From Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, 82152 Planegg-Martinsried, Germany
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ABSTRACT |
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The gene encoding human cystathionine Transsulfuration and reverse transsulfuration constitute part of
the metabolic interconversion of the sulfur-containing amino acids
cysteine and methionine (Fig. 1). The
forward pathway, the transformation of cysteine into homocysteine via
the intermediate L-cystathionine is catalyzed by the
sequential action of the enzymes cystathionine -lyase
was cloned from total cellular Hep G2 RNA. Fusion to a T7 promoter
allowed expression in Escherichia coli, representing the
first mammalian cystathionine
-lyase overproduced in a bacterial
system. About 90% of the heterologous gene product was insoluble, and
renaturation experiments from purified inclusion bodies met with
limited success. About 5 mg/liter culture of human cystathionine
-lyase could also be extracted from the soluble lysis fraction,
employing a three-step native procedure. While the enzyme showed high
-lyase activity toward L-cystathionine
(Km = 0.5 mM,
Vmax = 2.5 units/mg) with an optimum pH of 8.2, no residual cystathionine
-lyase behavior and only marginal
reactivity toward L-cystine and L-cysteine were detected. Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and
aminoethoxyvinylglycine. Propargylglycine inactivated human
cystathionine
-lyase much more strongly than trifluoroalanine, in
agreement with the enzyme's preference for C-
-S bonds.
Aminoethoxyvinylglycine showed slow and tight binding characteristics
with a Ki of 10.5 µM, comparable with
its effect on cystathionine
-lyase. The results have important
implications for the design of specific inhibitors for transsulfuration components.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lyase
(CBL)1 and cystathionine
-synthase (CGS) and has been identified in bacteria, fungi, and
plants. Conversely, reverse transsulfuration, catalyzed by the enzymes
cystathionine
-synthase and cystathionine
-lyase (CGL), is known
only in fungi and mammals (1, 2). Actinomycetes species present a
notable exception to this rule (1). The four enzymatic transsulfuration
components are all pyridoxal 5'-phosphate (PLP)-dependent
enzymes, but they pertain to different structural groups; CBL, CGS, and
CGL show extensive sequence homology and are members of the PLP
-family (Ref. 3; Fig. 2), while
cystathionine
-synthase is unrelated and belongs to the
-family.
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Fig. 1.
Overview of the transsulfuration
reactions. Enzymes catalyzing key steps are indicated. The
left panel represents reactions occurring in
bacteria, plants, and fungi; the right panel
shows those found in fungi and mammals.
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Fig. 2.
Best fit alignment of the amino acid
sequences of human CGL, rat liver CGL, yeast CGL, streptomyces CGL,
E. coli CGS, and E. coli CBL.
Identical residues are colored green, and those
identical within the group of CGLs are additionally boxed.
Nonidentical but homologous residues are colored
red. Catalytically critical residues and the domain
organization as deduced from the E. coli CBL crystal
structure (4) are indicated with arrows and bars,
respectively.
The high resolution crystal structures of Escherichia coli
CBL (4) and CGS (5), together with crystallographic (6) and kinetic
investigations (6-9) on inhibitors, allowed the suggestion and
evaluation of reaction mechanisms (4, 10). Both CBL and CGS are
homotetramers composed of ~40-45-kDa subunits and carry one PLP
cofactor per monomer covalently bound via a Schiff base to an active
site lysine. A similar situation has been found for CGL (1, 11, 12). In
the present paper, we extend our structure-function analyses to human
CGL (EC 4.4.1.1; unless otherwise stated, "CGL" refers to human
cystathionine -lyase). CGL catalyzes the second step in the reverse
transsulfuration pathway, i.e. the cleavage of the
L-cystathionine C-
-S bond, yielding
L-cysteine,
-ketobutyrate, and ammonia (Fig. 1). In
humans, the enzyme is linked to cystathioninuria, cystinosis, and
(although less frequently than cystathionine
-synthase (13))
homocystinuria (14). These metabolic disorders potentially result in
mental or physical impairments. In addition, malignant lymphoic cells
show markedly reduced levels of CGL and do not grow in media devoid of
L-cystine (14-16). Therefore, observations of
L-cyst(e)ine conversion by CGL from various species (1, 11,
14, 17, 18) suggested the possibility of exploiting the enzymatic
activity for cyst(e)ine depletion in blood (14). Alternatively,
intracellular inhibition of the enzyme could block a metabolic route
toward L-cyst(e)ine (14). Furthermore, since human hepatic
CGL activity levels are markedly reduced compared with those in rat
liver, CGL could be a rate-limiting factor in human liver glutathione
synthesis (through the limited availability of cysteine), which could
be ultimately responsible for a susceptibility to alcoholic liver
damage (Ref. 19 and references therein).
Despite the central metabolic role of CGL and its link to a broad
pathological spectrum, until now detailed mechanistic or structural
data on the human enzyme are lacking. Such studies have been severely
hampered by the small overall yields of native isolations (14). While
nonrecombinant CGL purifications from several species have been
described (neurospora (20), streptomyces (1), and rat (14, 17)), the
yeast enzyme so far is the only one that has been heterologously
expressed in E. coli (11). The reported CGL preparations
have not been exhaustively characterized, particularly with respect to
substrate specificities and the enzymes' responses toward inhibitors.
