New insights into the metabolism of organomercury compounds: Mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine γ-lyase

Article abstract of DOI:10.1016/j.abb.2011.11.002

Anthropogenic practices and recycling in the environment through natural processes result in release of potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-conjugate, such as the l-cysteine (Cys)-S-conjugate of methylmercury (CH₃Hg-S-Cys) or inorganic mercury (Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to l-methionine and l-cystine/l- cystathionine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-containing amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing l-amino acids, including l-methionine, l-cystine, and l-cystathionine as substrates. The current study extends this list to include mercuric S-conjugates, and shows that CH₃Hg-S-Cys and Cys-S-Hg-S-Cys are substrates and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH₃Hg-S-Hcy, are also inhibitors. Finally, we show that HgCl₂, CH₃Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible inhibitors of rat cystathionine γ-lyase. The present study broadens our knowledge of the biochemistry of mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze transformations of biologically important sulfur-containing amino acids.

Full text of DOI:10.1016/j.abb.2011.11.002

Archives of Biochemistry and Biophysics 517 (2012) 20–29
Contents lists available at SciVerse ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
New insights into the metabolism of organomercury compounds:
Mercury-containing cysteine S-conjugates are substrates of human glutamine
transaminase K and potent inactivators of cystathionine c-lyase
Christy C. Bridges a,1, Boris F. Krasnikov b, ,1, Lucy Joshee a, John T. Pinto b, André Hallen c, Jianyong Li d,
⇑
Rudolfs K. Zalups a, Arthur J.L. Cooper b,
⇑
a Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College Street, Macon, GA 31207, USA
b Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA
c Department of Chemistry and Biomolecular Sciences, Macquarie University, Balaclava Road, North Ryde, NSW 2109, Australia
d Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 August 2011
and in revised form 31 October 2011
Available online 10 November 2011
Anthropogenic practices and recycling in the environment through natural processes result in release of
potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates
in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-
conjugate, such as the
(Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to
L
-cysteine (Cys)-S-conjugate of methylmercury (CH3Hg-S-Cys) or inorganic mercury
-methionine and -cystine/ -cystathio-
L
L
L
Keywords:
Cystathionine c-lyase
Glutamine transaminase K
Kynurenine aminotransferase isozyme I
Mercury cysteine S-conjugate
Methylmercury cysteine S-conjugate
Sulfur-containing amino acids
nine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-con-
taining amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we
report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing
L-amino
acids, including -methionine, -cystine, and -cystathionine as substrates. The current study extends this
L
L
L
list to include mercuric S-conjugates, and shows that CH3Hg-S-Cys and Cys-S-Hg-S-Cys are substrates
and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH3Hg-S-Hcy,
are also inhibitors. Finally, we show that HgCl2, CH3Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible
inhibitors of rat cystathionine c-lyase. The present study broadens our knowledge of the biochemistry of
mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze
transformations of biologically important sulfur-containing amino acids.
Ó 2011 Elsevier Inc. All rights reserved.
Introduction
tion of contaminated fish and/or water [1–3]. Inasmuch as methyl-
mercury exposure can be detrimental to multiple tissues and
Environmental mercury may exist in elemental (Hg0), inorganic
(Hg2+) or organic (Hg(I), Hg(II)) forms [1]. When mercury vapor
(Hg0) and Hg(II) compounds in the atmosphere settle into bodies
of water, organic mercury (primarily monomethylmercury) is
formed by cobalamin-dependent methylation, which is mediated
by a variety of microorganisms [1]. For simplicity, monomethylmer-
cury will be referred to as methylmercury in the current manuscript;
however, it is important to note that methylmercury, in aqueous
solutions, often reacts with anions such as Clꢀ to form covalent
bonds [2].
organs, including the brain and kidneys, it is recommended that cer-
tain species of fish, particularly those at the top of the food chain, be
consumed sparingly, especially by pregnant women and young chil-
dren [4–8]. Despite this recommendation, 6–8% of women of child-
bearing age in the US may have unacceptably high levels of mercury
[7,8]. Because of high lipid solubility, methylmercury can readily
cross the placenta and accumulate in fetal tissues [1], thus raising
the possibility that a sizable population of newborns may be ex-
posed to mercury in utero.
Because of concerns about the toxicity of mercury, it is impor-
tant to identify the forms of mercury capable of being transported
by target organs and cells. Mercuric ions have a strong bonding
affinity for reduced sulfur atoms [9]. Consequently, mercuric ions
within biological systems are converted primarily to conjugates
of one or more sulfur (thiol)-containing biomolecules, such as
Methylmercury accumulates readily in tissues of numerous
aquatic organisms, especially large, predatory fish [1]. It is the pre-
dominant form of organic mercury in the environment and humans
are exposed to this organometallic species primarily via consump-
⇑
Corresponding authors. Fax: +1 914 594 4058.
E-mail address: Arthur_cooper@nymc.edu (A.J.L. Cooper).
These authors contributed equally.
1
0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2011.11.002

C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
21
Fig. 1. Space-filled models showing the structural similarity of two mercuric S-conjugates with commonly occurring sulfur-containing amino acids. Structural similarity
exists between methionine and the cysteine (Cys)-S-conjugate of methylmercury (CH3Hg-S-Cys), and between cystine and the Cys-S-conjugate of Hg2+ (Cys-S-Hg-S-Cys). The
models were generated with MolPOV 2.0 and POV Ray 3.5. Atoms: O, red; N, blue; C, dark gray (small); H, light gray; S, yellow; Hg, dark gray (large).
glutathione (GSH),2 L-cysteine (Cys), and L-homocysteine (Hcy).
Some of these mercury conjugates are similar structurally to certain
endogenous amino acids [10–12]. For example, the structure of the
Cys S-conjugate of methylmercury (i.e. CH3Hg-S-Cys) is similar to
that of methionine (Fig. 1). CH3Hg-S-Cys may be formed by direct
reaction of Cys with methylmercury. Alternatively, methylmercury
may react with GSH to form a glutathione S-conjugate (i.e. CH3Hg-
S-G), which can then be catabolized to CH3Hg-S-Cys by the sequen-
tial actions of c-glutamyltransferase and cysteinylglycinase/amino-
peptidase M located in the luminal membranes of intestinal and
renal epithelial cell [11,13] (Fig. 2). Due to the presence of a large
hydrophobic side group and structural similarity to methionine,
CH3Hg-S-Cys may be transported into cells via the amino acid trans-
porter, system L [13]. This transporter has an affinity for large hydro-
phobic amino acids, including L-methionine [13].
The similarity between CH3Hg-S-Cys and methionine has been
suggested by a number of authors [14–16] to be an example of
‘‘molecular mimicry’’ whereby CH3Hg-S-Cys is a mimic of methio-
nine at the site of the system L transporter. Molecular mimicry has
been defined as the phenomenon whereby one molecule/com-
pound can act as a structural and/or functional molecule of another
endogenous molecule/compound [11,15]. However, the concept
that cysteine S-conjugates of mercury are molecular mimics of sul-
fur-containing amino acids has been criticized by Hoffmeyer and
colleagues [17]. These authors’ definition of molecular mimicry is
restricted to isosteric compounds with similar electronic distribu-
tions [17]. Here, we simply point out the similarities between the
cysteine S-conjugates of mercury and other amino acids, including
sulfur-containing amino acids.
