A continuous spectrophotometric assay for human cystathionine beta-synthase.

Abstract

We report a new continuous spectrophotometric assay for human cystathionine beta-synthase (hCBS). This assay relies upon the finding that hCBS will take cysteamine in place of L-homocysteine, thereby producing thialysine. Thialysine is, in turn, decarboxylated by lysine decarboxylase, releasing CO2 that is monitored by the sequential action of phosphoenolpyruvate carboxylase and L-malate dehydrogenase. The decrease in absorbance at 340 nm is monitored as reduced nicotinamide adenine dinucleotide is consumed. Using this four-enzyme couple, we find that Km(app) = 1.2+/-0.2 mM for L-serine and 5.6+/-2.2 mM for cysteamine, with kcat = 1.3+/-0.1s(-1) for the formation of thialysine by hCBS. For comparison purposes, the same hCBS reaction was monitored via a radioactive single time point assay using 14C-(C-1)-labeled L-serine and cysteamine as substrates, counting the thialysine product, following ion exchange chromatography. This assay yielded Km(app) = 2.2+/-0.5 mM for L-serine and 6.6+/-2.2 for cysteamine, with kcat = 2.5+/-0.4 s(-1). These numbers indicate that, although it possesses a shortened carbon chain and lacks a carboxyl group, cysteamine displays a catalytic efficiency (kcat/Km) with hCBS that is within an order of magnitude of that observed with its natural thiol cosubstrate, L-homocysteine.

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A continuous spectrophotometric assay for human
cystathionine beta-synthase
Weijun Shena, Molly K. McGatha, Ruby Evandeb, David B. Berkowitza,*
aDepartment of Chemistry, University of Nebraska, Lincoln, NE 68588, USA
bDepartment of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA
Received 18 February 2005
Available online 20 April 2005
Abstract
We report a new continuous spectrophotometric assay for human cystathionine beta-synthase (hCBS). This assay relies upon the
finding that hCBS will take cysteamine in place of L-homocysteine, thereby producing thialysine. Thialysine is, in turn, decarbox-
ylated by lysine decarboxylase, releasing CO2that is monitored by the sequential action of phosphoenolpyruvate carboxylase and
L-malate dehydrogenase. The decrease in absorbance at 340 nm is monitored as reduced nicotinamide adenine dinucleotide is con-
sumed. Using this four-enzyme couple, we find that Kapp
kcat= 1.3 ± 0.1 s?1for the formation of thialysine by hCBS. For comparison purposes, the same hCBS reaction was monitored
via a radioactive single time point assay using14C-(C-1)-labeled L-serine and cysteamine as substrates, counting the thialysine prod-
uct, following ion exchange chromatography. This assay yielded Kapp
kcat= 2.5 ± 0.4 s?1. These numbers indicate that, although it possesses a shortened carbon chain and lacks a carboxyl group, cys-
teamine displays a catalytic efficiency (kcat/Km) with hCBS that is within an order of magnitude of that observed with its natural
thiol cosubstrate, L-homocysteine.
? 2005 Elsevier Inc. All rights reserved.
m¼ 1:2 ? 0:2 mM for L-serine and 5.6 ± 2.2 mM for cysteamine, with
m¼ 2:2 ? 0:5 mM for L-serine and 6.6 ± 2.2 for cysteamine, with
Keywords: Cystathionine beta-synthase; Continuous assay; Cysteamine; Thialysine; Lysine decarboxylase; Phosphoenolpyruvate carboxylase;
Malate dehydrogenase
Cystathionine beta-synthase (CBS;1EC 4.2.1.22) is a
pivotal enzyme in human sulfur metabolism [1,2]. The
transposition of the c-sulfur atom from essential dietary
L-methionine to the b-carbon of L-cysteine is achieved
through the sequential action of two PLP-dependent
enzymes, CBS and cystathionine c-lyase (CGL). CBS
converts L-serine and L-homocysteine into (L,L)-cystathi-
onine and water. It thereby also plays an important role
in the regulation of L-homocysteine levels. Since elevated
levels of total homocysteine correlate with cardiovascu-
lar disease [3–5], neural tube defects [6], and Alzheimer?s
disease [7], there is considerable biomedical interest in
this enzyme. Furthermore, it has recently been shown
that when CBS is exposed to physiological concentra-
tions of L-serine, L-cysteine, and L-homocysteine, 5%
of the cystathionine formed is from L-cysteine [8]. This
‘‘side reaction’’ releases H2S in place of H2O. Observa-
tions such as this suggest another potential role for
CBS in the brain, namely, biosynthesis of the neuro-
modulator H2S [9,10].