Inhibition of the transsulfuration enzymes is of interest because the
different enzymatic spectra displayed by different organisms (Fig. 1)
could be exploited for the development of new antibiotics and
herbicides. Inhibition studies were so far focused on CGS and CBL, the
enzymes of methionine biosynthesis in microorganisms and plants,
employing the mechanism-based inhibitors ,
,
-trifluoroalanine
(F3Ala; Ref. 7), propargylglycine (PG; Refs. 9, 21, 22),
and aminoethoxyvinylglycine (AVG; Refs. 6 and 8). Deeper insight into
the catalytic mechanisms and substrate recognition properties of CGL
should be helpful, e.g. for the development of pesticides
that are not harmful to humans.
Recently, the gene for human CGL has been cloned and sequenced (19). We
therefore set out to develop an expression system that would supply a
sufficient amount of the enzyme for mechanistic and structural
investigations. Herein, we describe the heterologous expression of
human CGL in E. coli and introduce a three-column purification protocol that yields large amounts of apparently homogenous gene product. The product's enzymatic properties and its
behavior toward inhibitors suggest possible guidelines for the
development of specific inactivators and warrant caution in the use of
certain substances for herbicides and antibiotics.
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EXPERIMENTAL PROCEDURES |
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Chemicals and Instrumentation-- Unless otherwise noted, all chemicals used were of analytical grade as supplied by Sigma/Fluka (Deisenhofen, Germany) or Merck AG (Darmstadt, Germany). F3Ala was a kind gift from AgrEvo (Frankfurt am Main, Germany). Human Hep G2 cells were kindly provided by Dr. P. Habenberger (Max-Planck-Institut für Biochemie, Martinsried, Germany). DNA oligonucleotides were ordered high pressure liquid chromatography-purified from MWG-Biotech GmbH (Ebersberg, Germany). Reverse transcriptase (RT) was obtained from Roche Molecular Biochemicals (Mannheim, Germany); Pfu DNA polymerase was from Stratagene (Heidelberg, Germany); and restriction enzymes and T4 DNA ligase were from New England Biolabs (Schwalbach, Germany). Chromatography columns were self-made and hand-packed with materials from Amersham Pharmacia Biotech (Uppsala, Sweden) and driven by peristaltic pumps and stirred gradient mixers. Automated dideoxynucleotide DNA sequencing (23) was performed in 25-µl polymerase chain reactions (PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit, Perkin Elmer, Überlingen, Germany) under supplier-recommended conditions and analyzed by Dr. M. Boicu (Max-Planck-Institut für Biochemie). N-terminal peptide sequencing was carried out by Dr. K.-H. Mann (Max-Planck-Institut für Biochemie). UV-visible spectra were recorded with a Beckman (München, Germany) DU 7500 diode array spectrophotometer. Kinetic data were analyzed with the program KaleidaGraph (Abelbeck Software, University of California, San Franscisco, CA). SDS gels were prepared and run according to Laemmli (24) and stained with Coomassie Brilliant Blue R-250. Analytical gel filtration experiments were performed with a 2.4-ml Superose-12 column on a SMARTTM system (Amersham Pharmacia Biotech).
Preparation of Total Cellular RNA, Cloning, and
Sequencing--
Total RNA from Hep G2 liver cells (ATCC-No. HB-8065)
was prepared according to Chirgwin et al. (25). Briefly,
after quantitative removal of the culture supernatant, 300 cm2 of confluent cells were lysed with 4 ml of denaturing
solution (4 M guanidinium thiocyanate, 25 mM
sodium citrate, 0.5% (w/v) N-lauroylsarcosine, 0.1 M 2-mercaptoethanol). The DNA was sheared by passing the
lysate 10 times through a pipette. After the addition of 0.1 g of
CsCl per ml of lysate, the solution was layered over a cushion of 9 ml
of 5.7 M CsCl in a SW28 tube (Beckman) and centrifuged at
22 °C overnight at 113,000 × g. The supernatant was
removed, and the RNA pellet was immersed overnight at 4 °C in 3 ml
of 5 mM EDTA, 0.5% (w/v) N-lauroylsarcosine,
and 5% (v/v) 2-mercaptoethanol. After the addition of 0.1 volume of 3 M sodium acetate, pH 5.2, the RNA was precipitated with 2.5 volumes of ethanol at 70 °C for 30 min. The total RNA was
centrifuged at 15,000 × g for 15 min, dried,
resuspended in water, and stored at
70 °C. The presence of RNA in
the required size range was confirmed by urea polyacrylamide gels with
16 and 23 S ribosomal RNA as standards. 100 pmol of an
oligo(dT)15 primer served for first strand cDNA
synthesis with 25 µg of total Hep G2 RNA in a 50-µl reaction. Other
components and reaction conditions were as suggested by the RT
supplier. Second strand cDNA synthesis and amplification of the
product were performed via polymerase chain reaction (26, 27) in
50-µl reaction volumes with 5 µl of the column-purified RT reaction mix as the template (30 polymerase chain reaction cycles: 30 s at
95 °C, 60 s at 55 °C, 12 min at 68 °C) and 100 pmol of
5'-overhang primers (forward primer,
5'-CGATGGGTACCATATGCAGGAAAAAGACGCCTCCTCACAAGG-3'; reverse primer,
GCGGTACCTCGAGTTACTACTGTGAATTCCACTTGGAGGGTGTGCTGCC-3'; sequences corresponding to the human CGL gene are italicized). The primers contained restriction enzyme recognition sites (forward primer NdeI; reverse primer XhoI; underlined) for
subsequent cloning into a pET22b(+) (Novagen, Madison, WI) expression
vector. The amplified cDNA and the vector preparation were digested
overnight with NdeI/XhoI at 37 °C and purified
via a 1% agarose gel. Ligation occurred overnight at 16 °C.
