Mechanism of cysteine-dependent inactivation of aspartate/glutamate/ cysteine sulfinic acid α-decarboxylases

Article abstract of DOI:10.1007/s00726-012-1342-7

Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

Full text of DOI:10.1007/s00726-012-1342-7

Amino Acids (2013) 44:391–404
DOI 10.1007/s00726-012-1342-7
ORIGINAL ARTICLE
Mechanism of cysteine-dependent inactivation of aspartate/
glutamate/cysteine sulfinic acid a-decarboxylases
•
Pingyang Liu Michael P. Torrens-Spence
•
•
Haizhen Ding Bruce M. Christensen
•
Jianyong Li
Received: 22 February 2012 / Accepted: 5 June 2012 / Published online: 21 June 2012
Ó Springer-Verlag 2012
Abstract Animal aspartate decarboxylase (ADC), gluta-
mate decarboxylase (GDC) and cysteine sulfinic acid
decarboxylase (CSADC) catalyze the decarboxylation of
aspartate, glutamate and cysteine sulfinic acid to b-alanine,
c-aminobutyric acid and hypotaurine, respectively. Each
enzymatic product has been implicated in different physi-
ological functions. These decarboxylases use pyridoxal
5-phosphate (PLP) as cofactor and share high sequence
homology. Analysis of the activity of ADC in the presence
of different amino determined that beta-alanine production
from aspartate was diminished in the presence of cys-
teine. Comparative analysis established that cysteine also
inhibited GDC and CSADC in a concentration-dependent
manner. Spectral comparisons of free PLP and cysteine,
together with ADC and cysteine, result in comparable
spectral shifts. Such spectral shifts indicate that cysteine is
able to enter the active site of the enzyme, interact with the
PLP-lysine internal aldimine, form a cysteine-PLP aldim-
ine and undergo intramolecular nucleophilic cyclization
through its sulfhydryl group, leading to irreversible ADC
inactivation. Cysteine is the building block for protein
synthesis and a precursor of cysteine sulfinic acid that is the
substrate of CSADC and therefore is present in many cells,
but the presence of cysteine (at comparable concentrations
to their natural substrates) apparently could severely inhibit
ADC, CSADC and GDC activity. This raises an essential
question as to how animal species prevent these enzymes
from cysteine-mediated inactivation. Disorders of cysteine
metabolism have been implicated in several neurodegen-
erative diseases. The results of our study should promote
research in terms of mechanism by which animals maintain
their cysteine homeostasis and possible relationship of
cysteine-mediated GDC and CSADC inhibition in neuro-
degenerative disease development.
Keywords Cysteine ꢀ Inactivation ꢀ PLP-containing
decarboxylase ꢀ Hypotaurine ꢀ b-Alanine ꢀ c-Aminobutyric
acid
Introduction
Animal glutamate decarboxylase (GDC), aspartate decar-
boxylase (ADC, also called aspartate a-decarboxylase or
aspartate 1-decarboxylase) and cysteine sulfinic acid
decarboxylase (CSADC) catalyze the decarboxylation of
a-carboxyl group of glutamate, aspartate and cysteine sulfi-
nic acid to produce c-aminobutyric acid (GABA), b-alanine
and hypotaurine, respectively; these amine products play
important role in living organisms. For example, GABA is
a chief inhibitory neurotransmitter and a regulator of neu-
ronal excitability in animals. Hypotaurine is the direct
precursor of taurine, a chemical that plays several physi-
ological functions in mammals (Holmes et al. 1992; Foos
and Wu 2002; Hayes and Sturman 1981). b-alanine is
one of the two precursors for the formation of carnosine
(b-alanyl-L-histidine), a peptide that plays an important
role as an intracellular buffer and antioxidant (Hipkiss
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-012-1342-7) contains supplementary
material, which is available to authorized users.
P. Liu ꢀ M. P. Torrens-Spence ꢀ H. Ding ꢀ J. Li (&)
Department of Biochemistry, Virginia Tech, Engel Hall 204,
Blacksburg, VA 24061, USA
e-mail: lij@vt.edu
B. M. Christensen
Department of Pathobiological Sciences,
University of Wisconsin-Madison, Madison, WI 53706, USA
123

392
P. Liu et al.
2009; Hama et al. 1971; Bellia et al. 2011). Carnosine has
also been demonstrated to enhance muscular performance
(Smith et al. 2009; Artioli et al. 2010; Smith et al. 2011).
As a result, b-alanine has been recommended as a benefi-
cial dietary supplement (Smith et al. 2009; Artioli et al.
2010).
specificity (Richardson et al. 2010). Insects do not have a
specific CSADC and there has been no report regarding the
ADC in mammals. In our recent study, however, we
demonstrated that insect ADC could use cysteine sulfinic
acid and cysteic acid as its substrates (Fig. 1). Accordingly,
insect ADC has the typical CSADC activity (Liu et al.
