Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes

Article abstract of DOI:10.1074/jbc.M117.817700

Cysteine S-nitrosation is a reversible post-translational modification mediated by nitric oxide (NO)-derived agents. S-Ni-trosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical NO-signaling pathway, little is understood about how much S-nitrosation affects protein function. Moreover, identifying physiologically relevant targets of S-nitrosation is difficult because of the dynamics of transnitrosation and a limited understanding of the physiological mechanisms leading to selective protein S-nitrosation. To identify proteins whose activities are modulated by S-nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant S-nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by S-nitrosation. We cloned, expressed, and purified four of these enzymes and observed that S-nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, 1-pyrroline-5-carboxylate dehydrogenase, catechol-O-methyltransferase, and D-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant S-nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by S-nitrosation in each enzyme.

Full text of DOI:10.1074/jbc.M117.817700

JBC Papers in Press. Published on February 26, 2018 as Manuscript M117.817700
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.817700
S-nitrosation Modifies Metabolic Enzyme Function
Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically
relevant metabolic enzymes
a
a
a
a,b
Joel J. Bruegger, Brian C. Smith, Sarah L. Wynia-Smith, Michael A. Marletta*
a
From the QB3 Institute, University of California, Berkeley, 356 Stanley Hall, Berkeley, California,
b
9
4720-3220, United States and the Department of Chemistry, and the Department of Molecular and Cell
Biology, University of California, Berkeley, 374B Stanley Hall, Berkeley, California 94720-3220, United
States
Running Title: S-nitrosation modifies metabolic enzyme function
To whom correspondence should be addressed: Michael A. Marletta, QB3 Institute, University of
California, Berkeley, 374B Stanley Hall, Berkeley, California 94720-3220. Telephone: (510) 642-8758 E-
mail: marletta@berkeley.edu
Keywords: S-nitrosylation, metabolomics, proteomics, nitric oxide, metabolism, enzyme kinetics.
ABSTRACT
directed mutagenesis, we identified the
predominate cysteine residue influencing the
observed activity changes in each enzyme. In
summary, using an integrated metabolomics
Cysteine S-nitrosation is a reversible
posttranslational modification mediated by nitric
oxide (•NO)-derived agents. S-nitrosation
approach,
we
have
identified
several
participates in cellular signaling and is associated
with several diseases such as cancer, cardiovascular
diseases, and neuronal disorders. Despite the
physiological importance of this nonclassical •NO
signaling pathway, little is understood about how
much S-nitrosation affects protein function.
Moreover, identifying physiologically relevant
targets of S-nitrosation is difficult because of the
dynamics of transnitrosation and a limited
understanding of the physiological mechanisms
leading to selective protein S-nitrosation. To
identify proteins whose activities are modulated by
S-nitrosation, we performed a metabolomics study
comparing wild-type and endothelial nitric oxide
synthase (eNOS) knockout mice. We integrated our
results with those of a previous proteomics study
that identified physiologically relevant S-nitrosated
cysteines, and we found that the activity of at least
physiologically relevant S-nitrosation targets,
including metabolic enzymes, which are inhibited
by this modification, and have found the cysteines
modified by S-nitrosation in each enzyme.
Nitric oxide (•NO) is an important
signaling molecule in vertebrate tissue that controls
physiological processes including vasodilation,
neurotransmission, and platelet aggregation (1-3).
•
NO is biosynthesized by the three mammalian
isoforms of nitric oxide synthase (NOS).
Endothelial (eNOS) and neuronal NOS (nNOS)
produce picomolar to nanomolar concentrations of
•
(
NO for cellular signaling, while inducible NOS
iNOS) produces •NO at cytotoxic concentrations
in the low micromolar range at sites of infection (4,
). The most thoroughly characterized •NO
5
2
1 metabolic enzymes might be regulated by S-
signaling pathway involves the enzyme soluble
guanylate cyclase (sGC) (6). •NO produced by
NOS freely diffuses into adjacent cells where it
activates sGC to increase the concentration of the
nitrosation. We cloned, expressed, and purified four
of these enzymes and observed that S-nitrosation
inhibits
phosphogluconate dehydrogenase (6PGD), Δ1-
pyrroline-5-carboxylate dehydrogenase
the
metabolic
enzymes
6-
secondary
messenger
cyclic
guanosine
monophosphate (cGMP) that activates downstream
signaling pathways.
(
(
ALDH4A1),
COMT),
catechol-O-methyltransferase
and
D-3-phosphoglycerate
dehydrogenase (PHGDH). Furthermore, using site-
1

