidence that lysine 119 is a general base. Arch. Biochem. Biophys. 427,
188–196
significance of the transsulfuration enzymes for H S production in murine
2
tissues. Antioxid. Redox Signal. 15, 363–372
7
. Nagahara, N., Okazaki, T., and Nishino, T. (1995) Cytosolic mercaptopy-
ruvate sulfurtransferase is evolutionarily related to mitochondrial rhoda-
nese. Striking similarity in active site amino acid sequence and the increase
in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-
directed mutagenesis. J. Biol. Chem. 270, 16230–16235
22. Koutmos, M., Kabil, O., Smith, J. L., and Banerjee, R. (2010) Structural
basis for substrate activation and regulation by cystathionine ␤-synthase
(CBS) domains in cystathionine ␤-synthase. Proc. Natl. Acad. Sci. U.S.A.
107, 20958–20963
23. Singh, S., Ballou, D. P., and Banerjee, R. (2011) Pre-steady-state kinetic
analysis of enzyme-monitored turnover during cystathionine ␤-synthase-
8
9
. Shibuya, N., Tanaka, M., Yoshida, M., Ogasawara, Y., Togawa, T., Ishii, K.,
and Kimura, H. (2009) 3-Mercaptopyruvate sulfurtransferase produces
catalyzed H S generation. Biochemistry 50, 419–425
2
hydrogen sulfide and bound sulfane sulfur in the brain. Antioxid. Redox 24. Taoka, S., and Banerjee, R. (2002) Stopped-flow kinetic analysis of the
Signal. 11, 703–714
reaction catalyzed by the full-length yeast cystathionine ␤-synthase.
J. Biol. Chem. 277, 22421–22425
. Singh, S., Padovani, D., Leslie, R. A., Chiku, T., and Banerjee, R. (2009)
Relative contributions of cystathionine ␤-synthase and ␥-cystathionase to 25. Jhee, K. H., Niks, D., McPhie, P., Dunn, M. F., and Miles, E. W. (2001) The
H S biogenesis via alternative transsulfuration reactions. J. Biol. Chem.
reaction of yeast cystathionine ␤-synthase is rate-limited by the conver-
sion of aminoacrylate to cystathionine. Biochemistry 40, 10873–10880
26. Taoka, S., Ohja, S., Shan, X., Kruger, W. D., and Banerjee, R. (1998) Evi-
dence for heme-mediated redox regulation of human cystathionine
␤-synthase activity. J. Biol. Chem. 273, 25179–25184
2
284, 22457–22466
1
1
0. Meier, M., Janosik, M., Kery, V., Kraus, J. P., and Burkhard, P. (2001)
Structure of human cystathionine ␤-synthase: a unique pyridoxal 5Ј-
phosphate-dependent heme protein. EMBO J. 20, 3910–3916
1. Taoka, S., Lepore, B. W., Kabil, O., Ojha, S., Ringe, D., and Banerjee, R. 27. Mudd, S. H., Levy, H. L., and Kraus, J. P. (2001) in The Online Metabolic
(
2002) Human cystathionine ␤-synthase is a heme sensor protein. Evi-
and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly,
W. S., Valle, D., Vogelstein, B., Kinzler, K. W., and Childs, B., eds) pp.
2007–2056, McGraw-Hill, New York
dence that the redox sensor is heme and not the vicinal cysteines in the
CXXC motif seen in the crystal structure of the truncated enzyme. Bio-
chemistry 41, 10454–10461
28. Jhee, K. H., McPhie, P., and Miles, E. W. (2000) Domain architecture of the
heme-independent yeast cystathionine ␤-synthase provides insights into
mechanisms of catalysis and regulation. Biochemistry 39, 10548–10556
29. Woehl, E. U., Tai, C. H., Dunn, M. F., and Cook, P. F. (1996) Formation of
the ␣-aminoacrylate immediate limits the overall reaction catalyzed by
O-acetylserine sulfhydrylase. Biochemistry 35, 4776–4783
1
1
2. Singh, S., Madzelan, P., and Banerjee, R. (2007) Properties of an unusual
heme cofactor in PLP-dependent cystathionine ␤-synthase. Nat. Prod.
Rep. 24, 631–639
3. Kabil, O., Weeks, C. L., Carballal, S., Gherasim, C., Alvarez, B., Spiro, T. G.,
and Banerjee, R. (2011) Reversible heme-dependent regulation of human
cystathionine ␤-synthase by a flavoprotein oxidoreductase. Biochemistry 30. Hwang, C. C., Woehl, E. U., Minter, D. E., Dunn, M. F., and Cook, P. F.
50, 8261–8263
(1996) Kinetic isotope effects as a probe of the ␤-elimination reaction
catalyzed by O-acetylserine sulfhydrylase. Biochemistry 35, 6358–6365
1
4. Weeks, C. L., Singh, S., Madzelan, P., Banerjee, R., and Spiro, T. G. (2009)
Heme regulation of human cystathionine ␤-synthase activity: insights 31. Borcsok, E., and Abeles, R. H. (1982) Mechanism of action of cystathionine
from fluorescence and Raman spectroscopy. J. Am. Chem. Soc. 131,
synthase. Arch. Biochem. Biophys. 213, 695–707
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