2012). The function of insect ADC in taurine production
may be as important (if not more) as its function in
b-alanine production, and the enzyme could have been
named CSADC if cysteine sulfinic acid were the first tested
substrate. Overlap in substrate specificity between insect
ADC and mammalian CSADC indicates that it is necessary
to critically compare the substrate specificity and bio-
chemical properties of these GDC-like enzymes. It has also
been reported that the activity of human GDC is negatively
affected by the presence of aspartate (Porter and Martin
1987). Aspartate-induced GDC inhibition suggests that
non-substrate amino acids might interfere with the catalytic
efficiency of GDC-like. Therefore, to more practically
comprehend the regulation of these enzymes, it is neces-
sary to understand conditions that affect the functions of
GDC-like protein family.
Although b-alanine plays several physiological roles in
mammals, ADC has not been identified in any animal
species except insects. In insects, b-alanine is used to
synthesize N-b-alanyldopamine (NBAD) and N-b-alanyl-
histamine. NBAD is oxidized easily by insect pheno-
loxidase and laccase, and its oxidized form can react with
nucleophilic groups on proteins, leading to protein cross-
linking. This has been considered as one of the primary
mechanisms involved in insect cuticle hardening or scler-
otization (Arakane et al. 2009; Moussian 2010; Morgan
et al. 1987; Hopkins et al. 1984; Kramer et al. 1984).
b-alanine has also been shown to inactivate the neuro-
transmitter histamine within the synaptic clefts of insect
photoreceptor cells (Hardie 1987; Stuart et al. 2007).
Therefore, b-alanine plays unique physiological functions
in insects.
Despite playing different physiological roles in animal
and insects, ADC, CSADC and GDC all use pyridoxal
5-phosphate (PLP) as cofactor and share fairly high
sequence identity ([40 %). Among these enzymes, GDC is
ubiquitous and therefore is considered the prototype. As a
result, the ADC and CSADC sequences in some sequenced
genomes often have been named GDC or GDC-like protein
without further differentiation. Our previous study deter-
mined that insect ADC and GDC have no overlap substrate
In this study, we expressed several GDC-like enzymes,
including human CSADC, mosquito GDC, mosquito ADC
and Drosophila ADC, and assessed the activities of these
enzymes to their natural substrates in the presence of other
proteogenic amino acids. We initially observed that in the
presence of similar concentrations of aspartate and cysteine
sulfinic acid, the b-alanine production (ADC activity) was
diminished and the reaction was driven primarily toward
hypotaurine production (CSADC activity). Both aspartate
Fig. 1 Reactions catalyzed by
insect ADC. a Aspartate
1-decarboxylation. b Cysteine
sulfinic acid
decarboxylation and cysteic
acid decarboxylation
123

Cysteine-dependent inactivation of a-decarboxylases
393
and cysteine sulfinic acid likely are present in many tissues
and cells. Subsequent screening of insect ADC with either
aspartate or cysteine sulfinic acid in the presence of each of
the other proteogenic amino acids revealed that cysteine
effectively inactivated the activity of insect ADC. Further
analysis determined that the activities of mosquito GDC
and human CSADC were also inactivated by cysteine in a
concentration-dependent manner.
liquid chromatography with electrochemical detection
(HPLC-ED). The mobile phase consisted of 50 mM
phosphate buffer (pH 3.5) containing 25 % acetonitrile at a
flow rate of 0.5 ml per minute. The oxidation potential of
the working was maintained at ?750 mV with an Ag/AgCl
reference electrode.
Time and dose dependence of cysteine inhibition
In this communication, we provide data that describe the
effect of cysteine on the activities of GDC-like proteins.
Specifically, we use insect ADC as a model enzyme to
discuss the mechanism of cysteine-dependent inactivation
of GDC-like enzymes.
on AeADC
ADC activity assays in the presence of different amino
acids identified cysteine as an effective inactivator of
AeADC activity. To assess the type of ADC inhibition by
cysteine, a series of solutions of 400 ll containing
6.25 mM aspartate and
a varying concentration of
Materials and methods
cysteine were freshly prepared in 100 mM phosphate
buffer (pH 7.0) and mixed with 100 ll of AeADC solution
to determine the effect of cysteine concentration on ADC
activity. The final concentration of the reaction contained
5 mM of aspartate and a series of cysteine concentrations
(0.05, 0.1, 0.2, 1, 2 and 5 mM). The enzyme preparation
contained 0.2 mg of AeADC and 1 mg of bovine serum
albumin (for enzyme stabilization) per milliliter and was
prepared in the same buffer. The reaction was initiated by
mixing 100 ll enzyme preparation into 400 ll aspartate
and cysteine solution. At 2-, 4-, 6-, and 8-min intervals,
50 ll of the reaction mixture was withdrawn and mixed
with 100 ll of 100 % ethanol to stop reaction, followed
by the described product derivatization and quantification
procedures.
Chemicals
All chemicals used in this report were from Sigma-Aldrich,
unless specified otherwise.
AeADC expression and purification
AeADC was expressed in BL21 and purified to homoge-
neity as described in a previous study (Richardson et al.
2010). A Bio-Rad protein assay kit was used to determine
protein concentration with bovine serum albumin as a
standard. AeADC was concentrated to 20 lg/ll in 20 mM
phosphate buffer (pH 7.0).