Fig. 2. Transformation of mercury species in mammalian tissues. Mercuric ions that
are ingested, absorbed or inhaled may bond with glutathione (GSH) and/or cysteine
(Cys) to form G-S- and Cys-S-conjugates of Hg2+ (G-S-Hg-S-G, Cys-S-Hg-S-Cys,
respectively) and methylmercury (CH3Hg-S-G, CH3Hg-S-Cys, respectively). G-S
-Hg-S-G and CH3Hg-S-G are processed further by
c-glutamyltransferase and
cysteinylglycinase to yield Cys-S-Hg-S-Cys and CH3Hg-S-Cys, respectively. Based on
endogenous concentrations of intracellular glutathione (1–10 mM), these cysteine S-
conjugates of mercury are likely to be among the major species of Hg present in
cellular systems. It should be noted that inhaled Hg0 is oxidized rapidly in blood and
tissues [18]. For convenience, methylmercuric species are depicted here as CH3Hg+
even though it is recognized that methylmercuric species are normally found bonded
to an anion or sulfur-containing molecule.
Following ingestion of methylmercury, there is evidence indi-
cating that some of this species undergoes biotransformation in
intracellular or extracellular compartments to yield inorganic mer-
cury [3]. Thus, thiol-S-conjugates of both Hg2+ and methylmercury
may be present in target cells and organs of biological systems.
Interestingly, the Cys-S-conjugate of Hg2+ (i.e. Cys-S-Hg-S-Cys),
2
Abbreviations used: CH3Hg-S-Cys, L-cysteine S-conjugate of methylmercury;
which is similar structurally to
nine) and Hcy-S-Hg-S-Hcy (similar to
at the luminal membrane of renal proximal tubular cells by the
amino acid transporter, system b0,+ [18–20]. System b0,+ is an
L
-cystine (Fig. 1) (and
L-cystathio-
CH3Hg-S-Hcy, L-homocysteine S-conjugate of methylmercury; Cys-S-Hg-S-Cys, L-
cysteine S-conjugate of inorganic mercury; DTT, dithiothreitol; GTK, glutamine
transaminase K; GSH, glutathione; G-S-Hg-S-G, glutathione S-conjugate of inorganic
mercury; GSSG, glutathione disulfide; Hcy-S-Hg-S-Hcy, L-homocysteine S-conjugate
L
-homocystine) is taken up
of inorganic mercury; KAT I, kynurenine aminotransferase isozyme I; KMB, a-keto- c-
methiolbutyrate; MPA, metaphosphoric acid; PLP, pyridoxal 50-phosphate; rhGTK,
recombinant human GTK.
important absorptive transporter of
L-cystine. In addition, the glu-
tathione S-conjugate of Hg2+ (G-S-Hg-S-G) is similar structurally to

22
C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
glutathione disulfide (GSSG) and may be a substrate/inhibitor of
proteins that interact with GSSG. GSSG is critical for post-transla-
tional modification of proteins involved in redox signaling [21,22].
Given that select thiol-S-conjugates of Hg2+ and methylmercury
are similar structurally to endogenous compounds, we hypothe-
sized that these conjugates may also function as substrates of cer-
tain intracellular enzymes (in addition to the membrane-bound
slow, rhGTK was shown to catalyze transamination of two posi-
tional isomers of b-naphthyl- -alanine [29], consistent with a large,
L
relatively open active site. Fig. 4 depicts a ribbon model of hGTK
monomer (the active enzyme is a homodimer) showing the posi-
tion of the pyridoxal 50-phosphate (PLP) coenzyme bound in Schiff
base linkage (internal aldimine) to a lysine residue in the active
site. Surface representations of the monomer reveal that the active
site is spacious (Fig. 4). Fig. 4 also illustrates the positions of all six
cysteine moieties within the monomer. It is readily apparent that
the active site does not contain a cysteine residue. One cysteine
residue (C127) is 6.8 Å away from the PLP coenzyme; other cys-
teine residues are 12.5–20.4 Å away from the coenzyme epicenter.
external enzymes
c-glutamyltransferase and cysteinylglycinase/
aminopeptidase M). To investigate this possibility we chose to
study the interaction of Cys- and Hcy-S-conjugates of Hg2+ and
methylmercury with kidney glutamine transaminase K [GTK; also
known as kynurenine aminotransferase isozyme I (KAT I)] and cys-
tathionine
c-lyase (c-cystathionase). The rationale for these inves-
Transamination of
L-cystathionine, L-lanthionine, and L-cystine
tigations evolved from the observation that rat kidney GTK utilizes
in vitro yields -keto acids that can non-enzymatically cyclize to
a
a
-amino acid and
a
-ꢀketo acid substrates of the general structure
ketimines (reviewed in [24,30]). Some of these compounds and
their reduced forms (cyclic amines) have been reported to be pres-
ent in brain, plasma and/or urine and may be neuroactive (dis-
cussed in [24]). Furthermore, GTK activity is present in human
brain [31]. Therefore, GTK may be responsible for generating sul-
fur-containing neuroactive cyclic ketimines in human brain. Be-
cause the active site of rhGTK is large [28], it is also possible that
the enzyme will catalyze transamination in human tissues with
thiol-S-conjugates of Hg (Fig. 3, Eq. (1) where n = 1 or 2, and
Y = CH3HgS- or Cys–S–HgS–). This hypothesis is supported by the
finding that Se-methyl-L-selenocysteine is a substrate of rhGTK
[29] and this conjugate and other selenocysteine Se-conjugates,
many of which contain large aromatic moieties, are substrates of
Y(CH2)nCH(NH3+)CO2 and Z(CH2)nC(O)CO2ꢀ, respectively, where
n = 1 or 2 and Y (or Z) is generally a hydrophobic or uncharged
moiety that can be relatively large [23,24] (Fig. 3, Eq. (1)). (An
exception is cystine and some other sulfur-containing amino acids
where Y possesses a terminal charged grouping.) Thus, for exam-
ple, rat kidney GTK catalyzes transamination with
(as the name implies), -methionine and -phenylalanine, and their
corresponding -keto acids [23,24]. Glutamine transaminases
purified from bovine kidney [25] and brain [26] exhibit activity to-
ward -glutamine, -methionine and -phenylalanine, but also to-
ward large sulfur-containing amino acids (e.g. -cystine,
homolanthionine and -cystathionine). Highly purified rat kidney
GTK exhibits activity toward -cystine, -homocystine, -cystathio-
nine and -lanthionine when phenylpyruvate is used as the co-sub-
L-glutamine
L
L
a
L
L
L
L
L-
L
L
L
L
rat GTK [32]. On the other hand, whereas Se-methyl-
teine and -methionine are relatively good substrates of rhGTK,
the closely related amino acid, -selenomethionine, is an extremely
poor substrate (<0.1% as affective as -methionine) [29]. Thus, due
L-selenocys-
L
L
strate (amine acceptor) [27].
L
X-ray crystallographic studies of recombinant human GTK/KAT
I (rhGTK) have shown that this enzyme has a remarkably open con-
figuration at the active site. The enzyme substrate binding site pos-
sesses a striking crown of aromatic residues that adorns the
relatively large active site [28]. Although turnover is somewhat
L
to these subtleties in binding geometries it was not certain a priori
that the enzyme would catalyze transamination of thiol-S-conju-
gates, despite the relatively open active site. The present work,
however, does indeed show that rhGTK catalyzes transamination
Fig. 3. Specificity of glutamine transaminase K (GTK) and cystathionine
depicted in Eq. (1), where n = 1 or 2, and Y and Z can range in size from H- (e.g.
methionine, n = 2) or C6H5- (phenylpyruvate, n = 1;
-phenylalanine, n = 1). Cystine [Y or Z = –SSCH2CH(NH3+)CO2ꢀ, n = 1] and some other sulfur-containing amino acids, in
which Y or Z possesses a terminal charged grouping, are also substrates. Cystathionine -lyase catalyzes several -elimination, b-elimination and replacement reactions.