0003-2697/$ - see front matter ? 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2005.03.051
*Corresponding author. Fax: +1 402 472 9402.
E-mail address: dbb@unlserve.unl.edu (D.B. Berkowitz).
1Abbreviations used: CBS, cystathionine beta-synthase; Hcy,
homocysteine; PLP, pyridoxal phosphate; CGL, cystathionine gam-
ma-lyase; Tris, tris(hydroxymethyl)aminomethane; LDC, lysine
decarboxylase; PEP, phosphoenolpyruvate; PEPC, phosphenolpyru-
vate carboxylase; MDH, L-malate dehydrogenase; SAM, S-adeno-
sylmethionine; SDS–PAGE, sodium dodecyl sulfate polyacrylamide
gel electrophoresis; Tris, tris(hydroxymethyl)aminomethane; Bis-Tris,
bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane.
ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 342 (2005) 103–110
www.elsevier.com/locate/yabio

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Human CBS (hCBS) is a modular enzyme, compris-
ing an N-terminal heme domain and a C-terminal S-
adenosylmethionine (SAM)-binding regulatory domain,
both flanking a central catalytic core that carries the
essential PLP cofactor [1,2]. In contrast to the mamma-
lian enzyme, the yeast enzyme possesses neither a heme
cofactor nor a SAM binding site. Full-length hCBS is a
tetramer of 63 kDa subunits and tends to aggregate,
hampering crystallization efforts. Limited proteolysis
trims full-length hCBS to a catalytic core (ca. 45 kDa)
that exists as a dimer, shows higher activity than full-
length enzyme, and is not subject to SAM regulation
[11,12]. The truncated version of the protein, lacking
the C-terminal 143–145 amino acids, has been expressed
and similarly results in an active dimer of 48 kDa sub-
units [13,14]. The truncated form of CBS is observed
when liver cells or mice are exposed to proinflammatory
agents and appears to be an adaptive response for
increasing flux through the transsulfuration pathway
in response to oxidative challenge [15]. Two X-ray crys-
tal structures for hCBS have been reported, both for the
truncated dimer [16,17]. The assays described herein are
performed with the highly active hCBS-DC143 dimer.
Heretofore, the human CBS enzyme has typically
been assayed by quantitating the formation of radioac-
tively labeled [14C]cystathionine from labeled L-serine
(Fig. 1), at a fixed time point, following separation of
the two amino acids by ion exchange chromatography,
much as originally described by Mudd et al. [13,18–
20]. Variants of this assay, in which the labeled amino
acids are separated by paper chromatography [21,22]
or thin-layer chromatography [23], have also been re-
ported. A complementary time point assay relies on
the release of radiolabeled water from a-tritiated L-ser-
ine by CBS [23]. A colorimetric time point assay, based
upon the serine sulfhydrylase activity of mammalian
CBS, in vitro, has also been described [24].
Although the14C radioisotopic time point assay is by
far the most common hCBS assay employed, the tedious
nature of this assay is widely acknowledged [2]. This has
served to motivate efforts to develop continuous assays
for CBS. In elegant work, Aitken and Kirsch [25,26]
have recently described the first two such assays for
yeast CBS (yCBS). The forward reaction is monitored
by coupling with two enzymes, namely recombinant
cystathionine beta-lyase and lactate dehydrogenase
(commercially available). The reverse reaction can be
monitored by capturing the homocysteine formed with
Ellman?s reagent. However, this assay is less convenient,
because the reverse reaction is thermodynamically
highly unfavorable (DGrev? +8.5 kcal/mol).