Assays of ADC and CSADC activities of AeADC
in the presence of different amino acids
Spectral changes of AeADC or PLP in the presence
of cysteine
Prior to enzyme activity assays, concentrated enzyme
preparations were diluted with phosphate buffer containing
1 mg of bovine serum albumin (for enzyme stabilization).
Amino acid solutions in this manuscript were adjusted to
pH 7.0 before use. The typical reaction mixtures of 50 ll,
containing 2 lg of purified AeADC, and 5 mM of aspartate
or 5 mM of cysteine sulfinic acid, were prepared in
200 mM phosphate buffer (pH 7.0) in the absence or
presence of 5 mM each of the proteogenic amino acids (the
final concentration for tyrosine was 2 mM due to its low
solubility). The reaction mixtures were incubated at 25 °C
and the reaction was stopped by addition of two volumes of
100 % ethanol 10 min after incubation. The mixture was
then treated with an equal volume of o-phthaldialdehyde
(OPT) reagent as described in a previous method (Rich-
ardson et al. 2010). Product formation was based on the
detection of b-alanine-OPT derivative by reverse-phase
Initial data indicated that inhibition of AeADC by cysteine
seemed to be due to its interaction with PLP cofactor,
which likely led to the noticeable spectral changes of the
enzyme. To evaluate the possible interaction of aspartate,
cysteine sulfinic acid, or cysteine and AeADC, 10 ll of
5 mM aspartate, cysteine sulfinic acid or cysteine was first
mixed with 80 ll of 200 mM phosphate buffer (pH 7.0),
followed by rapid addition of 10 ll of 20 lg/ll of AeADC
and spectral analysis. The spectral changes of the AeADC
and amino acid mixtures from 300 to 480 nm were moni-
tored at each 45-s interval for a 10-min period using an
Aligent 8453 UV/visible spectrophotometer. Data were
processed using the Aligent UV/visible ChemStation soft-
ware. To determine the possible interaction of free PLP
with aspartate, cysteine sulfinic acid or cysteine, free PLP
was prepared in phosphate buffer and mixed with each of
the compounds (with a final concentration of PLP and
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P. Liu et al.
cysteine at 0.2 and 0.5 mM) and the possible spectral
changes of the PLP and cysteine mixture were monitored
from 300 to 480 nm as those described for AeADC and
amino acid mixtures.
Results
The presence of cysteine sulfinic acid on the activity
of AeADC to form beta-alanine
Effect of cysteine on Anopheles gambiae GDC
(AnGDC), HuCSADC and AeADC Q377L activities
When AeADC was mixed with aspartate, accumulation of
b-alanine was observed in the reaction mixture, but when
the same concentration of cysteine sulfinic acid was also
incorporated into the reaction mixture, production of
b-alanine was diminished and instead a high level of
hypotaurine was accumulated (Fig. 2a, b). When the con-
centration of aspartate was increased to fourfold of cysteine
sulfinic acid in the reaction mixture, the ADC activity was
still inhibited (Fig. 2c) suggesting that AeADC favored
cysteine sulfinic acid as its substrate, or cysteine sulfinic
acid could much better compete with aspartate for binding/
The inhibition of AeADC by cysteine was verified. This
questions if cysteine has similar effect on other GDC-like
proteins. To assess the general effect of cysteine on GDC-
like enzymes, AnGDC, HuCSADC and AeADC Q377L (a
mutation showing different substrate selectivity) (Liu et al.
2012) were expressed as AeADC and the effect of cysteine
on their activity was analyzed similarly as described for
AeADC. Typically, reaction mixtures of 50 ll, containing
2 lg of purified AnGDC or HuCASDC and 5 mM of glu-
tamate or 5 mM of cysteine sulfinic acid, were prepared in
200 mM phosphate buffer (pH 7.0) in the absence or
presence of 5 mM of cysteine. The other conditions,
including incubation time, product derivatization and
product quantitation, were identical to those described for
AeADC activity assays, except for the increase of aceto-
nitrile (50 %) in the mobile phase during detection GABA.
Computer modeling
Enzyme activity assays and spectral changes of AeADC in
the presence of cysteine indicated that cysteine could
enter into the active site of AeADC and interact with the
internal aldimine, leading to the formation of the PLP–
cysteine complex. To elucidate the structural basis of
cysteine inhibition, AeADC structural model was gener-
ated using the atomic coordinates of HuCSADC (PDB:
2JIS) as templates. AeADC shares 52 % sequence identity
with HuCSADC overall and 56 % identity between their
PLP-binding domains. The predicted AeADC structural
model was then used to assess the structural basis of
cysteine binding through molecular docking (Trott and
Olson 2010). AutoDockTools and Autodock Vina were
used to evaluate the interaction of the substrate and the
enzyme (Trott and Olson 2010). The structure of cysteine
for docking was prepared using PRODRG (Schuttelkopf
and van Aalten 2004). Cysteine is one of the preferred
substrates for animal kynurenine aminotransferases and
these enzymes are not inactivated by cysteine. To further
understand and clarify the mechanism of cysteine inhibi-
tion to GDC-like proteins, the predicted AeADC structural
model was compared with an Aedes aegypti kynurenine
aminotransferase structural model with cysteinyl aldimine
(PDB: 2R5C) (Han et al. 2008). The active site for cys-
teine is found in one of the biological dimers (Han et al.