Individual colonies were obtained from E. coli XL1-Blue
cells electroporated with the ligation mixture, grown on LB plates
supplemented with 100 µg/ml ampicillin (LB-Amp100), and propagated in
liquid LB-Amp100 medium for plasmid preparations. Potentially positive
clones were identified through restriction digests and verified by
automated Sanger dideoxynucleotide sequencing. In a similar fashion,
the CGL gene was cloned behind a Ptac promoter in the pCYB1
expression vector (New England Biolabs).
Heterologous Expression in E. coli--
40 µl of
electrocompetent BL21(DE3) E. coli cells were electroporated
with 0.5 µg of plasmid DNA at 18,000 V/cm (Electroporator 1000, Stratagene). Transformants were rescued in 1 ml of LB for 1 h at
37 °C and used to inoculate an overnight 100-ml LB-Amp100 starter
culture. 3 ml of the starter culture were propagated in 3-liter
Erlenmeyer flasks with 650 ml of LB-Amp200. After shaking at room
temperature to an A595 of ~0.7, 1 mM isopropyl--D-thiogalactopyranoside was
added, and the cultures were maintained at room temperature for another
12 h. Cells were harvested by centrifugation in a Beckman JS4.2
rotor at 4200 rpm for 30 min, resuspended in 7 ml/1 liter original
culture of resuspension buffer (50 mM KPi
buffer, pH 8.0, 0.5 mM PLP, and 2 mM EDTA), and
stored at
70 °C until further processing. 15% polyacrylamide SDS
gels showed a strong expression band at about 45 kDa (Fig.
3b), which was roughly
distributed in a 10:90 ratio between soluble and insoluble fractions,
respectively, as estimated by the Coomassie stain.
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Purification under Native Conditions--
Cells from the 6-liter
overnight culture were thawed and, after the addition of 0.1 mM phenylmethylsulfonyl fluoride, incubated with 0.3 mg/ml
hen egg white lysozyme for 30 min at room temperature. Cell lysis and
DNA shearing were completed by 15-min sonification on ice using the
50% pulsed maximum output of a Branson (Danbury, CT) sonifier equipped
with a macrotip. All subsequent steps were performed with the protein
on ice or at 4 °C. The lysate was cleared for 45 min at 20,000 rpm
in a Beckman JA25.50 rotor. The supernatant was applied to a 300-ml
DEAE-Sepharose FF column, equilibrated with buffer A (20 mM
KPi buffer, pH 8.0, 0.1 mM PLP, 2 mM EDTA). A linear 1-liter gradient run at 8 ml/min to
buffer A plus 0.5 M ammonium sulfate eluted CGL at about
0.1 M salt. Fractions containing CGL were identified via
enzymatic assays (see below) and SDS-polyacrylamide gels. The DEAE pool
was adjusted to 1.0 M ammonium sulfate, loaded on a 100-ml
phenyl-Sepharose HP column, equilibrated with buffer A plus 1.0 M ammonium sulfate, and eluted at 4 ml/min with a linear 600-ml gradient to buffer A. Peak fractions were identified as before,
pooled, and concentrated to 20 mg/ml using an Amicon 30-kDa ultrafiltration membrane (Millipore, Eschborn, Germany). The
concentrated pool was applied to a 300-ml Sephacryl S200 size exclusion
column and chromatographed in 10 mM KPi, pH
8.0, 10 µM PLP at a flow rate of 1 ml/min. The product
was located in the fractions as before, pooled, and concentrated to 20 mg/ml with Centriprep-30 concentrators (Amicon). Concentrated pools
were shock-frozen in liquid nitrogen for storage at 70 °C. The
catalytic activity of the final product did not degrade for several
months at this temperature, and the protein diluted to 6 mg/ml was
stable at 4 °C for at least 14 days. A typical purification run
yielded ~5 mg of CGL per liter of original culture. The efficiency of individual purification steps is summarized in Table
I.
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Preparation of Inclusion Bodies and Renaturation-- The CGL-expressing cells were harvested and lysed as before. The pellet from the cell lysis was resuspended in 50 mM sodium phosphate, 300 mM NaCl, pH 7.8, 0.5% N,N-dimethyldodecylamine-N-oxide, sonicated (Branson, macrotip, 80% pulsed, 80% output for 15 min), and again centrifuged. Washing was repeated once with N,N-dimethyldodecylamine-N-oxide and twice without detergent. 150 mg of purified inclusion bodies were dissolved in 10 ml of solubilization buffer (6 M guanidinium hydrochloride, 150 mM NaCl, 2 mM EDTA, 20 µM PLP, 5 mM 1,4-dithiothreitol, 100 mM Tris/HCl, pH 8.5), slowly dripped overnight at 4 °C into a 500-ml reservoir of renaturation buffer (150 mM NaCl, 2 mM EDTA, 20 µM PLP, 5 mM 1,4-dithiothreitol, 100 mM Tris/HCl, pH 8.5), and subsequently dialyzed against three changes of 4 liters of (a) 150 mM NaCl, 2 mM EDTA, 20 µM PLP, 100 mM Tris/HCl, pH 8.5, (b) 50 mM NaCl, 2 mM EDTA, 10 µM PLP, 100 mM Tris/HCl, pH 8.5, and (c) 50 mM NaCl, 2 mM EDTA, 10 µM PLP, 20 mM Tris/HCl, pH 8.5. The soluble supernatant was then passed over a 50-ml DEAE-Sepharose FF column at 4 ml/min with a 400-ml linear gradient from 50 mM NaCl, 2 mM EDTA, 10 µM PLP, 20 mM Tris/HCl, pH 8.5, to the same buffer containing 0.5 M ammonium sulfate. Analysis of the fractions was performed as before. Similar renaturation trials were attempted using renaturation buffers with 1.2 M L-arginine and direct dialysis against guanidinium-free buffers.