Herein, a complementary coupled continuous assay
for hCBS is described (Fig. 2). The assay takes advanta-
ges of two key observations: (i) cysteamine can be
substituted for L-homocysteine as the thiol cosubstrate
in the hCBS reaction and (ii) thialysine, the product of
that b-replacement reaction, is an alternative substrate
for lysine decarboxylase (LDC). While there were hints
in the literature that CBS and LDC could employ such
unnatural substrates [27,28], these activities were not
well established. Decarboxylation of L-thialysine would
release CO2which could presumably be captured in a
Claisen-type condensation mediated by PEPC. This
would yield oxaloacetate, itself a substrate for malate
dehydrogenase (MDH), which permits convenient mon-
itoring of the reaction at 340 nm, associated with con-
sumption of NADH. Note that the MDH step is
particularly well suited to acting as the final step in this
coupled enzyme assay as, even at pH 8.0, Keqfor this
reaction is approximately 104.1[29], indicating that oxa-
loacetate reduction by NADH is exergonic by some
5.6 kcal mol?1at room temperature.
Materials and methods
Materials
14C-(C-1)-labeled L-serine was purchased from Amer-
ican Radioactive. L-Serine, (D,L)-homocysteine, cyste-
amine, pyridoxal 5-phosphate, phosophoenolpyruvate
(PEP), EDTA, Tris, Bis–Tris, and NADH were from
Sigma–Aldrich. Dowex 50 W · 8 cation exchange resin
(100–200 mesh, hydrogen form) and Econo-columns
were purchased from Bio-Rad. The Ecolite scintillation
cocktail from ICN Biomedicals was used.
Enzymes
L-Malatedehydrogenase(porcineheart)was fromSig-
ma [Nominally: 470 U/mg protein (reported by Sigma
using NADH (0.14 mM), oxaloacetate (2.5 mM) in
Fig. 1. Standard radioisotopic time point assay for measuring hCBS activity. Chromatographic separation of the radiolabeled cystathionine product
from the unreacted labeled serine substrate is required.
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KPO4buffer (50 mM, pH 7.5) at 25 ?C); Found: 210
U/mg protein, wherein one unit oxidizes 1 lmol NADH
(0.4 mM) per minute in the presence of oxaloacetate
(3 mM) at pH 8.0 (200 mM Tris–HCl) and 37 ?C]. Phos-
phoenopyruvate carboxylase (PEPC, Zea mays) was
from Calbiochem [Nominally: 7.4 U/mg protein (re-
ported by Calbiochem using PEP (2 mM), MgSO4
(18 mM), NaHCO3(100 mM), NADH (0.15 mM), and
MDH (50 U/mL) at pH 8.0 (50 mM Tris–HCl) at
30 ?C); Found: 2.1 U/mg protein, wherein one unit cata-
lyzes the formation of 1 lmol of oxaloacetate from PEP
(2 mM), MgCl2(1 mM), NaHCO3(10 mM) at pH 8.0
(200 mM Tris–HCl) at 37 ?C. Oxaloacetate formation
was followed by its reduction with NADH (0.4 mM), as
catalyzed by MDH (2.1 U/0.1 mL)]. Lysine decarboxyl-
ase from Hafnia alvei was purified by a modification of
the published procedure [30,31]. From 84 g of wet cells,
3.6 mgofprotein(homogeneousbySDS–PAGE)wasob-
tained with a specific activity of 115 U/mg of protein.
LDC activity was measured by a modification of the
method of Palcic [32,33]. One LDC unit decarboxylates
1 lmol of
(200 mM Tris–HCl). Protein concentration was deter-
mined bytheLowry method [34].Therecombinantdimer
hCBS-DC143 was expressed as a GST fusion protein in
Escherichia coli and purified according to the procedure
by Taoka et al. [13,20]. The purified dimer displayed a
specific activity of 10 U/mg, wherein one CBS unit cata-
lyzes the formation of 1 lmol of (L,L)-cystathionine per
minute, from L-serine (30 mM) and (D,L)-homocysteine
(60 mM) at pH 8.0 (200 mM Tris–HCl) and 37 ?C.