Some of the more important reactions that are pertinent to the current study are shown here. The enzyme catalyzes a -elimination reaction with -cystathionine (Eq. (2))
and with -homoserine (Eq. (3)). In both cases, the -keto acid product is -ketobutyrate. Cystathionine -lyase also catalyzes a b-elimination reaction with -cystine (Eq. (4)).
In this case, the -keto acid product is pyruvate.
c
-lyase. Transamination reactions between amino acids and
a-keto acids catalyzed by GTK are
a-ketobutyrate, n = 2) to relatively large hydrophobic moieties such as CH3S- (KMB, n = 2;
L-
L
c
c
c
L
L
a
a
c
L
a

C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
23
Fig. 4. Ribbon model of rhGTK monomer showing pyridoxal 50-phosphate in the active site. The diagram shows a relatively large active site. Cysteine residues are
emphasized. Atoms: S, yellow; H, light gray. The model was produced using the UCSF Chimera program (http://www.cgl.ucsf.edu/chimera/).
of large sulfur-containing amino acids as well as the Cys-S-conju-
3-propanediol), dithiothreitol (DTT), GSH, sulfur-containing amino
acids, -glutamine, -phenylalanine, -homoserine, phenylpyruvate,
sodium -keto- -methiolbutyrate [sodium -keto-( -methyl-
thio)butyrate; KMB] were obtained from Sigma, St. Louis, MO. 2,4-
Dinitrophenylhydrazine was purchased from MP Biochemicals, Ir-
vine, CA.
gates of Hg2+ and methylmercury.
L
L
L
We next turned our attention to cystathionine
zyme catalyzes a -lyase reaction with -cystathionine, generating
-ketobutyrate, cysteine (eliminated fragment) and ammonia
c-lyase. This en-
a
c
a
c
c
L
a
(Fig. 3, Eq. (2)). L-Homoserine is also a c-lyase substrate, generating
a-ketobutyrate and ammonia (Fig. 3, Eq. (3)). In this case the
hydrolysis reaction is balanced by elimination of H2O. Thus, the
net reaction does not include an H2O term. The enzyme can also
Enzymes
catalyze a b-lyase reaction with L-cystine (Fig. 3, Eq. (4)), generat-
ing pyruvate, the persulfide analogue of cysteine (thiocysteine) and
ammonia [33,34].
Highly purified rhGTK [1.8 mg/ml in 20% glycerol, 10 mM potas-
sium phosphate buffer (pH 7.4); specific activity 18 U/mg in the
standard reaction assay mixture, see below] was obtained by the
Rat liver cystathionine c-lyase has a cysteine residue within the
method of Han et al. [38]. Cystathionine
24 U/mg, 20 mM -homoserine as substrate) was purified from
rat liver as described by Pinto et al. [39]. One unit of enzyme activ-
ity is the amount of enzyme that generates 1 mol of product per
c-lyase (specific activity
active site [35] that is highly susceptible to inactivation by a num-
ber of sulfhydryl reagents including p-chloromercuribenzoate,
iodosobenzoate and N-ethylmaleimide [36,37]. Therefore, we con-
sidered the possibility that Cys-S-conjugates of mercury, particu-
larly Cys-S-Hg-S-Cys, would interact with this sulfhydryl in the
L
l
min at 37 °C under standard reaction conditions.
active site of cystathionine
Cys-S-Hg-S-Cys is a strong irreversible inhibitor of cystathionine
-lyase.
c-lyase. The present work shows that
Enzyme activity measurements
c
The standard GTK assay mixture (50
ammediol buffer (pH 9.0), 5 mM KMB and 20 mM
[40]. After incubation at 37 °C (5–10 min), 150 l of 1 M NaOH was
added and the absorbance at 322 nm due to phenylpyruvate enol
322nm = 16,000 Mꢀ1 cmꢀ1) was determined within 5 min against
l
l) contained 100 mM
L
-phenylalanine
Materials and methods
l
Reagents
(e
a blank consisting of complete reaction mixture lacking enzyme.
The activity of rhGTK in a reaction mixture containing 100 mM
potassium phosphate buffer (pH 7.4) in place of ammediol buffer
in the standard reaction mixture is ꢁ50% that exhibited at pH 9.0
Methylmercuric chloride (CH3HgCl) and HgCl2 were obtained
from Aldrich, Milwaukee, WI. Mercuric S-conjugates were generated
by mixing HgCl2 with Cys or Hcy in a 1:2.5 ratio. Methylmercury S-
conjugates were generated by mixing CH3HgCl with Cys or Hcy in a
1:1.25 ratio. The conjugates were incubated for five minutes at room
temperature prior to use. Ammediol (2-amino-2-methyl-1,
[29]. The standard cystathionine c-lyase assay mixture (50 ll) con-
tained 100 mM potassium phosphate buffer (pH 7.4) and 20 mM
L-homoserine [39]. After incubation at 37 °C (10 min), the reaction

24
C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
was stopped by addition of 20
zine in 2 M HCl. After further incubation at 37 °C for 10 min, 130 ll
of 1 M NaOH was added and the absorbance was read within 5 min
at 430 nm against a blank consisting of reaction mixture lacking
l
l of 5 mM 2,4-dinitrophenylhydra-
16
14
12
10
8
L
-homoserine (or enzyme) (e430nm a-ketobutyrate 2,4-dini-
trophenylhydrazone, 15,000 Mꢀ1 cmꢀ1). To measure aminotrans-
ferase activity of GTK toward various amino acids (other than the
mercury S-conjugates) the reaction mixture (50
noted, contained 100 mM potassium phosphate buffer (pH 7.4),
1.0 mM -amino acid (50 nmol) and 0.4 mM phenylpyruvate
(20 nmol) and GTK. In the case of -cystine the concentration was
0.4 mM and the buffer was 50 mM sodium pyrophosphate, pH
9.2. The reaction mixture was incubated at 37 °C and the amount
of phenylpyruvate remaining was determined (as described above)
against a blank containing complete reaction mixture lacking en-
zyme. A similar procedure was used to measure the rhGTK-cata-
lyzed transamination of mercury S-conjugates, except that the
concentration of phenylpyruvate was 0.2 mM. Blanks contained
the complete reaction mixture (including mercuric conjugates)
without enzyme.
ll), except where
6
L
4
L
2
0
Fig. 5. rhGTK-catalyzed transamination of phenylpyruvate with glutamine and
various sulfur-containing amino acids. Except where indicated, the reaction
l) contained 1 mM amino acid, 0.4 mM phenylpyruvate, 100 mM
potassium phosphate buffer (pH 7.4) and enzyme (2.15 mU). In the case of
the concentration was 0.4 mM and the buffer was 50 mM sodium pyrophosphate
(pH 9.2). After incubation for 1 h at 37 °C the amount of phenylpyruvate remaining
in solution was determined relative to a blank reaction mixture lacking enzyme;
n = 3.
mixture (50
l
L-cystine
Spectrophotometric determinations were conducted with a Te-
can Infinite M1000 96-well plate spectrophotometer (Tecan, Dur-
ham, NC) or with a SpectraMax 96-well plate spectrophotometer
(Molecular Devices, Sunnyvale, CA).