Protein Determination--
Protein concentrations were measured
by the method of Bradford (28) with a bovine serum albumin standard
curve. For calculation of kinetic constants, CGL concentrations were
determined by the 280-nm absorption using the calculated molar
extinction coefficient 280 = 30,470 M
1 cm
1 and a subunit molecular
weight of 44,534.
Determination of the pH Optimum-- Solutions of 0.8 mM L-cystathionine in 40 mM borate buffer at pH 7.8 and 9.4 were titrated against each other to build a pH profile. 985 µl of these buffers were mixed with 10 µl of 0.1 M DTNB in ethanol and 5 µl of enzyme (6 mg/ml). The linear portion of the reaction time course was monitored at 30 °C for 60 s through the development of a 412-nm absorption. Subsequent kinetic analyses were performed at the determined optimum pH of 8.2 for the native enzyme preparation.
Michaelis-Menten Kinetics--
Detailed kinetic analyses were
only performed with CGL purified under native conditions. Time courses
for the CGL reaction were monitored at 30 °C for 1 min using the
above direct colorimetric assay with DTNB. The 1-ml reactions contained
985 µl of 40 mM borate buffer, pH 8.2, adjusted to
L-cystathionine concentrations between 0.03 and 3.0 mM, 5 µl of CGL (6 mg/ml), and 10 µl of 0.1 M DTNB in ethanol. After rapid mixing, the development of
the 412-nm absorption was followed for 1 min. Kinetics were linear for
this time and throughout this concentration range. Data were plotted in
reciprocal form according to Eadie and Hofstee (29, 30) to extract
values for Km and Vmax. To
evaluate the substrate specificity of CGL, identical experiments were
performed in which L-cystathionine was replaced by
L-cystine or L-cysteine. L-Cysteine
levels were also monitored in a discrete fashion as described below.
L-Cystine conversion was followed by the above DTNB assay
and another continuous examination employing lactate dehydrogenase to
detect -ketoacids produced in the reaction via oxidation of NADH
(20).
Monitoring of Cysteine Levels--
L-Cysteine was
quantitated according to Gaitonde (31). 80-µl samples were removed
from CGL reactions (borate buffer with L-cystathionine and
enzyme) and mixed with 30 µl of 20% trichloroacetic acid, and the
precipitated protein was removed by centrifugation. 80 µl of the
terminated reactions were mixed with equal amounts of acetic acid and
ninhydrin reagent (250 mg of ninhydrin dissolved in 6 ml of acetic acid
and 4 ml of hydrochloric acid), boiled for 10 min, and diluted with 1 ml of 95% ethanol. Development of the pink color was monitored at 560 nm. The amount of L-cysteine was calculated using the
published molar extinction coefficient, 560 = 2.6 × 104 M
1 cm
1. A
standard curve with known amounts of L-cysteine in reaction buffer, treated in the same way as the reactions, served to verify the
linearity of the response in the relevant concentration range. References contained all buffer and reaction components with the exception of L-cysteine or L-cysteine-producing
precursors. Under the given conditions, L-homocysteine,
L-cystine, and L-methionine do not show a
similar response (31).
Inhibition Kinetics-- Inhibition of CGL by AVG was detected in 30-min time courses, monitored by the DTNB assay. Reaction buffers contained saturating amounts of L-cystathionine (2.5 mM) in 40 mM borate buffer, pH 8.2, and varying levels of inhibitor. The resulting progress curves were analyzed according to Cha (32, 33), Morrison (34), and Morrison and Walsh (35).
Inactivation of CGL by PG and F3Ala was studied by reacting
2.5 mM L-cystathionine in 40 mM
borate buffer, pH 8.2, with set amounts of CGL, preincubated for given
times at 37 °C with varying amounts of the inhibitors. Reactions
were monitored for 60 s in the continuous DTNB assay. From the
activity remaining at various times after incubation with set amounts
of F3Ala, the half-times of inactivation
(t1/2) were determined. A Kitz-Wilson analysis (Ref.
36; inverse inhibitor concentration against t1/2)
was used to extract the inhibition constant, Ki, and
the rate of inactivation, kinact. To analyze the
inactivation by PG, normalized activities, remaining after given times
of incubation, were plotted against the relative concentrations of PG
and enzyme active sites.
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RESULTS |
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Cloning and Recombinant Expression of Human CGL-- After 30 cycles of polymerase chain reaction and restriction digestion, a single product of the expected size (1218 base pairs) was visible on ethidium bromide-stained agarose gels (Fig. 3a). The cloned full-length CGL gene corresponded to one of the published sequences (19). Previously, similar cloning strategies have met with a second product with an internal 132-base pair deletion compared with the rat liver CGL gene (19). We could not detect such a heterogeneity in the present RT assays. CGL shows homology throughout its entire length not only to CGL from other species but also to CGS and CBL (Fig. 2), two other enzymes of the transsulfuration pathways, and from the crystal structures of E. coli CBL (4) and CGS (5), specific functions can be attributed to the domains and key residues of the protein and its relatives. The observed deletion in the shorter CGL mRNA species would correspond to a severe trimming of the PLP binding domain including several catalytically indispensable residues (Fig. 2). Presumably, an enzyme with the detected deletion would be inactive.