2008).
Fig. 2 The ADC or CSADC activity of AeADC in the presence of
cysteine sulfinic acid or aspartate. a The ADC activity of AeADC with
5 mM of aspartate. b The CSADC activity of AeADC with 5 mM of
cysteine sulfinic acid. c The ADC activity of AeADC with 5 mM of
aspartate in the presence of 5 mM of cysteine sulfinic acid. d The
ADC activity of AeADC with 20 mM of aspartate in the presence of
5 mM of cysteine sulfinic acid
123

Cysteine-dependent inactivation of a-decarboxylases
395
catalysis. The same results were obtained when DmADC
was analyzed in the same manner (not shown).
activity was not significantly affected by any of the tested
amino acids (Fig. 3a). When AeADC was incubated with
aspartate in the presence of each of 19 remaining proteo-
genic amino acids, its ADC activity was diminished in the
presence of cysteine. No other amino acids showed any
significant effect on either increase or decrease of its ADC
activity (Fig. 3b). The selective cysteine-dependent
Effects of amino acids on AeADC activities
When AeADC was incubated with cysteine sulfinic acid in
the presence of each of 20 proteogenic acids, its CSADC
Fig. 3 The effect of amino
acids on the activities of
AeADC. a The CSADC activity
of AeADC with the addition of
different amino acid. b The
ADC activity of AeADC with
the addition of different amino
acid. Each 50 ll reaction
mixture contained 5 mM of
substrate (either aspartate or
cysteine sulfinic acid), 200 mM
phosphate buffer (pH, 7.0),
0.4 lM of PLP and 5 mM of
amino acid addition. The
reaction was triggered by
adding 2 lg AeADC, incubated
at 25 °C for 10 min and stopped
by adding two volumes of
100 % ethanol
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396
P. Liu et al.
inhibition of the ADC activity, but not the CSADC activity
of AeADC, raises an essential question regarding the
chemical mechanism of such selective inhibition.
mixtures containing AeADC, 5 mM of aspartate and 1 mM
of cysteine were stopped at 2, 4, 6 and 8 min after incu-
bation (Fig. 4b). This suggests that the interaction of cys-
teine with AeADC actually led to progressive and
irreversible inactivation of the enzyme. The kinetics of
AeADC inactivation by cysteine was calculated to be
Kobs = -0.364/min. The inactivation efficiency can reach
almost 100 % beyond 10 min of incubation (not shown).
Prolonged pre-incubation of cysteine with AeADC will
completely kill the ADC activity of the enzyme (please see
supplemental figure 1).
Time and concentration dependence of cysteine
inhibition
When AeADC was mixed with solutions containing 5 mM
of aspartate and a varying concentration of cysteine, the
level of ADC activity inhibition was approximately pro-
portional to the concentrations of cysteine in the reaction
mixtures (Fig. 4a), suggesting somewhat a competitive
nature of the inhibition. Based on the specific activity
calculated during a 10-min incubation period, the presence
of 2 mM of cysteine or above resulted in 80 % inhibition
of ADC activity (Fig. 4a). However, a progressive decrease
in the specific activity was observed when the reaction
The time and concentration dependency of cysteine
inactivation of the AeADC activity differed from the cys-
teine inactivation of CSADC activity. When AeADC was
first incubated in 5 mM of cysteine for 5 or 10 min and
then mixed with cysteine sulfinic acid, the CSADC activity
of the AeADC was greatly decreased compared to a
Fig. 4 The dose and time
dependence of cysteine
inhibition on ADC activity.
a Different concentrations of
cysteine were tested on the
inhibition of ADC activity.
b The velocity of ADC activity
was measured at different time
point when the reaction mixture
contained 5 mM of aspartate
and 1 mM of cysteine
123

Cysteine-dependent inactivation of a-decarboxylases
397
Fig. 5 The effect of cysteine
pre-incubation on the CSADC
activity of AeADC; 10 ll of
25 mM cysteine was added into
each 50 ll reaction mixture
containing 2 lg AeADC,
200 mM phosphate buffer (pH
7.0) and 0.4 lM of PLP for
0 min (a), 5 min (b) or 10 min
(c) at 25 °C. The reaction was
triggered by adding 10 ll of
25 mM of cysteine sulfinic acid
and incubated at 25 °C for
10 min before it was stopped
cysteine-free control reaction. The level of cysteine-
AeADC inactivation increased proportionally to cysteine
pre-incubation time (Fig. 5). Under the applied conditions,
the specific activity was 2.2 lmol/min/mg for the 10-min
cysteine-pre-incubated ADC as compared to 5.5 lmol/min/
mg for the enzyme without cysteine pre-incubation. These
results indicate that cysteine sulfinic acid competes effec-
tively with cysteine to bind to AeADC, which prevents or
greatly slows down the interaction of cysteine with the
enzyme so that the rate of product formation was not
noticeably affected during a 10-min incubation period (see
Fig. 3a). In contrast, cysteine competed more effectively
than aspartate to react with AeADC when mixed with the
enzyme at the same time, leading to a progressive inacti-
vation of the enzyme and diminished product formation
(see Fig. 3b).
of the enzyme (Fig. 6a, b). When 0.5 mM of cysteine was
mixed with AeADC solution, progressive increase and
decrease of the 335-nm peak and the 405-nm peak,
respectively, were observed (Fig. 6c). Increasing the con-
centration of cysteine in the mixture increased the rate of
spectral change (not shown). When aspartate and cysteine
(each at 0.5 mM final concentration) was mixed with the
enzyme, a similar increase of the 335-nm peak and
decrease of the 405-nm peak were observed (Fig. 6d).