L-lysine (2 mM) per minute at pH 8.0
pH Profiles of reporting enzymes
MDH. The pH profile of MDH was obtained via the
continuous UV assay described above but using a
100 mM Tris/100 mM Bis–Tris–HCl buffer across the
pH range (6.5–8.5) tested. Percentage activity vs pH
was plotted.
PEPC. The pH profile was carried out using the cou-
pled UV assay described above, except that 100 mM
Tris/100 mM Bis–Tris–HCl buffer was used across the
pH range (6–8.5) tested.
LDC. For the pH profile of LDC the postworkup
dye-based time point assay of Phan et al. [35] was used.
LDC (0.06 U) was incubated with L-lysine (4 mM) at
37 ?C in 100 mM Tris/100 mM Bis–Tris–HCl at the
specified pH. The reaction was quenched after 0, 2,
4, 6, 8, and 10 min with 1 M K2CO3and then heated
for 5 min at 40 ?C with 2,4,6-trinitrobenzenesulfonic
acid. The bis–sulfonamide adduct so formed was ex-
tracted with toluene, and its absorption was measured
at 340 nm. Relative LDC activity was assessed by com-
paring the slopes of linear fits of the absorbance vs
time data thereby obtained across the pH range
studied.
CBS. The pH profile was obtained from a typical
radioactive time point (single time point at 30 min) assay
(vide infra) with the unnatural substrate cysteamine
(30 mM) and
L-serine (30 mM) in 100 mM Tris/
100 mM Bis–Tris–HCl at 37 ?C across the pH range
studied. Following the standard separation procedure,
the concentration of thialysine formed (measured via
Fig. 2. Continuous coupled assay for hCBS. Substitution of cysteamine for the usual thiol substrate L-homocysteine results in the formation of a new
CBS product, thialysine. Decarboxylation of thialysine by LDC releases CO2that is captured by PEPC, giving oxaloacetate. Oxaloacetate, in turn, is
reduced by NADH through the action of MDH. The consumption of NADH with time is conveniently monitored in a UV/vis spectrophotometer.
Spectrophotometric assay for cystathionine beta-synthase / W. Shen et al. / Anal. Biochem. 342 (2005) 103–110
105

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liquid scintillation counting) was determined and plot-
ted against pH.
Titration experiments for the individual reporting
enzymes
These assays were monitored using a Shimadzu UV-
2401PC UV/visible spectrophotometer, outfitted with a
quartz multimicrocell (features 16 parallel microwells
of 100 lL each). Titration experiments were performed
with 30 mM L-serine, 30 mM cysteamine, 1 mM MgCl2,
2 mM PEP, and 0.1 mM PLP and CBS (2.8 lg) at pH
8.0 (200 mM Tris–HCl) and 37 ?C (water-jacketed cell
holder/circulating water bath for temperature control,
100 lL final volume). The following reporting enzyme
concentrations were sampled: LDC (0, 0.15, 0.30, 0.45
to 0.60 thialysine U), PEPC (0, 0.04, 0.08, 0.16, 0.20,
0.28, 0.45, 0.60 U), and MDH (0, 0.5, 1.0, 2.11 U).
[Note: The zero point here is not actually zero, as we
have found that the commercial PEPC (product No.
524810, lot No. B51536) contains MDH (i.e., 0.45 PEPC
U contain 0.03 MDH U)].