HPLC measurement of L-methionine and KMB
Results
The HPLC system consisted of a liquid chromatograph equipped
with an 8-channel coulometric array (CoulArray) detector (ESA,
Inc., Chelmsford, MA) [39,41]. The enzyme activity in the reaction
Sulfur-containing amino acids are substrates of rhGTK
Fig. 5 shows that the sulfur-containing amino acids,
L-methio-
mixtures (50
metaphosphoric acid (MPA). The resulting 5% w/v MPA homoge-
nates were injected directly onto a Bio-Sil ODS-5S, 5- m particle
ll) was terminated by addition of 15 ll of 25% w/v
nine, -cystine, -cystathionine, and -lanthionine are substrates
L
L
L
of rhGTK when 0.4 mM phenylpyruvate is used as an amine accep-
tor. Except as noted, the buffer was 100 mM potassium phosphate
(pH 7.4). After incubation of the reaction mixture containing
2.15 mU of enzyme at 37 °C for 1 h the amount of phenylpyruvate
l
size, 4.0 ꢂ 250 mm, C18 column (Bio-Rad, Life Science Research
Group, Hercules, CA) and eluted with a mobile phase consisting
of 50 mM NaH2PO4, 50
nitrile (pH 2.52) at a flow rate of 1 ml/min. All buffers, following
preparation, were routinely degassed, filtered through a 0.2-
lM octane sulfonic acid, and 1% (v/v) aceto-
remaining was determined. Under the conditions of the assay,
methionine (1.0 mM), -cystine (0.4 mM; 50 mM sodium pyro-
phosphate buffer, pH 9.2), -cystathionine (1.0 mM) and -lanthio-
nine (1.0 mM) are about 12–30% as effective as -glutamine
L-
L
l
m
L
L
Millipore nylon filter, and the pH re-adjusted, if necessary. PEEK™
(polyetheretherketone) tubing was used throughout the HPLC sys-
L
(1.0 mM) as substrates. (Cystine has very limited solubility at pH
7.4, but is more soluble at higher pH values. Even at pH 9.2, how-
ever, the maximum concentration in the assay mixture is about
0.4 mM. The higher pH is not a major limitation for enzyme activity
as GTK has a pH optimum of ꢁ8.5–9.0) [23,38].
tem, and a 0.2-
to protect both column and flow cells, respectively, from any par-
ticulate matter. A Rheodyne injection valve with a 5- l sample
lm PEEK™ filter was placed pre- and post-column
l
loop was used to manually introduce samples. The 8-channels of
the CoulArray detector were set at 100, 200, 300, 400, 500, 600,
700 and 800 mV, respectively. Elution times (min) and detection
potential ranges (mV) are: MPA (1.6), L-methionine (9.6, 600–
800 mV) and KMB (11.1, 600–800 mV). Other sulfur-containing
compounds eluted at times that did not interfere with analysis of
In order to provide additional evidence for the ability of rhGTK to
catalyze transamination of sulfur-containing amino acids, 0.5 mM
KMB was used as an
a-keto acid substrate in place of phenylpyru-
vate, and the -methionine generated by transamination was deter-
L
mined by HPLC with CoulArray detection. The advantage of this
technique is that redox-active compounds can be characterized di-
rectly without the need to derivatize them and they can be identified
by both their column retention times and the voltage required to
produce a signal (i.e. the voltage required to oxidize (remove an elec-
tron from) the analyte). As a result of the presence of a sulfur ether
moiety, which is readily oxidizable under the HPLC conditions, the
L-methionine or KMB.
Data analyses
For measurements of the effect of HgCl2 and CH3HgCl on the
rhGTK-catalyzed transamination of L-phenylalanine with KMB, dif-
ferences between two means were analyzed using the Mann–
Whitney U test. For measurements of inactivation of cystathionine
substrate (KMB) and product (
sive and detectable by coulometry. Except in the case of
the reaction mixture (50 l) contained 100 mM potassium phos-
L-methionine) are both redox respon-
L
-cystine,
l
c-lyase by various mercury-containing compounds, differences
phate buffer (pH 7.4), 1.0 mM amino acid, 0.5 mM KMB and
2.15 mU rhGTK. After incubation for 60 min at 37 °C the reaction
among means were analyzed using a two-way Analysis of Vari-
ance, followed by Tukey’s post hoc testing. Each set of respective
data was analyzed first with the Kolmogorov–Smirnov test for nor-
mality, followed by the Levene test for homogeneity of variances. A
p-value of <0.05 was considered statistically significant. Data are
presented as the mean SD. Except where noted, measurements
were carried out at least in triplicate.
was terminated by the addition of 12.5
methionine content of the mixture was measured relative to mix-
tures that lacked enzyme (blanks). (The concentration of -cystine
was 0.4 mM and the buffer used was 50 mM sodium pyrophosphate,
pH 9.2.) The amount of -methionine generated (nmol; average of
ll of 25% w/v MPA. The L-
L
L

C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
25
two determinations) with various amino substrates was as follows:
-glutamine (16.0), -cystine (1.4), -cystathionine (0.9), -lanthio-
nine (0.6). The formation of -methionine in reaction mixtures con-
taining -glutamine was accompanied by an approximately
enzyme exposed to Hg2+ or CH3HgCl, rhGTK (ꢁ2.6 mU) was incu-
L
L
L
L
bated in complete reaction mixture (50 ll) lacking KMB in the
L
presence or absence of 1 mM CH3HgCl. After incubation for 1 h at
L
37 °C, transamination was initiated by addition of 2.5 of
ll
equimolar loss of KMB.
100 mM KMB. After a further incubation for 30 min at 37 °C, the
formation of phenylpyruvate was measured. The amount of phe-
nylpyruvate formed by rhGTK that was exposed to 1 mM CH3HgCl
for 1 h was 11.7 2.1% relative to the control (100 13%) (n = 6). A
similar experiment was carried out with HgCl2. In this case, the
amount of phenylpyruvate formed by rhGTK that was exposed to
1 mM HgCl2 for 1 h was 6.9 1.5% relative to the control
(100 10%) (n = 6).
There are at least two possible explanations for the findings of
residual activity in rhGTK exposed to CH3HgCl or Hg2+. First, bind-
ing of the mercury compound causes 100% irreversible inactiva-
tion, but 5–10% of the enzyme population is resistant to binding
of the mercury compound and remains fully active. Secondly,
100% of the enzyme is strongly modified within about 20 min
and all modified enzyme species are active, albeit at a reduced effi-
ciency. To distinguish between the two possibilities, 0.6 mU of en-
The values for rhGTK-catalyzed transamination of L-cystine and
L-cystathionine with KMB are somewhat lower than those obtained
with phenylpyruvate as an
ously demonstrated for bovine [26] and rat kidney [27] glutamine
transaminases, the critical issue here is that rhGTK catalyzes trans-
a-keto acid substrate. As was previ-
amination of
able -keto acid acceptors. Thus, because CH3Hg-S-Cys and Cys-S-
Hg-S-Cys are similar to -methionine and -cystine/ -cystathionine
L-methionine, L-cystine and L-cystathionine with suit-
a
L
L
L
insofar as both conjugates contain sulfur and a relatively large side
grouping, we considered the possibility that CH3Hg-S-Cys and Cys-
S-Hg-S-Cys would be substrates and/or inhibitors of rhGTK.
Inhibition of rhGTK by mM concentrations of HgCl2 and CH3HgCl
As discussed below, cystathionine
c-lyase is strongly inhibited
zyme was incubated for 1 h at 37 °C in 40
phosphate buffer (pH 7.4) containing 0, 0.5, 1 or 2 mM HgCl2.
Thereafter, -phenylalanine and KMB were added to yield final con-
centrations of 20 mM and 5 mM, respectively (final volume 50 l).
ll of 100 mM potassium
by
lM (or less) concentrations of mercury-containing compounds.