However, the addition of 0.5 mM of cysteine and cysteine
sulfinic acid did not result in significant change in the
spectra of the enzyme within a 10-min period (Fig. 6e).
Effects of different amino acids on the UV/visible
spectrum of free PLP
Free PLP has a peak absorbance around 390 nm and a
small peak (or a shoulder) at 330 nm at pH 7.0. When
cysteine was mixed with free PLP, a progressive decrease
of the 390-nm peak and increase of the 335-nm peak were
observed (Fig. 7a). These spectral changes are similar to
those observed in AeADC and cysteine mixture except that
the decrease was at 405 nm for the enzyme as opposed to
the decrease at 390 nm for free PLP (Fig. 6c). In contrast,
no apparent spectral changes of the PLP were observed
when aspartate or cysteine sulfinic acid was mixed with
free PLP (Fig. 7b). These results indicate that cysteine
forms complex with PLP under physiological pH
conditions.
Interaction of aspartate, cysteine sulfinic acid
or cysteine with AeADC
AeADC contains a PLP cofactor that is covalently linked
with a conserved lysine residue as an internal aldimine.
This internal aldimine produces absorbance peaks with
kmax around 335 and 405 nm. The AeADC spectrum
remains unchanged after 2 h of incubation at 25 °C. This
indicates that the AeADC is stable at 25 °C. When either
aspartate or cysteine sulfinic acid (each at 0.5 mM final
concentration) was mixed with AeADC in phosphate buffer
at pH 7.0, there were no obvious changes of the spectrum
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P. Liu et al.
Fig. 6 The UV/visible spectra of AeADC under different conditions.
a The spectra of AeADC with the addition of aspartate (0.5 mM final
concentration). b The spectra of AeADC with the addition of cysteine
sulfinic acid (0.5 mM final concentration). c The spectra of AeADC
with the addition of cysteine (0.5 mM final concentration). d The
spectra of AeADC with the addition of aspartate and cysteine (each
0.5 mM final concentration). e The spectra of AeADC with the
addition of cysteine sulfinic acid and cysteine (each 0.5 mM final
concentration). The total reaction volume is 100 ll; 10 ll of 20 lg/ll
AeADC was incubated with 0.5 mM of different compounds at 25 °C
for 10 min in 200 mM phosphate buffer (pH 7.0). The change of
spectra was monitored in a period of 10 min and each recoding was
taken at an interval of 45 s
Reaction of cysteine with the internal aldimine
react with the internal aldimine, form cysteine–PLP com-
plex and inhibit their activities in the same manner. When
AnGDC was mixed with glutamate in the presence of
cysteine, the inhibition of GDC was clearly observed
(Table 1) and such inhibition was approximately propor-
tional to the concentration of cysteine in the reaction
in AnGDC, HuCSADC, and AeADC Q377L
Cysteine is chemically similar to glutamate, aspartate and
cysteine sulfinic acid. It was predicted that cysteine is
likely able to enter the active sites of GDC and CSADC,
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Cysteine-dependent inactivation of a-decarboxylases
399
Fig. 7 The UV/visible spectra of free PLP under different conditions.
a The spectra of free PLPwith the addition of cysteine. The total reaction
volume is 100 ll; 0.2 mM of PLP was incubated with 0.5 mM of
cysteineat25 °Cfortenminin200 mMphosphatebuffer(pH7.0). Each
spectrum was taken at an interval of 45 s. b The spectra of free PLP
under different conditions. The total reaction volume is 100 ll. Red line
shows the spectrum of 0.2 mM of PLP in 200 mM phosphate buffer (pH
7.0)at10 min. Dark blue line shows thespectrum of0.2 mMof free PLP
and 0.5 mM of aspartate in 200 mM phosphate buffer (pH 7.0) at
10 min. Light blue line shows the spectrum of 0.2 mM of free PLP and
0.5 mMof cysteinesulfinic acidin200 mMphosphatebuffer(pH7.0) at
10 min. Orange line shows the spectrum of 0.2 mM of free PLP and
0.5 mM of cysteine in 200 mM phosphate buffer (pH 7.0) at 10 min
(color figure online)
Fig. 8 The effect of cysteine on the AeADC Q377L. 10 ll of 25 mM
of cysteine was added into each 50 ll reaction mixture containing
2 lg AeADC Q377L, 5 mM of cysteine sulfinic acid, 200 mM
phosphate buffer (pH 7.0) and 0.4 lM of PLP. The reaction was
stopped and derivatized after 10 min of incubation at 25 °C. The
production of hypotaurine for control group (a) and cysteine-addition
group (b) was tested and compared. The electrochemical detection
was the same as previously
Table 1 The activities of wild-type AeADC, AeADC Q377L,
AnGDC and HuCSADC with or without cysteine addition
same time, the activity of the enzyme was decreased, which
is different from that observed from AeADC (Table 1).