Continuous coupled spectrophotometric assay for CBS
The assay was carried out in a 100-lL well of the
multimicrocell containing 2 mM PEP, 0.1 mM PLP,
0.6 mM NADH, 1 mM MgCl2, 30 mM L-serine and
CBS (2.8 lg/mL),with
(0.52 U = 0.31 thialysine U), PEPC (0.45 U), and
MDH (2.11 U) in 200 mM Tris buffer, pH 8.0, at
37 ?C. The reaction was initiated by the addition of
30 mM cysteamine. Note: The buffer itself (comprising
approximately 40% of the final cuvette volume) was
purged with Ar for 15 min prior to use to remove exog-
enous CO2. The multimicrocell was sealed with parafilm,
following addition of cysteamine. A control well, lack-
ing CBS, was run in parallel. The decrease of absor-
bance at 340 nm was recorded. Rates were routinely
corrected for background CO2 (typically <10 mAbs/
min). [Note that while this procedure works well for
the removal of background CO2from buffered solutions
prepared in the laboratory, biological samples might
contain appreciable amounts of bicarbonate. In such
cases, it may be necessary to purge the sample in the
presence of carbonic anhydrase to drive the bicarbonate
to gaseous CO2, which would then be removed by the in-
fused Ar stream.]
couplingenzymesLDC
Radioactive time point assays for CBS
The reaction mixture, containing 60 mM (D,L)-homo-
cysteine (or 30 mM cysteamine), 30 mM14C-1 labeled
L-serine (specific activity typically 20,000 cpm/lmol),
112 lg/mL CBS, 2.5 mM EDTA, and 0.1 mM PLP in
100 mM Tris–HCl buffer (0.5 mL; pH 8.0) was incu-
bated at 37 ?C for 30 min. The reaction was then termi-
nated by addition of 0.5 mL 10% trichloroacetic acid.
Half of the reaction mixture (0.5 mL) was loaded onto
an Econo-column packed with Dowex 50 W · 8 cationic
exchange resin (2 g dry resin per column), and the col-
umn was washed with 20 mL dd water. Then, unreacted
L-serine was eluted with 40 mL 0.4 N HCl. After wash-
ing again with 20 mL water, the product was eluted with
4 · 4 mL of 4 N NH4OH. Each basic fraction (4 mL)
was diluted with scintillation cocktail (Ecolite, 15 mL)
and counted (Packard Model 1900 CA Tricarb scintilla-
tion counter), after standing overnight. The control
reaction, lacking CBS, was run with every set of assays.
When the concentration of radiolabeled L-serine was
varied (i.e., for Kmdeterminations), separate controls
were run for each serine concentration. However, in
cases where the thiol (cysteamine or homocysteine) con-
centration was varied, a single control experiment suf-
ficed, as tests indicated an invariant background count
in the ‘‘second elution peak’’ regardless of (cold) thiol
concentration.
Results and discussion
To assess the feasibility of the coupled CBS assay de-
picted in Fig. 2, a pH/rate profile of each enzyme in the
couple was determined. Where possible, a single enzyme
assay was used here to insure that the pH effects ob-
served reflect properties of the enzyme being interro-
gated. Single-enzyme assays were employed for CBS,
LDC, and MDH, whereas PEPC was coupled with
MDH. In all cases, a mixed Tris (pKa= 8.1)/Bis–Tris
(pKa= 6.5) buffer was chosen to build-in buffering
capacity across the pH range under study, without the
need to alter buffer composition. The results are
presented in Fig. 3 and indicate a relatively broad pH
Fig. 3. pH profile of hCBS and the reporting enzymes for the coupled
assay.
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Page 5

optimum for LDC, stretching from pH 6 to 8. The other
three enzymes display peak activity at pH 8 ± 0.5. Given
that most reported kinetic data for CBS are at pH P8,
these results are promising.
It was thus decided to fix the pH at 8.0 for this cou-
pled spectrophotometric assay and to titrate the three
reporting enzymes up to levels where they would not
be partially rate-limiting. In Fig. 4, the response of the
overall rate to changes in the individual reporting en-
zyme levels is plotted. It is worth noting that this cou-
pled assay can be conveniently run in a commercial
multimicrocell (Shimadzu) that features 16 parallel
100-lL miniwells. Beginning with MDH (Fig. 4A) and
working backward toward LDC (Fig. 4C), the reporting
enzymes were brought up to kinetically saturating levels,
one at a time.