Expression of CGL in E. coli under control of a T7 promoter
was, at various culture conditions, reproducibly accompanied by the
formation of insoluble inclusion bodies. In order to enhance solubility
by reducing the expression level, fermentation was performed at
isopropyl--D-thiogalactopyranoside concentrations of
0.1-1 mM, at varying temperatures (18, 25, 37 °C), and
with the gene under control of a Ptac-promoter in several
E. coli strains (BL21(DE3), XL1-Blue, and DH5
). Neither
alone nor in combination did these factors significantly influence the
degree of solubility of CGL. Induction of the Ptac
construct produced a soluble ~50-kDa band on SDS gels, which was
characterized as E. coli tryptophanase (not shown), while no
overexpression corresponding to CGL was observed. We do not have an
explanation for our failure to express CGL with the latter promoter
system. All further results therefore refer to enzyme produced with the
T7-promoter system.
Purification and Biophysical Characterization--
In order to
have at hand large amounts of native CGL for detailed mechanistic and
structural studies, we developed a swift purification procedure for the
soluble fraction of the protein (Table I). The product was over 95%
pure as judged from Coomassie-stained SDS-polyacrylamide gels (Fig.
3b). The integrity of the purified samples was attested by
their strong activity toward the in vivo CGL substrate
L-cystathionine (see below) and by UV-visible spectra showing the expected maxima at 280 nm (protein) and 427 nm
(protein-bound PLP) (Fig. 4). Considering
the similar protein sizes of the rat and human enzymes and the
abundance of tryptophan residues (rat liver CGL: 2; human CGL: 3), the
280 nm/427 nm ratio of human CGL (7.5) is comparable with the value of
5 seen with rat liver CGL (17). The absorption spectra of the enzyme
were virtually indistinguishable in borate, phosphate, or Tris buffers.
An additional minor maximum was observed at 494 nm (Fig. 4). When CGL
apoenzyme was prepared by extensive dialysis against buffer containing
L-alanine, both long wavelength absorption maxima
disappeared, suggesting that they are both PLP-related and not due to
contaminants (Fig. 4). This conclusion is in agreement with the unique
band on SDS gels and the unequivocal N-terminal sequence of the
product. However, when PLP was reincorporated into the CGL apoenzyme by
dialysis and gel filtration, the 494-nm absorption band did not
reappear.
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Elution times of the purification product on an analytical Superose-12 size exclusion column were identical within experimental error to those of CGS from E. coli (monomer molecular mass ~40 kDa; not shown) which has been proven to exist in tetrameric form (10, 37). This shows that, analogous to the latter enzyme, CGL forms a homotetramer under native conditions, consistent with previous reports from other sources (1, 11, 12).
Because of the large amount of CGL deposited in insoluble form, we also
attempted denaturing purification with subsequent in vitro
folding of the protein. Fast dilution into guanidinium-free buffers
yielded a soluble protein, verified as CGL by N-terminal sequencing.
However, instead of the strong absorption at 427/494 nm due to the PLP
cofactor, UV-visible spectra of the backfolded fraction showed a
smaller absorption at 326 nm. The pH optimum was observed at pH 8.7, almost half a pH unit higher than that of the native protein (see
below) and was not as clearly defined (Fig.
5a), while the catalytic
efficiency, defined by
Vmax/Km, of the folded enzyme
(Km = 0.8 mM,
Vmax = 0.4 unit/mg) was about 1 order of
magnitude smaller than that of the native product (see below). The
almost parallel lines obtained in double reciprocal plots (Fig.
5b; see below) for the native and the folded preparations
suggest that only a minor fraction of the renatured samples was
correctly folded. We refrained from additional purification steps
because of the small expected yields. All of the following analyses
refer to the native purified CGL.
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Kinetic Analyses--
The pH optimum of the CGL reaction with
L-cystathionine was determined in borate buffer and located
at pH 8.2 (Fig. 5a). Contrary to previous reports for rat
CGL (12), we have not found pronounced differences when other buffers
(Tris, phosphate) were used, consistent with the spectral results. All
subsequent kinetic data were recorded in borate buffer, pH 8.2. Km and Vmax values for human CGL with respect to the in vivo substrate
L-cystathionine were extracted from double reciprocal plots
(Fig. 5b; Km = 0.5 mM,
Vmax = 2.5 units/mg), and
kcat was calculated to 1.9 s1.
Interestingly, despite their highly conserved features (Fig. 2) and
similar catalytic efficiencies with respect to the in vivo substrate L-cystathionine, the CGL enzymes from different
species exhibit order-of-magnitude differences in their
Km and Vmax values (Table
II).
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CGL from yeast (11) and rat liver (14) show an appreciable CBL-like
activity, i.e. they are able to cleave both the C--S and
C-
-S bonds of L-cystathionine. In contrast, with human
CGL and the substrate L-cystathionine, the appearance of
total product sulfhydryl groups (detected in the DTNB assay) perfectly
matched the developing L-cysteine levels (detected by
Gaitonde's method (31); Fig.
6a), showing that the
substrate was almost exclusively split at the C-
-S bond.