We recently proposed that an active site glutamine
residue (Q377) in AeADC plays a major role in its ability to
use aspartate as substrate, because mutation of the active
site Q377 to leucine (L377) diminished its ADC activity
with no apparent effect on its CSADC activity. When the
mutant AeADC was mixed with both cysteine and cysteine
sulfinic acid at an equal molar concentration, the CSADC
activity of the mutant AeADC was considerably reduced
(Fig. 8; Table 1).
Enzyme
Substrate
Specific activity
(lmol/min/mg)
Control
With cysteine
(5 mM)
AeADC
wild-type
Aspartate
7.39 0.6 1.59 0.2
Cysteine sulfinic acid 6.70 0.8 6.37 0.5
Aspartate 2.14 0.2 Not detectable
Cysteine sulfinic acid 5.82 0.4 0.6
Glutamate 1.07 0.2 Not detectable
AeADC
Q377L
AnGDC
HuCSADC Cysteine sulfinic acid 4.89 0.5 3.61 0.2
Analysis of the interaction between cysteine
and PLP-dependent enzymes through molecular
docking
The assaying conditions are described in ‘‘Materials and methods’’
mixtures (not shown). Both human CSADC and AeADC
use cysteine sulfinic acid as substrate, but human CSADC
has no activity on aspartate. When human CSADC was
mixed with both cysteine sulfinic acid and cysteine at the
To assess the mechanism of cysteine-dependent inactiva-
tion against GDC-like proteins, cysteine was used as the
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P. Liu et al.
Fig. 9 Computational analysis of interactions between cysteine and
AeADC or HuCSADC. a The relative position of cysteine and the
PLP at the active site of predicted AeADC structure. b A model
showing the relative position of cysteinyl aldimine and the active site
residues of AeKAT1. The homology model of AeADC are shown as
red and pink ribbons. The structural model was generated with Swiss
model using the atomic coordinates of HuCSADC (PDB: 2JIS) as
templates. The crystal structural model of AeKAT1 is shown as blue
and cornflower blue ribbons. The distances are indicated with green
dashed lines (color figure online)
ligand to perform the docking experiment in AeADC. The
orientation of cysteine within AeADC active site and its
interaction with the active site residues were compared
with those of cysteinyl aldimine in a kynurenine amino-
transferase that uses cysteine as a substrate (Han et al.
2008; Han et al. 2009). The optimum docking position
(predicted by Autodock Vina) was based on chemical
potential that takes into account both the bound confor-
mation preference and the free energy of binding (i.e., the
ligand in the position has the lowest energy) (Trott and
Olson 2010). The orientation of cysteine was slightly dif-
ferent at the active site between the two enzymes (Fig. 9a,
b). Distances between the a-N atom or sulfhydryl group of
the substrates and the C40 atom of the lysine–PLP or PLP
were labeled. The results showed that the sulfhydryl group
incoming substrate to form external aldimine), resulting in
the formation of cysteine-PLP aldimine and 2) the aldimine
intermediate then undergoes intramolecular cyclization
(nucleophilic addition) through the sulfhydryl group to
form a stable covalently linked complex (Fig. 10), leading
to the irreversible inactivation of the enzyme. In addition to
the above findings, we demonstrate that the cysteine
inactivation of GDC-like proteins is dependent on the rel-
ative affinities of cysteine and natural substrates of the
GDC-like proteins. This is clearly illustrated by the varied
efficiency of cysteine-dependent inactivation of ADC,
CSADC and ADC Q377L (due presumably to enhanced
inactivator binding). Our results lead to an intriguing
question as to how living species prevent the inactivation
of GDC-like proteins by cysteine. This manuscript should
serve as a useful reference toward comprehensive under-
standing of the regulation of GDC-like proteins in vivo.
This work also highlights a possible research direction
regarding the effect of the disorder of cysteine regulation
on the physiological functions of GDC-like proteins. The
toxicity of excess cysteine has been demonstrated in rabbits
and rats (Andine et al. 1991; Lehmann 1987; Lehmann
et al. 1993).
0
˚
was cycled back, making it within 5 A to the C4 atom of
the PLP in AeADC (Fig. 9a). Notice that the cysteine does
not form Schiff base with PLP in the predicted model of
AeADC, making the distance between the sulfhydryl group
and the imine carbon slightly longer than it should have
been. In contrast, the cysteine was stretched at the active
site of the kynurenine aminotransferase; the sulfhydryl
group and carboxyl group of cysteinyl aldimine interact
with the surrounding residues (Q44, R405, and N193),
physically restraining the sulfhydryl group from interacting
with the external aldimine (Fig. 9b).