Having established conditions under which the cou-
pled assay is dependent on the activity of hCBS, we next
evaluated the dependence of the assay on the concentra-
tion of hCBS itself (Fig. 5A). Particularly noteworthy
Fig. 4. Titration of the reporting enzymes: (A) variation of added
MDH concentration with LDC (0.52 U) and PEPC fixed (0.45 U).
Note that a substantial rate is seen with no added MDH, as commercial
PEPC (Calbiochem) already contains MDH activity (approximately
0.03 U per 0.45 U PEPC). (B) Variation of PEPC concentration with
LDC (0.52 U) and MDH (2.1 U) fixed. (C) Variation of LDC
concentration with PEPC (0.45 U) and MDH (2.1 U) fixed. All assays
are performed in 100 lL microwells containing 2.8 lg hCBS at pH 8
and 37 ?C, as described under Materialsand methods. Rates are plotted
as absolute values, as one observes the decrease in absorbance at
340 nm as NADH is consumed in the MDH step.
Fig. 5. (A) Response of the coupled UV assay to hCBS concentration.
Shown are the primary data for NADH oxidation with time, under
typical assay conditions [30 mM L-serine, 30 mM cysteamine, 1 mM
MgCl2, 2 mM PEP, 0.1 mM PLP, PEPC (0.45 U), LDC (0.52 U), and
MDH (2.1 U) in 200 mM Tris buffer, pH 8.0] with increasing hCBS
concentration [rate increases in the order: 0 (control; open triangles),
0.7 (filled circles), 1.4 (open squares), 2.1 (filled triangles), 2.8 (open
diamonds), and 3.5 lg (filled diamonds); all in a 100-lL final volume].
(B) Data replotted as rate vs [CBS].
Spectrophotometric assay for cystathionine beta-synthase / W. Shen et al. / Anal. Biochem. 342 (2005) 103–110
107

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here is the continuous nature of this assay, in sharp con-
trast to the single product concentration vs time infor-
mation that is gleaned from the radioisotopic assay
normally employed for hCBS. In Fig. 5B, the slopes of
each of the primary traces are replotted as a function
of the concentration of hCBS. A linear dependence of
observed rate upon hCBS concentration is observed.
Since the new assay employs an unnatural cosub-
strate, cysteamine, the efficacy of the latter was tested
in a single time point radiometric assay, analogous to
the standard Mudd assay with homocysteine [18]. The
ion exchange column profile (Fig. 6A) reveals baseline
separation for the natural radiolabeled amino acid sub-
strate (L-serine) and product ((L,L)-cystathionine) under
the conditions of this assay. Since L-thialysine is more
basic than (L,L)-cystathionine, it is also readily separated
from L-serine (Fig. 6B).
Next, Michaelis–Menten kinetic analyses for both
the natural (L-serine/homocysteine) substrate pair and
the unnatural (L-serine/cysteamine) pair were con-
ducted, using this single time point assay. The catalytic
efficiencies and individual kinetic constants thereby
obtained are collected in Table 1. The corresponding
kinetic parameters from the literature (for homocyste-
ine) and from the continuous UV assay reported herein
(for cysteamine) are also included for comparison. The
values that we obtain for Km(L-Ser), Km(L-homocyste-
ine), kcat, and the catalytic efficiency (kcat/Km) for hCBS
(entry 3) fall within the range of the values previously
reported by others (entries 1 and 2). For the new thiol
cosubstrate, cysteamine, we obtain a kcatthat is 3- to 6-
fold lower than that observed with homocysteine (en-
tries 4 and 5). The Kmvalues obtained for cysteamine
are 1.5- to 1.8-fold higher than that seen for homocys-
teine. However, the Kmvalue observed for L-serine is 2-
to 4-fold lower with the unnatural second substrate
than with homocysteine itself. Taken together, these
data indicate that cysteamine is a useful unnatural sub-
strate for CBS, with a catalytic efficiency (kcat/Km) that
either is comparable to that seen with homocysteine (if
one uses the L-serine Kmvalues) or is within an order of
magnitude of it (if one uses the thiol cosubstrate Km
values).