By contrast our results show that rhGTK, despite the fact that both
enzymes contain cysteine residues, is much more resistant to inhi-
bition. Fig. 6 shows the time course for phenylpyruvate production
L
l
After an additional 30-min incubation at 37 °C, the amounts of
phenylpyruvate formed were 8.96 0.66, 1.42 0.42, 0.76 0.26,
and 0.52 0.20 nmol, respectively (n = 3). In another series of
experiments, 0.6 mU of enzyme was incubated for an hour at
at 37 °C in a reaction mixture (50
ll) containing 2.6 mU rhGTK,
5 mM KMB, 20 mM -phenylalanine, and 100 mM potassium phos-
L
phate buffer (pH 7.4). The transamination reaction is freely revers-
ible (Fig. 3, Eq. (1)). Thus, the slower reaction rate at 60 min
relative to the initial rate is presumably a result of the back reac-
37 °C in 40
taining 2 mM HgCl2. Thereafter, 5
and the mixture was incubated for 30 min at 37 °C. Then, 10
of 100 mM -phenylalanine and 5 l of 100 mM KMB were added
(final volume 55 l). After an additional 30-min incubation at
l
l of 100 mM potassium phosphate buffer (pH 7.4) con-
ll of 100 mM DTT was added
tion competing with the forward reaction. When 10
lM HgCl2 or
ll
10 M CH3HgCl was included in the reaction mixture no difference
l
L
l
in the rate of phenylpyruvate formation compared to the control
was noted (data not shown). However, when 1 mM HgCl2 or
1 mM CH3HgCl was included in the reaction mixture, transamina-
tion of L-phenylalanine was inhibited at all time points relative to
the control that lacked mercury compound (Fig. 6).
One possible explanation for the findings is that slow binding of
Hg2+ and CH3HgCl to the enzyme occurred over a 20 min period
followed by establishment of a greatly slowed transamination rate
between 30 and 60 min. However, due to the nature of the plot
shown in Fig. 6 it was difficult to discern whether there was still
some residual enzyme activity at 60 min or at later time points.
Therefore, to investigate the possibility of residual activity in
l
37 °C, phenylpyruvate was measured. The amount of phenylpyru-
vate formed was 7.12 0.16 nmol (n = 3). Thus, after addition of
a 6.25-fold molar excess of DTT over that of HgCl2, the inhibition
by Hg2+ was largely (ꢁ80%) reversed.
The data suggest that reaction with mM concentrations of Hg2+
ions or CH3HgCl is a relatively slow process, but that the interac-
tion eventually substantially lowers the activity of rhGTK. The
activity, however, can be largely restored by treatment with DTT.
Possibly, covalent binding of the mercury species to one or more
cysteine sulfhydryls alters the conformation of the enzyme so that
the enzyme cannot bind substrate or binds substrate much less
effectively. The DTT restores the original sulfhydryl groups with
recovery of activity. Alternatively, S-Hg exchange involving DTT re-
sults in an Hg conjugate that can no longer bind to the enzyme. As
shown below, the results are in marked contrast to those obtained
Phenylalanine standard
1 mM HgCl2
40
30
20
10
0
+
1 mM CH
3Hg
with cystathionine
c-lyase, where the enzyme is rapidly and irre-
versibly inactivated by
l
M (or lower) concentrations of HgCl2,
Cys-S-Hg-S-Cys and CH3Hg-S-Cys. It should be noted that the use
of mM concentrations of mercuric species is not biologically rele-
vant. The experiments were designed to show the markedly differ-
ent susceptibility of cystathionine
c-lyase to various forms of
mercury compared to GTK despite the fact that both enzymes uti-
lize sulfur-containing amino acids as substrates and possess cys-
teine residues.
0
20
40
60
rhGTK-catalyzed transamination of CH3Hg-S-Cys and Cys-S-Hg-S-Cys
Time (minutes)
As noted above, CH3Hg-S-Cys and Cys-S-Hg-S-Cys were hypoth-
esized to be substrates of GTK. In order to provide evidence for this
hypothesis, enzyme-catalyzed disappearance of phenylpyruvate
was measured in a reaction mixture containing 2.6 mU rhGTK,
0.2 mM phenylpyruvate, 1 mM mercury S-conjugate and 100 mM
potassium phosphate buffer (pH 7.4). The corresponding
Fig. 6. Effect of HgCl2 and CH3HgCl on the rhGTK-catalyzed transamination of
phenylalanine with KMB. The reaction mixture (50 l) contained 20 mM
L
-
-
l
L
phenylalanine, 5 mM KMB, 2.6 mU of enzyme, 100 mM potassium phosphate
buffer (pH 7.4) in the presence or absence of 1 mM HgCl2 or CH3HgCl. Samples were
incubated at 37 °C and phenylpyruvate formation was measured at the times
shown (n = 3).

26
C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
homocysteine S-conjugates were included for comparison. Lower
concentrations of phenylpyruvate were included in this experi-
ment compared to those used to determine the substrate specific-
ity toward physiologically important sulfur-containing amino
acids (Fig. 5). Significant disappearance of absorbance of phenyl-
pyruvate (enol) relative to the blank should be more readily appar-
ent at lower concentrations of phenylpyruvate. The low
phenylpyruvate concentration in this experiment (0.2 mM) should
not be limiting for enzyme activity because rat kidney GTK (and
presumably rhGTK) has a high affinity for phenylpyruvate [23].
No significant rhGTK activity was detected with 1 mM Hcy-S-
Hg-S-Hcy (Fig. 7). The enzyme appears to have some activity with
the homocysteine derivative, CH3Hg-S-Hcy, but the activity value
did not quite reach significance. On the other hand, significant
enzymatic activity was found with the cysteinyl derivatives, Cys-
S-Hg-S-Cys and CH3Hg-S-Cys (Fig. 7).
tion. A feature of this type of reaction is that the apparent Km value
of one of a pair of substrates is strongly dependent on the nature
and concentration of the other substrate.
Next, we determined the effect of various mercury S-conjugates
on rhGTK-catalyzed transamination of
Accordingly, reaction mixtures (50 l) containing enzyme (2 mU),
various concentrations of -phenylalanine, 5 mM KMB, and
L-phenylalanine with KMB.
l
L
100 mM potassium phosphate buffer (pH 7.4) were incubated for
30 min at 37 °C in the presence or absence of mercury S-conjugate
(i.e. Cys-S-Hg-S-Cys, Hcy-S-Hg-S-Hcy, CH3Hg-S-Cys or CH3Hg-S-
Hcy) and the amount of phenylpyruvate determined. The relative
amounts of phenylpyruvate formed at three concentrations of L-
phenylalanine, ranging from 10 mM (a concentration slightly above
the Km value) to 50 mM (a concentration considerably above the Km
value) in the presence and absence of various mercury S-conjugates
are shown in Table 1. This table shows that, under the conditions of
the assay, the mercury S-conjugates, at a concentration of 0.5–1 mM,
are strongly inhibitory. Because Cys-S-Hg-S-Cys and CH3Hg-S-Cys
are substrates of rhGTK (Fig. 7) part of the inhibition noted with
Inhibition of rhGTK activity by Cys-S-Hg-S-Cys, Hcy-S-Hg-S-Hcy,
CH3Hg-S-Cys and CH3Hg-S-Hcy
these two compounds must be competitive with respect to
ylalanine. If the inhibition, however, were strictly competitive then
the relative inhibition would be less as the concentration of -phen-
L-phen-
Because CH3Hg-S-Cys and Cys-S-Hg-S-Cys have similarities
with methionine and cysteine/cystathionine, respectively, and are
substrates of rhGTK, they were predicted to be inhibitors of the
L
ylalanine was increased relative to the concentration of conjugate.