Cysteine is indispensible in animals as a building block
for protein synthesis and a precursor for the production of
glutathione and taurine. This is particularly true for young
individuals as growth is always accompanied with active
protein synthesis, which requires adequate supplies of
cysteine and which may explain in part the relatively high
levels of cysteine in young individuals. The plasma
cysteine concentration ranges from 80 to 200 lmol/l in
healthy individuals (Stipanuk et al. 2006) and cysteine
tends to decrease along the aging process (Hildebrandt
et al. 2002; Droge 2005). Cysteine is reactive and poten-
tially toxic because oxidation of cysteine in the presence of
oxygen may produce reactive oxygen species, but under
Discussion
This is the first detailed report concerning the potential
effect of cysteine on the regulation of animal ADC,
CSADC and GDC. Our study determined that (1) cysteine
can enter into the active site of the above enzymes and
undergo transaldimination (i.e., the replacement of e-amino
group of internal aldimine with the amino group of an
123

Cysteine-dependent inactivation of a-decarboxylases
401
Fig. 10 The proposed interaction between free PLP and amino acids.
a The Schiff base between amino acid and PLP; b 4-thiazolidine-
carboxylic acid derivative; c the cyclic compound between histidine
and PLP; d the intramolecular cyclization to form the 4-thiazolidin-
ecarboxylic acid derivative
other conditions it may help scavenge radical species if the
reactive compounds are already formed (Bourdon et al.
2005; Miki and Funato 2012). Disorders of cysteine regu-
lation often result in disease conditions (Olney et al. 1990).
Relative to the control children, children with autism had
lower plasma cysteine concentration (James et al. 2004). In
addition, cysteine, together with its metabolic derivative
homocysteine, is defined as exotoxin (Coyle and Puttfarc-
ken 1993). For example, malfunction of cysteine dioxy-
genase (the primary cysteine catabolism enzyme) can lead
to elevated cysteine levels that have been associated with
several major neurodegenerative diseases (Heafield et al.
1990; Coyle and Puttfarcken 1993; McCaddon et al. 1998;
Olanow 1993; Diaz-Arrastia 2000). The association of
cysteine disorders with neurodegenerative diseases is more
related to the aging process (Zecca et al. 2004) and the
precise role of cysteine disorder in neurodegenerative dis-
eases remains to be established.
cysteine (Stipanuk 2004). Accordingly, it seems reasonable
to suggest that a deficiency of the enzyme could potentially
lead to the cysteine accumulation that in turn may affect
the biosynthesis of GABA and taurine due to the cysteine-
dependent inactivation of GDC and CSADC, respectively.
The inactivation of decarboxylase by cysteine may explain
in part the tight regulation of cysteine in mammals and
other species.
Many decarboxylases contain PLP as a cofactor that
forms an internal aldimine with a conserved lysine residue.
The internal aldimine is essential to all PLP-dependent
decarboxylation (Oliveira et al. 2011). Formation of inter-
nal aldimine between active site lysine and PLP involves
the formation of a carbinolamine intermediate and sub-
sequent dehydration to form imine (internal aldimine).
Recent studies determined that a conserved active site
cysteine residue (C446 in HuGDC67; C455 in HuGDC66)
that promotes the dehydration of the carbinolamine inter-
mediate is essential for the formation of the internal
aldimine in human GDC (Oliveira et al. 2011). We initially
speculated that free cysteine might form disulfide bond with
this active site cysteine, resulting in a progressive inacti-
vation of the enzyme. However, the relative rapid rate of
AeADC inactivation by cysteine excludes such possibility,
because formation of disulfide bond between free cysteine
and residue cysteine requires time-consuming oxidation.
In addition, the hepatic CDO activity increases while
CSADC activity decreases as the intake of sulfur-contain-
ing amino acids elevates (Stipanuk et al. 2002). Cysteine
dioxygenase catalyzes the oxidation of cysteine to cysteine
sulfinic acid that is then decarboxylated by CSADC in the
taurine biosynthesis pathway (McCoy et al. 2006). Cys-
teine dioxygenase has important medical implications and
is generally considered as an important enzyme regulating
123

402
P. Liu et al.
When AeADC was incubated with its substrate or non-
Schiff base formed between amino acids and PLP were
done in either basic or organic solutions (Heyl et al. 1948).
Like any other amino acid, the formation of imine between
cysteine and PLP should be a minor pathway, but the
subsequent intramolecular nucleophilic addition of the
sulfhydryl group toward the imine (which apparently pro-
ceeds easily) helps to eliminate the imine complex, which
likely breaks the equilibrium and drives the reaction toward
imine formation. This may explain the rather progressive
accumulation of the cyclized cysteine–PLP complex,
because intramolecular cyclization to form the thiazolidine
ring through the sulfhydryl group should be fairly rapid if it
were not due to the rate-limiting step of imine formation.
Insect ADC shares high sequence identity with mam-
malian CSADC and both can use cysteine sulfinic acid as a
substrate. Aspartate could inactivate GDC by converting
the holoenzyme to apoenzyme as a result of the transami-
nation of aspartate (Porter and Martin 1987), but glutamate
could not affect insect ADC (Fig. 3). When AnGDC was
mixed with an equal molar concentration of cysteine and
glutamate, its GDC activity was diminished (Table 1). The
CSADC activity of insect ADC was not significantly
affected by cysteine, unless cysteine was pre-incubated
with the enzyme. In contrast, when HuCSADC was added
to a mixture of an equal molar concentration of cysteine
and cysteine sulfinic acid, the production of hypotaurine
was diminished (Table 1). These results indicate that cys-
teine is an inactivator of decarboxylases that use aspartate,
glutamate and cysteine sulfinic acid as their substrates.