Fig. 6. Dowex 50 column profiles for the separation of radiolabeled L-
serine (first peak) from radiolabeled hCBS product (second peak). In
(A) 30 mM L-homocysteine was used as thiol cosubstrate providing
(L,L)-cystathionine as product. On the other hand, (B) shows the result
of substituting cysteamine for L-homocysteine, yielding L-thialysine as
the hCBS product. In each case, the assay was run in duplicate, and a
control containing all components but hCBS was run. The reactions
were run at 37 ?C for 30 min, processed, chromatographed, and
counted as described under Materials and methods.
Table 1
Summary of kinetic parameters for C-terminal-truncated hCBS
Km(L-Ser) (mM)
Km(L-Hcy) (mM)
Kmcysteamine (mM)SA (lmol h?1mg?1)
kcat(s?1)
kcat/Kmf(mM?1s?1)
(1) Refs. [1,13]
(2) Ref. [11]
(3) Mudd assay (L-Hcy)a
(4) Mudd assay (cysteamine)b
(5) Continuous UV assay
18 ± 8
2.7
4.9 ± 0.6c
2.2 ± 0.5d
1.2 ± 0.2d
9.7 ± 4.3
0.5
3.7 ± 0.5e
750 ± 33010 ± 4.4
6.7
7.5 ± 0.3c
2.5 ± 0.4d
1.3 ± 0.1d
0.6
2.6
1.5
1.1
1.0
566 ± 20c
188 ± 26d
93 ± 4
6.6 ± 2.2e
5.6 ± 2.2e
aIn this assay, CBS activity is monitored by counting of radioactive (L,L)-cystathionine, as derived from14C-L-serine and L-homocysteine, as
described.
bIn this assay, CBS activity is monitored by counting of radioactive L-thialysine, as derived from14C-L-serine and cysteamine, as described under
Materials and methods.
cIn these runs, the (D,L)-homocysteine concentration was fixed at 60 mM.
dIn these runs, the cysteamine concentration was fixed at 30 mM.
eIn these runs, the L-serine concentration was fixed at 30 mM.
fKmfor L-Ser (available for all assays) is used to compare the ‘‘catalytic efficiency’’ function across the table.
108
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In summary, a continuous UV assay for hCBS is re-
ported. Nearly all previous assays of hCBS have used
the natural substrates
L-serine and
The former is used to generate a Michael acceptor in
the active site, in the form of a PLP-bound amino acry-
late intermediate. The latter acts as a thiol nucleophile to
capture that electrophilic intermediate, in a Michael
sense. We reasoned that cysteamine might serve as a
homocysteine surrogate, being a comparable nucleo-
phile and being only one methylene unit short of the nat-
ural substrate, though lacking the carboxyl group.
As is demonstrated by steady state kinetic analysis
(see the data in Table 1), cysteamine does indeed serve
as a very good ‘‘second substrate’’ for hCBS, thereby
generating the targeted unnatural enzymatic product L-
thialysine. This reaction can then be effectively linked
to a three-enzyme couple for L-lysine decarboxylase
activity [32,33], because L-thialysine, in turn, is an excel-
lent substrate for LDC. This coupled hCBS assay af-
fords several conveniences in comparison to the
radioactive time point assay commonly employed here-
tofore to assess hCBS activity. Specifically, the need to
quench the reaction, take aliquots, and do chromatogra-
phy is obviated, and a much more continuous readout of
product concentration with time is obtained.
L-homocysteine.
Acknowledgments
The authors thank the American Heart Association
(AHA GIA 0350404N) for support of this research.
D.B.B. thanks the Alfred P. Sloan Foundation for a fel-
lowship. M.K.M. acknowledges support from a Univer-
sity of Nebraska for a UCARE (Undergraduate
Creative Activities & Research Experiences) Fellowship.
We are grateful to Professor Ruma Banerjee for provid-
ing the hCBS-DC143 clone and for useful conversations.
Kannan R. Karukurichi and Roberto de la Salud-Bea
are thanked for LDC purification.
References
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