This was not the case for these two conjugates and also for Hcy-S-
Hg-S-Hcy and CH3Hg-S-Hcy. In fact, within experimental error there
was little change in the degree of inhibition exerted by the mercury
standard
by rhGTK. We initially carried out experiments to determine the
Km for -phenylalanine exhibited by rhGTK under conditions used
L-phenylalanine – KMB transaminase reaction catalyzed
L
S-conjugates from 10 to 50 mM L-phenylalanine (Table 1). This find-
for the inhibition studies. Fitting of data to the Michaelis–Menten
equation showed that the Km exhibited by rhGTK for KMB in the
presence of 40 mM L-phenylalanine (in which the KMB concentra-
tions were varied between 1 and 20 mM) and 100 mM potassium
ing suggests a strong noncompetitive component. However, it was
not possible to carry out kinetic experiments with concentrations
of mercury S-conjugates at concentrations >1 mM because this is
the limit of the solubility of these conjugates. Moreover, the non-lin-
earity of the reaction over the 30 min incubation (Fig. 6) complicates
interpretation of data. The exact type of inhibition remains to be
determined, but it is worth noting that the mercury conjugates ap-
pear to bind more strongly to rhGTK (strong inhibition at 61 mM)
phosphate buffer (pH 7.4) at 37 °C is 1.8 0.6 mM (Sigma plot;
n = 3 for each value of
exhibited toward -phenylalanine in the presence of 5 mM KMB
(in which the -phenylalanine concentration was varied between
5 and 20 mM) was 7.3 1.3 mM (average of four separate experi-
ments in which n P 3 for the determination of each value of ).
The Km for -phenylalanine noted here is higher than that reported
previously for rhGTK at pH 7.5 (1.7 mM) [38]. It should be noted
that the previous study utilized 16 mM -ketobutyrate as the
v). A similar analysis showed that the Km
L
L
than
L
-phenylalanine (Km ꢁ7.6 mM).
v
L
Inhibition of rat liver cystathionine
c-lyase by lM concentrations of
a
a-
HgCl2, Cys-S-Hg-S-Cys and CH3Hg-S-Cys
keto acid substrate. Aminotransferases catalyze a ping-pong reac-
As noted above, cystathionine
c-lyase catalyzes a b-elimination
reaction with -cystine in a reaction that generates pyruvate
L
([33,34]; Fig. 3, Eq. (4)). Therefore, we considered the possibility
that the enzyme might be able to catalyze pyruvate formation from
Cys-S-Hg-S-Cys and CH3Hg-S-Cys. However, we were unable to de-
tect pyruvate formation (as the 2,4-dinitrophenylhydrazone deriv-
ative) within the sensitivity of the assay (ꢁ0.2 nmol), when 1 mU
1.6
1.4
1.2
a
1.0
0.8
0.6
0.4
0.2
0.0
cystathionine c-lyase was incubated for 1 h at 37 °C in a reaction
Table 1
Inhibition of rhGTK-catalyzed transamination of L-phenylalanine with KMB by Cys-
and Hcy-S-conjugates of Hg2+ and methylmercury.
Inhibitor
L
-Phenylalanine concentration
10 mM 20 mM
Relative phenylpyruvate formation (%)
ND
50 mM
S
S
S
S
None
[100]
[100]
[100]
S
S
CH3Hg-S-Cys (0.5 mM)
CH3Hg-S-Hcy (0.5 mM)
Cys-S-Hg-S-Cys (1 mM)
Hcy-S-Hg-S-Hcy (0.5 mM)
72 26
49 11
55 11
63 25
40
29
35
4
8
4
50
33
50
3
6
7
31
43
4
5
Fig. 7. Substrate specificity of rhGTK toward Cys- and Hcy-S-conjugates of Hg2+ and
methylmercury. The reaction mixture (50 l) contained 0.2 mM phenylpyruvate,
1.0 mM mercury S-conjugate, 100 mM potassium phosphate buffer (pH 7.4) and
2.6 mU of enzyme. After incubation for 2 h at 37 °C the disappearance of
phenylpyruvate was measured in comparison to a blank lacking enzyme; ND not
detectable. aNot significantly different from the blank values (n = 5).
l
The reaction mixture contained varying concentrations of
KMB, 100 mM potassium phosphate buffer (pH 7.4) and enzyme (2 mU) in a final
volume of 50 l in the presence or absence of mercury S-conjugate. After incubation
for 30 min at 37 °C, phenylpyruvate formation was determined. N = 4 or 5.
L-phenylalanine, 5 mM
l

C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
27
mixture (50
and 100 mM potassium phosphate buffer, pH 7.4.
As also noted above, rat liver cystathionine -lyase was previ-
ously shown to be highly susceptible to inactivation by a number
of sulfhydryl reagents including p-chloromercuribenzoate. This
reaction and that with HgCl2 are formally depicted below, where
P = protein. Thus, it was predicted that HgCl2 would also be a
l
l) containing 1 mM Cys-S-Hg-S-Cys (or CH3Hg-S-Cys)
concentration (0.2
of cystathionine -lyase and the fact that the enzyme is a homotetr-
-lyase
monomers in the incubation mixture was 75 fmol. Thus, the mer-
cury-containing compounds were in large excess over cystathionine
lM) was 10 pmol. Based on the specific activity
c
c
amer (subunit Mr ꢁ44,000), the amount of cystathionine
c
c-lyase in these experiments. Another point of concern is whether
the mercury-containing compounds interfere with the 2,4-dini-
strong inactivator of cystathionine
be the case (see below).
c-lyase and this was found to
trophenylhydrazone assay for the quantitation of pyruvate (b-lyase
reaction) or
a-ketobutyrate (standard cystathionine b-lyase reac-
tion). In control experiments it was shown that the presence of
2 mM HgCl2 or 2 mM Cys-S-Hg-S-Cys had no effect on the yield of
PSH þ ClHgAr ! PSHgAr þ HCl
a-keto acid 2,4-dinitrophenylhydrazone when standard cystathio-
nine -lyase reaction mixtures (0.05 ml) were spiked with 10 nmol
pyruvate and incubated at 37 °C for 1 h followed by a further 10 min
c
PSH þ ClHgCl ! PSHgCl þ HCl
The previous findings with p-chloromercuribenzoate [36] and
the present findings with HgCl2 indicate that the active site cys-
teine is reactive. It was then of interest to determine whether this
reactive cysteine residue can interact with mercury cysteine conju-
gates that are substrate analogues. If the conjugates bind simply as
substrate analogues then it is expected that they will be reversible
inhibitors. However, if they can bind in such a manner as to come
in close contact with this reactive cysteine residue then it was pre-
dicted that the mercury cysteine conjugates would be strong irre-
versible inhibitors of the enzyme. This was found to be the case.
When the effect of various mercury-containing compounds on
incubation with the 2,4-dinitrophenylhydrazine reagent.