L-Cysteine was degraded at least 2 orders of magnitude
slower than L-cystathionine at respective concentrations of
0.55 mM (Fig. 6b). Similarly,
L-cystine degradation was not detectable through product
-ketoacids (lactate dehydrogenase assay), while the DTNB assay
showed a very small response (Fig. 6b). Both reactions,
degradation of L-cystine and L-cysteine, involve the splitting of C-
-S bonds. Therefore, we observe a high
reaction specificity of human CGL toward C-
-S bonds.
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Slow Binding Inhibition by AVG-- Reaction curves obtained with CGL in the presence of both AVG and saturating amounts of L-cystathionine displayed a slow decrease of the reaction rate. They finally reached a steady state velocity, which was dependent on the concentration of the inhibitor. In order to exclude the possibility that decomposition or modification of AVG by CGL caused the slow arrival at the steady state levels, CGL was preincubated with AVG before the addition of the substrate. Subsequent activity assays indicated the same steady state velocities. Thus, AVG shows slow binding inhibition (38) of CGL.
Three simple mechanisms could account for the observed slow binding
inhibition (Refs. 34 and 35; Table III):
(I) slow binding of inhibitor, I, to the enzyme, E; (II)
rapid binding of I to E followed by a slow isomerization
step to EI*; and (III) slow isomerization of E
into a state E* capable of rapidly binding I. In order to
gain insight into the mechanism in the present case, the slow coupling
of CGL and AVG was further characterized by recording reaction progress
curves in the presence of varying amounts of inhibitor over prolonged
times. These progress curves showed no dependence of the initial
reaction velocity, vo, on the inhibitor
concentration. The time traces were fitted to Equation 1 (Table III) to
extract apparent reaction rate constants, kobs. A plot of the obtained kobs values against the
inhibitor concentration (Fig.
7a) resulted in a straight
line without any indication of saturation kinetics. These observations
suggest the direct, slow establishment of a tight, inactive
EI complex without a rapid preequilibrium (mechanism I,
Table III). For this mechanism, kobs is composed
as shown in Equation 2 (Table III), in which k2
represents the dissociation rate. Fitting of
kobs to Equation 2 yields a rate constant,
k2, of 3.4 × 103
s
1 and a value of 10.5 µM for the
dissociation constant, Ki. For
k1, a value of 324 M
1
s
1 was calculated using the equation
Ki = k2/k1.
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The above analysis assumes the simplest mechanism for slow binding
inhibitions, i.e. slow formation of an EI
complex. An indication that mechanism I does not perfectly explain the
present data was obtained by replotting 1/(kobs k2) versus 1/[I], which did not result in the expected straight line but showed hyperbolic behavior. The latter observation favors the fast formation of a weak
EI complex that slowly isomerizes to a tight EI*
complex (mechanism II, Table III). This mechanism is indeed found for
most slow binding inhibition reactions (34, 35, 38). The seeming
discrepancy with the results favoring mechanism I can be explained,
assuming that in the present case Ki (describing the
initial EI complex formation) is much greater than the
overall dissociation constant Ki* (describing the
entire reaction to EI*). If, as in the present analysis, the
inhibitor concentration is varied in the range of
Ki* (µM), one works with inhibitor concentrations much lower than Ki (in the millimolar range). Under these conditions, the rate equation for
kobs for mechanism II simplifies (35) from
Equation 3 (Table III), with k4 as the
dissociation rate of EI*, to Equation 4 (Table III), which
is indistinguishable from Equation 2. It is therefore correct to
analyze the data in the present case based on the equation for
mechanism I.
The binding of AVG was also monitored via UV-visible spectra, showing a decrease of the pyridoxaldimine absorbance at 427 nm and the appearance of a new chromophore at 330 nm (Fig. 4). In order to elucidate whether the observed inhibition by AVG was reversible, AVG-inhibited enzyme was dialyzed extensively against AVG-free buffer. Almost full enzymatic activity (more than 90% of the initial activity) could be restored. The regain of the enzymatic activity was accompanied by the reappearance of the 427-nm pyridoxaldimine absorbance and the disappearance of the 330-nm absorption maximum for the AVG-inhibited enzyme. These results suggest that AVG acts on CGL by slow and tight, but reversible, binding to the active site PLP cofactor, thereby changing the absorption characteristics of the cofactor.
Suicide Inhibition by PG and F3Ala--
Contrary to
AVG, PG inactivated the enzyme faster than the manual mixing time for
the samples. The irreversible inactivation of CGL by PG has been
previously studied using the rat enzyme (39), and the following
mechanism was proposed. Abstraction of a -proton leads to an allene
that is capable of Michael addition to an enzyme active site
nucleophile (Fig. 8c). The
very fast inactivation of CGL by PG was therefore not surprising, since the covalent coupling takes place at the
-carbon, i.e.
the site of attack of CGL in the physiological substrate
L-cystathionine. Because of the rapidity of the
inactivation, we determined the activity levels remaining after a
10-min incubation of CGL with varying amounts of inhibitor. When the
data are represented relative to the concentration of active sites
(Fig. 7b), it can be seen that 1 eq of PG inactivates 1 eq
of active sites. This stoichiometry is in contrast to the results with
the rat enzyme, where two active sites were inactivated by 1 PG eq
(39). Still, PG shows extremely strong inhibition of CGL, since almost
every PG molecule leads to the inactivation of a CGL active site (Fig.
7b).