There has been no report discussing cysteine as an inacti-
vator of PLP-dependent decarboxylases in literature,
although cysteine was reported to inhibit the activity of
crayfish GDC (Grossfeld 1985). Therefore, our data
regarding the potential regulation of these enzymes by
cysteine fill some knowledge gap in this area.
substrate amino acids, there was no noticeable change of
the spectrum of AeADC. However, the apparent spectral
changes of AeADC in the presence of cysteine clearly
indicate the interaction of cysteine with enzyme-bond PLP.
Amino acids react with PLP and the interaction of cysteine
and PLP results in the formation of imine and subsequent
intramolecular cyclization of the site chain to form a stable
complex (Fig. 8) (Schonbeck et al. 1975; Buell and Hansen
1960). The incorporation of cysteine to PLP led to pro-
gressive 390-nm peak decrease and 335-nm peak increase
that corresponded to the changes observed from AeADC
with cysteine, except that the peak for AeADC was at
405 nm instead of 390 nm. The formation of thiazolidinic
compound between PLP and cysteine was demonstrated by
nuclear magnetic resonance (Abbott and Martell 1970).
The spectral characteristics of the cysteine–PLP derivative,
isolated from cysteine-treated AeADC (see supplemental
figure 2), is also consistent with that of thiazolidinic species
reported in literature.
It has generally been considered that PLP can react with
the amino group of amino acids, leading to imine pro-
duction, but there has been no detailed discussion about the
pathway in vivo. Although the reaction likely proceeds
under physiological conditions, the equilibrium unlikely
favors the production of their imine complex between free
PLP and any given amino acids. Nucleophilic addition to
the carbonyl carbon is an acid-promoted reaction. To
undergo nucleophilic addition to the carbonyl carbon of
PLP, however, the weak nucleophilic amino group needs to
be unprotonated. This may explain some increase in imine
formation between PLP and amino acids at relative basic
conditions in vitro (although the carbonyl carbon of PLP
should be more reactive at relatively acidic condition)
(Heyl et al. 1948). Because the amino group of any given
amino acids is in general positively charged, imine for-
mation between PLP and amino acids should be a minor
pathway in vivo (no PLP would have been available for
decarboxylases and aminotransferase otherwise). Indeed, if
aldehydes could react very easily with the amino group of
amino acids and other biological amines, it would be a
disaster as numerous potentially toxic imine complexes
would have circulated in the body of living species
(including humans). This leads to a question why cysteine
readily reacts with PLP.
Our study provides solid evidence indicating that cys-
teine could inactivate GDC-like proteins through its reac-
tion with the enzyme-bound PLP. PLP serves as the
cofactor for many aminotransferases and decarboxylases;
*4 % of all classified enzymatic activities are PLP
dependent (Percudani and Peracchi 2003). Based on the
reaction mechanisms, however, one may argue that if
cysteine can inactivate GDC-like proteins, why many other
PLP-containing enzymes do not seem to be affected by this
amino acid. Moreover, some aminotransferases actually
use cysteine as one of their preferred substrates (Han et al.
2008, 2009). In reality, a number of factors likely limit the
ability of cysteine to form cyclized complex with protein-
linked PLP. First, cysteine may not be able to enter the
active site of many PLP-containing proteins. Second, cys-
teine may enter into the active site, but its amino group
may not be able to be positioned at close proximity to the
imine bond of the internal aldimine to initiate
Although the pKa of the amino group is around 9 or
above, there should be a very small fraction of any given
amino acid with its amino group unprotonated at physio-
logical pH. It is reasonable to predict that, when PLP is
mixed with different amino acids, an insignificant level of
Schiff base might be formed and the reaction is in dynamic
process (association–dissociation) in the solution under
physiological pH (this explains why studies regarding the
123

Cysteine-dependent inactivation of a-decarboxylases
403
transaldimination. Third, even the amino group of cysteine
can interact with the internal aldimine and replace the
amino group of e-amino group of active site lysine to form
external aldimine, and the active site residues may restrict
the flexibility of the sulfhydryl group of cysteine from
undergoing nucleophilic addition to form a complex with
PLP. For example, kynurenine aminotransferases can use
cysteine as a substrate (Han et al. 2009, 2008), but the
interactions of several active site residues with the carboxyl
group and sulfhydryl group restrict the flexibility of the
thiol group, thereby physically obstructing it from partici-
pating in nucleophilic addition to PLP (Fig. 10).
regulation of cysteine in vivo and if inactivation of GDC-
like proteins due to disorders of cysteine metabolism could
eventually lead to neurological symptoms. These appar-
ently deserve further investigations.
Acknowledgments The authors thank Dr. Pablo Sabrado (Virginia
Tech) for the use of Aligent 8453 UV/visible Spectrophotometer. This
work is supported by NIH grant AI 19769.
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