The present findings suggest that positioning a mercuric S-conju-
gate as a substrate analogue in close proximity to the active site of
cystathionine c-lyase facilitates modification of the cysteinyl moi-
ety within the enzyme active site. Despite the fact that the S–Hg
bond has a high stability constant (ꢁ0.22 kJ/mol) [9,43], the coordi-
nation bonds of mercury are kinetically quite labile [43–45]. Thus,
the greater potency of Cys-S-Hg-S-Cys compared to HgCl2 as an inac-
tivator of cystathionine
c-lyase (Fig. 7) may be due to binding of Cys-
S-Hg-S-Cys as an analogue of
L
-cystathionine/ -cystine followed by a
L
rapid S-Hg exchange reaction with a protein-bound cysteine moiety
within the active site of the enzyme.
the activity of purified rat liver cystathionine
mined, significant inhibition of cystathionine
when M amounts of these compounds were added to the standard
reaction mixture containing 20 mM -homoserine (Fig. 8). ( -Homo-
serine (Km = 20 mM) [42] is used in routine analytical assays for cys-
tathionine -lyase (Fig. 3, Eq. (3).) Interestingly, the extent to which
c
-lyase was deter-
c-lyase occurred
In a separate set of experiments, 2.1 mU of cystathionine
was incubated for 1 h at 37 °C in the presence of 100 mM potassium
phosphate buffer (pH 7.4) and 20 M of either HgCl2 or Cys-S-Hg-S-
Cys (final volume 20 l). At the end of the incubation 1 M potassium
phosphate (pH 7.4) and 100 mM -homoserine were added, such
that the final volume was 50 l and the concentrations of buffer
and -homoserine were 100 mM and 20 mM, respectively. After an
additional 30-min incubation at 37 °C, -ketobutyrate formation
c-lyase
l
L
L
l
l
c
L
inactivation occurred with the various mercury-containing com-
pounds was as follows: Cys-S-Hg-S-Cys > HgCl2 > CH3Hg-S-Cys. Pos-
sibly, the difference between Cys-S-Hg-S-Cys and CH3Hg-S-Cys
reflects the fact that Cys-S-Hg-S-Cys bears closer resemblance to
l
L
a
was measured. Only 5% of the activity relative to a control (2.1 mU
of enzyme incubated in phosphate buffer in the absence of mercury
compound) was detected for enzyme that had been exposed to
HgCl2, whereas no activity could be detected with enzyme that
had been exposed to Cys-S-Hg-S-Cys. In both cases, addition of
DTT to a final concentration of 5 mM did not restore any enzyme
activity, even after incubation for 5 h at 37 °C. However, the choice
of DTT in this experiment may not have been optimal. For example,
it is known that in the case of bacterial MerB (organomercurial lyase;
catalyzes the reaction: RHg(I) ? RH + Hg(II)), DTT binds to the Hg-S
enzyme adduct to form a stable 3-coordinate Hg complex [46,47].
Therefore, the experiment was repeated to determine whether
20 mM cysteine or 20 mM GSH (final concentration) in place of
DTT could reactivate enzyme that had been inactivated by HgCl2
or Cys-S-Hg-S-Cys. No activity was restored after incubation of
HgCl2- or Cys-S-Hg-S-Cys-inactivated enzyme for two hours at
the substrates
Cys. CH3Hg-S-Cys resembles methionine, which is not a cystathio-
nine -lyase substrate. It should be noted that the amount of mer-
cury present in the 50-
L-cystathionine and L-cystine than does CH3Hg-S-
c
ll
reaction mixture at the lowest
37 °C in the presence of 20 mM L-cysteine or 20 mM GSH.
Discussion
As mentioned in the Introduction, L-methionine, L-cystine and L-
cystathionine are substrates of rat kidney GTK. Consistent with this
finding, the present work shows that these sulfur-containing ami-
no acids are also substrates of the human counterpart (rhGTK)
(Fig. 5). Moreover, this work also shows that the sulfur-containing
mercury conjugates Cys-S-Hg-S-Cys and CH3Hg-S-Cys are transam-
inase substrates of this enzyme (Fig. 7). The ability to transaminate
these conjugates presumably is a result in part of the relatively
open active site and the ability of the enzyme to utilize large amino
acid substrates. However, under the conditions of our assay these
mercury conjugates are not as active as the ‘‘natural’’ in vivo sub-
Fig. 8. Inactivation of cystathionine
pounds. The reaction mixture (50
potassium phosphate (pH 7.4) and, where indicated, Hg-containing compound.
c
-lyase by various mercury-containing com-
ll) contained 20 mM L-homoserine, 100 mM
Enzyme (0.08 mU; 75 fmol of enzyme subunit) was added last. After incubation for
1 h at 37 °C,
a-ketobutyrate was measured by the 2,4-dinitrophenylhydrazine
method. The control contained complete reaction mixture plus enzyme, but no
added Hg-containing compound. ⁄Significantly different from the same treatment
group exposed to 0.2
ture) (P < 0.05). ⁄⁄Significantly different from the same treatment group exposed to
0.2 or 2.0 M Hg-containing compound (P < 0.05) (n P 3).
lM Hg-containing compound (10 pmol/50 ll reaction mix-
l
strates L-glutamine, L-cystine, L-methionine and L-phenylalanine.

28
C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
The question arises as to whether these transamination reac-
dehydrogenases [53,54], contain cysteine residues that, when mod-
ified, can significantly alter enzyme activity. Thiol/disulfide bonds
are critical for maintaining the structural, catalytic and allosteric
integrity of a vast number of enzymes and signal proteins. In addi-
tion, they are key components involved in the maintenance of redox
balance and redox-sensitive reaction pathways, and in mediating
protein signaling events [21,22,55–57]. Binding of mercury com-
pounds to sulfhydryl centers of exposed cysteine residues may lead
to reversible and/or irreversible inhibition of the enzyme, enzyme
denaturation and/or protein aggregation. For example, a recent
study provides data suggesting that HgCl2 and CH3HgCl irreversibly
inhibittheactivityof arylamineN-acetyltransferase-1, anintracellu-
lar enzyme involved in the biotransformation of aromatic and het-
erocyclic amines [58]. Similarly, the current study indicates that
HgCl2 and CH3HgCl are able to inhibit the intracellular enzymes,
tions with Cys-S-Hg-S-Cys and CH3Hg-S-Cys are biologically rele-
vant. Transamination of Cys-S-Hg-S-Cys and CH3Hg-S-Cys was
demonstrated at 1 mM concentration, but it is doubtful that this
concentration could be attained in vivo even after severe mercury
poisoning. Moreover, there will be strong competition with endog-
enous amino acid substrates naturally present at much higher con-
centrations. However, aminotransferases generally exhibit Km
values in the mM or tens of mM range and this is true of GTK
([38]; present work). Thus, it is possible that GTK is not fully satu-
rated with endogenous amino acid substrates and that some bind-
ing of Cys-S-Hg-S-Cys and CH3Hg-S-Cys will occur even when these
conjugates are present at low concentrations. There is precedent
for transamination of a metabolite at very low levels in vivo despite
the presence of much higher levels of other endogenous amino acid
substrates. For example, neuroactive kynurenate is obtained by
transamination of kynurenine. Two aminotransferases have been
extensively studied as contributing to the formation of kynurenate
from kynurenine in vivo, namely KAT I and KAT II. As noted above,
GTK and, much more potently and irreversibly, cystathionine
lyase. In previous work it was shown that after inactivation of crys-
talline rat liver cystathionine -lyase by p-chloromercuribenzoate,
c-
c
activity could only be partially restored by addition of 2,3-dimerca-
KAT I is identical to GTK. KAT II is identical to glutamate – a-ami-
ptopropionate [36].
noadipate aminotransferase (for a review of the role of KATs in the
brain see [48]). The concentration of kynurenine and its transami-
nation product kynurenate in the brain are in the 400 nM range
and 1 nM range, respectively [48,49]. Thus, kynurenate can be gen-
erated in brain through transamination of kynurenine despite the
low levels of precursor kynurenine and high levels of endogenous
alternative substrates – e.g. glutamine (KAT I) and glutamate (KAT
II). Thus, we suggest that it is entirely possible that Cys-S-Hg-S-Cys
and CH3Hg-S-Cys even at nM concentrations could be transaminat-
ed in vivo.