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In contrast to PG, F3Ala has to react at its -carbon,
explaining its much slower inhibition of CGL. Due to the slower
reaction, we could conveniently study inactivation by F3Ala
with a manual assay. CGL was incubated with F3Ala, and
aliquots were removed at several time points to determine residual CGL
activity. A biphasic behavior was observed for the activity plotted
against the incubation time; a fast initial inactivation was followed
by a slower one for longer incubation times. This behavior is often
observed for irreversible inactivation processes and is due to the
consumption of the inhibitor (40). Therefore, the initial inactivation
rates were used to determine the inhibition constant,
Ki (0.27 mM), and the rate of
inactivation, kinact (0.027/min), for
F3Ala from a Kitz-Wilson plot (Ref. 36; Fig.
7c).
Absorption spectra of CGL inactivated by PG or F3Ala showed
no decrease of the absorption at 427 nm; i.e. the PLP
cofactor in the inactivated enzyme forms a protonated pyridoxaldimine
like with its substrate. However, neither with PG nor with
F3Ala did dialysis against inhibitor-free buffer lead to a
recovery of the enzymatic activity, confirming that an irreversible,
covalent modification of the enzyme underlies the inactivation reactions.
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DISCUSSION |
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An Efficient Expression and Purification System for Human CGL-- Detailed mechanistic and structural investigations on human CGL are of prominent importance due to the enzyme's role in various metabolic disorders and its connection to certain forms of leukemia. We have therefore established the recombinant expression in E. coli and a native protocol for the facile production of milligram amounts of homogeneous, active enzyme. This achievement represents the first recombinant purification of a mammalian transsulfuration component.
The native purified protein showed an unexpected UV-visible spectrum
with an additional absorption band at 494 nm. The additional 494-nm
maximum could be due to some active site heterogeneity maintained
throughout the purification with an unidentified E. coli
factor forming a relatively stable quinonoid PLP derivative. Because
the 494-nm absorption band was missing when the holoenzyme was
reconstructed from free PLP and apoenzyme, it is unlikely that it
indicates different cofactor environments formed within the same
protein. The latter phenomenon has been seen with rat liver CGL, where
fluorescence studies of PLP-derivatives (41) showed the presence of two
differentially designed PLP-binding sites with 10-fold different
affinity constants for the cofactor. Similarly, an
2
2 composition has been reported for CGS
from E. coli, in which
and
signify identical
polypeptide chains differing in their net charge and their reaction
rates with PG (42).
Human CGL Displays Strong Substrate and Reaction
Specificities--
For yeast CGL, specific activities toward
L-cystine and L-cysteine amounting to 78 and
10%, respectively, relative to the L-cystathionine-directed activity have been reported (11).
Similarly, the streptomyces enzyme seems to be quite active toward
L-cystine (Ref. 1; Table II), and from data published for
rat liver CGL similar conclusions can be drawn (14, 17). In contrast,
human CGL displays a surprising substrate specificity and a clear
preference of C-S over S-S bond breakage; L-cysteine and
L-cystine are converted orders of magnitudes more slowly
than the natural substrate L-cystathionine. Furthermore,
L-cystathionine is split almost exclusively in a CGL-specific manner, in contrast to the behavior of yeast CGL, which
also seems to harbor pronounced CBL activity (11). One reason for the
failure of human CGL to convert L-cystine could be a high
regiospecificity together with the difficulty of polarizing a bond
between like atoms, which would hinder the cleavage of the S-S bond
under release of L-cysteine. The different behavior of the
yeast enzyme indicates a lower regiospecificity that enables the enzyme
to attack the C--S bond of L-cystine or
L-cysteine.
Considering the low in vivo concentrations of L-cysteine and L-cystine (18), it is unlikely that CGL will participate considerably in their in vivo conversion. The present findings therefore call into question the role of CGL in certain forms of cystinosis (accumulation of L-cystine) and the applicability of CGL for cyst(e)ine depletion in order to inhibit the growth of leukemic cells (14).
Implications for Inhibitor Design Based on AVG-- Because different classes of organisms display different spectra of transsulfuration enzymes, the enzymatic components in plants and microorganisms are attractive targets for the development of antibiotics and herbicides (6, 37), e.g. through interference with methionine biosynthesis. The reactions of transsulfuration enzymes with three mechanism-based inhibitors, AVG, PG, and F3Ala, are summarized in Table IV.
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It constitutes a tremendous task to design inhibitors that can distinguish between the homologous active sites of CGL, CGS, and CBL. Any prospective inhibitors should be tested for their reactivity toward CGL, in order to exclude adverse effects in humans. We therefore addressed the reactivity of CGL toward AVG, an antimicrobial agent naturally produced by streptomycetes (43). Slow binding inhibition has been documented for AVG acting on E. coli and plant CBL, and the molecule was therefore favored as a model substance for the development of novel herbicides (6). In the present analysis, the enzymatic progress curves again show a slow establishment of an enzyme-inhibitor species, and strong inhibition of CGL was observed with AVG concentrations in the order of the total enzyme concentration. AVG therefore qualifies as a slow-tight binding inhibitor also for CGL.