Although more detailed studies are required to firmly establish
the mechanism, the present findings of inhibition of rhGTK by HgCl2
and CH3HgCl have the characteristics of a slow, but reversible non-
competitive inhibition. There is a good precedent for such an occur-
rence. Frasco et al. [59] showed that human butyrylcholinesterase,
which does not possess a cysteine residue sensitive to thiol reagents,
is slowly, but reversibly, inhibited (minutes) by mM amounts of
HgCl2 in a noncompetitive manner. Human butyrylcholinesterase
crystallized in the presence of HgCl2 was found to contain two Hg
binding sites with variable Hg occupancy. No Hg was found in the ac-
tive site or was bound to cysteine sulfur [59]. As noted above, the
crystal structure of rhGTK is known [28]. Subsequent studies from
our group will focus on rhGTK crystallized in the presence of HgCl2
and mercury S-conjugates. As also noted above, mM concentrations
of CH3HgCl and HgCl2 are not biologically relevant. Nevertheless, the
results are helpful because they indicate that the compounds will be
useful in X-ray crystallographic studies of rhGTK. Moreover, they
highlight the different susceptibilities of rhGTK and cystathionine
Transamination of Cys-S-Hg-S-Cys and CH3Hg-S-Cys are pre-
dicted to yield the corresponding mercury-containing
a-keto acids.
However, it is not clear to what extent these -keto acids might
a
accumulate in vivo given the large pool of GSH (mM) in most tis-
sues and the potential for Hg–S exchange. If the exchange is rela-
tively slow, then it is possible that the
a-keto acids derived from
Cys-S-Hg-S-Cys and CH3Hg-S-Cys may contribute to the toxic ef-
fects in vivo in individuals exposed to organic mercury via food in-
take, especially since GTK is of relatively high specific activity in
the kidney [23], and the kidney is a major site of accumulation of
c-lyase to mercury-containing compounds as discussed below.
HgCl2, Cys-S-Hg-S-Cys and CH3HgCl strongly inhibit cystathio-
Cys-S-Hg-S-Cys and CH3Hg-S-Cys [13]. It is possible that if the
keto acid analogues of Cys-S-Hg-S-Cys and CH3Hg-S-Cys are pro-
duced in vivo they may react with/inhibit -keto acid-utilizing en-
zymes thereby contributing to the toxicity. However, the predicted
mercury-containing -keto acids have not yet been characterized
a
-
nine c-lyase at lM, or lower, concentrations (Fig. 8). This inhibition
is irreversible for HgCl2 and Cys-S-Hg-S-Cys (and probably also for
CH3Hg-S-Cys). These findings are markedly different from those
noted for rhGTK, where strong inhibition by HgCl2 and CH3HgCl re-
quires mM concentrations. Moreover, inhibition of rhGTK by HgCl2
was shown to be reversible by addition of DTT. [The reversibility of
the inhibition by CH3HgCl was not investigated.] As noted above,
a
a
and their biological and toxicological properties must await further
studies. Finally, if there is some Hg–S exchange between GSH and
the
a
-keto acid analogues of Cys-S-Hg-S-Cys and CH3Hg-S-Cys,
the sensitivity of cystathionine c-lyase to inhibition by HgCl2 is
mercaptopyruvate may be one of the products formed. Mercapto-
pyruvate is a donor of sulfane sulfur to a suitable acceptor in a
reaction catalyzed by mercaptopyruvate sulfurtransferase [50].
Addition of sulfane sulfur to cysteine residues has the potential
to modify protein structure and function [50].
probably due to a reactive cysteine in the vicinity of the active site.
Again, there is a good precedent in the work of Frasco et al. [59].
These authors showed that, unlike human butyrylcholinesterase,
Torpedo californica acetylcholinesterase possesses a cysteine resi-
due sensitive to sulfhydryl reagents, and is irreversibly inhibited
In addition, our data show that Cys-S-Hg-S-Cys and CH3Hg-S-Cys
(and the corresponding Hcy conjugates) are strongly inhibitory rel-
ative to the substrate phenylalanine. However, the exact kinetic
mechanismremains to be elucidated. One possibility is that the mer-
cury S-conjugates react covalently (participate in Hg–S exchange)
with one or more cysteine residues in rhGTK. Reaction with the cys-
teinyl moiety (C127) near the active site (or possibly with more dis-
tant cysteines) may account for the apparent noncompetitive type of
inhibition observed during our kinetic studies. Planned X-ray crys-
tallographic studies of rhGTK crystallized in the presence of either
HgCl2 or Cys-S-Hg-S-Cys should provide additional information.
It is important to note that the activity of many enzymes,
including, for example, certain aminotransferases [51,52] and
in a pseudo-first-order process by lM amounts of HgCl2.
The earlier discovery that mercury species bond avidly with thiol-
containing biomolecules to form S-conjugates such as Cys-S-Hg-S-
Cys and CH3Hg-S-Cys [9] was instrumental in expanding our under-
standing of the toxicity of mercury compounds within target organs
and cells. When these S-conjugates of mercury act as molecular ana-
logues of endogenous, sulfur-containing amino acids, they broaden
the scope of the toxicological consequences of mercury exposure. In-
deed, the present study shows that Cys-S-Hg-S-Cys is a potent irre-
versible inhibitor of cystathionine
Cys and CH3Hg-S-Cys are aminotransferase substrates and reversible
inhibitors of GTK. In rodents, the specific activity of cystathionine
lyase is highest in the liver followed by kidney, with much lower
c-lyase, while both Cys-S-Hg-S-
c-

C.C. Bridges et al. / Archives of Biochemistry and Biophysics 517 (2012) 20–29
[7] A.F. Castoldi, T. Coccini, L. Manzo, Rev. Environ. Health 18 (2003) 19–31.
29
levels in other organs [60]. On the other hand, the specific activity of
GTK is highest in kidney, but the enzyme is also notably active in liver
and, to a lesser extent, in brainand other organs [23,24]. Thus, the dif-
ferences inorgantoxicity ofCys-S-Hg-S-Cys versusCH3Hg-S-Cysmay
be related, in part, to differences in the way the compounds interact
with enzymes/transporters. For example, CH3Hg-S-Cys, but not Cys-
S-Hg-S-Cys, has been shown to cross the blood–brain barrier via the
amino acid transporter, system L [61,62]. Given that the human brain
contains large amounts of cystathionine [63], the neurotoxicity of
CH3Hg-S-Cys may be related, in part, to its transport through the
blood–brain barrier and its subsequent targeting of cystathionine
metabolism/turnover/function in the human brain. These mercury-
containing organosulfur conjugates are the forms to which humans
would be most often likely exposed following consumption of con-
taminated fish that are high in the food chain. Thus, the metabolic
burden of processing inorganic or organic forms of mercury, such
as monomethyl S-conjugates, would be expected to occur within tis-
sues of autotrophic species and/or animals lower in the food web that
are initially exposed to mercury from the atmosphere or that settled
in water.
Clearly human exposure to mercury-containing compounds has
numerous consequences, not only at the organ level, but also at the
cellular/molecular level. Our results emphasize the fact that mercury
not only binds covalently and indiscriminately to thiol moieties in
proteins, but once conjugated with sulfur-containing amino acids
and peptides, particularly cysteine and GSH, forms compounds that
can interact with enzymes, thereby potentially broadening the pro-
file associated with overall mercury toxicity. These compounds
may represent the major portion of organomercury that contributes
to the metabolic burden following consumption of mercury-contam-
inated foods.
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This work was supported by grants from the National Institutes of
Health RO1 [ES8421](to AJLC), RO1 [ES5980](to RKZ), RO1 [ES11288]
(to RKZ), NS062836 (to JL), RO3 [ES15511](to CCB), R15 [ES19991](to
CCB) and CA111842 (to JTP).
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