From the proposed scheme for CGL reacting with AVG (Fig. 8b;
Ref. 6), a molecular mechanism (mechanism II) can be deduced as
follows. In the first step, AVG binds to the active site PLP cofactor
by formation of a Schiff base bond. The resulting complex corresponds
to a weak EI complex, because the linkage can be readily resolved by reversion of the transaldimination reaction. Competing with
reverse transaldimination, the bound AVG can also face -proton abstraction by the active site lysine and subsequent proton transfer to
C-4', leading to a much more stable ketimine complex, corresponding to
EI*. This mechanism was proposed by Clausen et
al. (6), based on the crystal structure of E. coli CBL
complexed with AVG. As most of the CBL active site residues are
conserved in CGL (Fig. 2), a similar mechanism for the latter enzyme
seems likely. A relatively stable ketimine complex also explains the
appearance of a 330-nm absorption band under a concomitant decrease of
the 427-nm absorption. The ketimine formation is reversible, as was shown by the reactivation of AVG-inhibited CGL accompanied by a
decrease of the 330-nm absorption and recovery of the 427-nm absorption. Therefore, the mechanism of inactivation observed for most
,
-unsaturated inhibitors acting on PLP-dependent
enzymes, i.e. covalent modification of the cofactor or
active site residues, can be ruled out for the inactivation of CGL
by AVG.
Independent from the mechanistic considerations, the earlier assumption that AVG shows low reactivity against mammalian transsulfuration enzymes (6, 44) is not correct. The overall Ki (i.e. Ki*) of the AVG-CGL couple (10.5 µM) is only 10-fold larger than that of the AVG-CBL pair (1.1 µM; Ref. 6), indicating that AVG shows weak discriminatory power between these two enzymes. Furthermore, AVG still qualifies as a very strong inhibitor of CGL, because Ki* is about 2 orders of magnitude smaller than Km for the natural substrate L-cystathionine. AVG therefore seems to be unsuited as a lead compound for the development of antibiotics or herbicides.
Suicide Inactivators as Lead Substances for Inhibitor
Design--
It was thought that mechanism-based
inhibition/inactivation (45) might provide the selectivity needed for
the development of highly specific inhibitors. Suicide inhibition of
CGL and homologous enzymes by F3Ala and PG has been
demonstrated previously (7, 39, 46-48). The inhibition reactions
showed very low partition ratios, i.e. a strong tendency
toward inactivation instead of turnover, rendering these compounds very
efficient inhibitors. The substrate and reaction specificity observed
for human CGL and the present PG/F3Ala analyses suggest
that mechanism-based inhibitors may indeed be developed that are
specific enough to distinguish CBL from CGL. PG is a much better
inactivator of CGL than F3Ala, and the relative
reactivities of the two inactivators qualitatively match those of the
L-cystathionine/L-cystine substrate pair (see
above). Strikingly, the reactivities of PG and F3Ala are
reversed when acting on CBL (Refs. 7 and 22; Table IV). In this respect
it is interesting that ,
,
-trifluoromethylalanine was found to
be a much stronger inhibitor of rat CGL than F3Ala (49),
corroborating the idea to base the design of specific CGL and CGS
inhibitors on
-halogenated
-amino acids.
A possible explanation for the higher reactivity of PG compared with
F3Ala with CGL can be deduced from the postulated reaction mechanism (Fig. 8a). After initial -proton abstraction by
the active site lysine residue, which is bound to the PLP cofactor in
the internal aldimine, a ketimine intermediate absorbing at 330 nm is
formed by reprotonation at C-4'. The ketimine intermediate subsequently
suffers
-proton abstraction by this lysine residue. Another
(not yet identified) active site base abstracts a proton from the
inhibitor amino group during the preceding transaldimination and
subsequently acts as the proton donor in the breakage of the C-
-S
bond. This base functionality may be exploited by PG to form a covalent
enzyme-inhibitor complex through its activated
-carbon atom (Fig.
8c). In contrast, F3Ala does not provide a reactive
-carbon atom, and the only base in the enzyme that may be
reactive toward the
-carbon could be the active site lysine (Fig.
8d). In CBL, this lysine residue protonates the leaving group at C-
and thereafter is suitably oriented for the Michael addition, whereas the corresponding CGL lysine presumably protonates C-4' (Fig. 8a) and thus is in an unfavorable orientation for
the reaction at C
.
Taken together, the present studies suggest that the design of specific
inhibitors for PLP-dependent enzymes of the -family based on irreversible inhibitors seems more promising than that based
on reversible ones. The higher specificity of the former compounds is
presumably manifested in the steps following the initial
-proton
abstraction. Therefore, the development of inhibitors that discriminate
between CGS and CGL seems especially difficult, since both enzymes
attack at the C-
of their substrates. However, inhibition of CGS can
be achieved with compounds mimicking the first substrate, different
activated forms of
homoserine.2 The different
leaving groups used by plant and microbial CGS should allow us to
discriminate between these enzymes. A similar strategy should be
possible for CGL/CGS distinction, at least for specific inhibition of
CGS, and yield potent inhibitors when combined with the
enzyme-activated irreversible inhibition approach.
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FOOTNOTES |
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* This work was supported by stipends from the Boehringer Ingelheim Fonds (to C. S.), the Max-Planck Gesellschaft (to P. S.), and the Deutsche Forschungsgemeinschaft (to M. C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed.
2 T. Clausen, M. C. Wahl, A. Messerschmidt, R. Huber, J. Fuhrmann, B. Laber, W. Streber, and C. Steegborn, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
CBL, cystathionine
-lyase;
AVG, L-aminoethoxyvinylglycine;
CGL, cystathionine
-lyase;
CGS, cystathionine
-synthase;
DTNB, 5,5'-dithiobis(2-nitrobenzoic acid);
F3Ala,
,
,
-trifluoroalanine;
PLP, pyridoxal 5'-phosphate;
PG, D,L-propargylglycine;
RT, reverse
transcriptase.
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REFERENCES |
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