S-nitrosation Modifies Metabolic Enzyme Function
Another important, though less well
understood, signaling mechanism involving •NO is
cysteine S-nitrosation. S-nitrosation is a post-
translational modification of cysteine residues by
which an S-nitrosothiol is initially formed via a one-
electron oxidation. Once formed, S-nitrosothiols
can be transferred through an intermediate
transnitrosating agent such as S-nitrosoglutathione
subjected to on-resin trypsin digestion and bound
peptides containing the S-nitrosated cysteine
residue were eluted by oxidation with performic
acid to generate sulfonic acids at the S-nitrosated
cysteine residue followed by LC-MS/MS analysis
to identify S-nitrosated protein cysteine residues.
Efficient cysteine blocking with MMTS is essential
to the success of this approach as any unblocked
cysteine residue will also be bound by the mercury
resin and increase the background of the assay.
(
GSNO) (7). Many studies have corroborated the
influence of S-nitrosation on protein and tissue
function, as well as the variation of S-nitrosation
profiles in disease states. However, the roles of S-
nitrosation in cellular signaling pathways remain
poorly understood (8-11). If S-nitrosation
participates in cellular signaling and is not simply a
result of nitrosative stress, the cellular levels of
protein S-nitrosation must be tightly controlled
However, the functional relevance of the
vast majority of these observed eNOS-dependent S-
nitrosation targets is unknown. Some of the S-
nitrosated cysteine residues could be highly
reactive toward NO-derived agents simply due to
increased cysteine sulfur nucleophilicity; however,
S-nitrosation may also be involved in functional
roles. For example, an S-nitrosated protein may
serve as a transnitrosating agent, conferring
specificity to a target cysteine of another protein.
One example of this functionality is the
transnitrosation of caspase-3 by thioredoxin (14). S-
nitrosation could also play a role in protein
localization and cellular transport (24). Due to
changes in surface electrostatics, S-nitrosation can
potentially disrupt or promote the formation of
oligomers or other protein complexes. Lastly, S-
nitrosation can directly inhibit or stimulate
enzymatic activity.
(
12). In this sense, S-nitrosation is often compared
to O-phosphorylation, where a kinase transfers a
phosphoryl moiety to an acceptor amino acid from
an ATP donor molecule, which then changes the
function or activity of the enzyme target (13).
Phosphatases then act to reverse the process by
hydrolytically removing the phosphoryl group.
Similarly, in S-nitrosation, a NO-derived agent
+
transfers •NO (formally NO ) to a cysteine residue,
potentially altering protein function. Unlike
phosphorylation, however, many key details remain
unknown.
While there is a growing body of evidence
that S-nitrosation has
mechanisms, relatively few validated protein
targets of S-nitrosation have been characterized
a
role in signaling
In
this
study,
complementary
-
/-
metabolomics of wild-type and eNOS mice is
reported. Analysis of the metabolic differences in
conjunction with the previously published
proteomics study led to the identification of 21
metabolic enzymes that may be modulated by S-
nitrosation (23). These metabolic enzymes were
found to be S-nitrosated in the previous proteomics
(
14-17). Targets of S-nitrosation identified while
using exogenous NO-donors are difficult to
establish as physiologically relevant due to the high
reactivity and low specificity of NO-derived agents.
This is also complicated because the in vivo
mechanisms of S-nitrosation and primary NO-
donor sources are unknown (18-22). To eliminate
the problems associated with the use of an
exogenous •NO donor, Ischiropoulos and
coworkers (23) identified sites of protein S-
nitrosation through a proteomic comparison of
study
with
upregulated
substrate
and
downregulated product (or vice versa) in the
metabolomics study reported here. Four of the 21
enzyme
targets
(6-phosphogluconate
1
dehydrogenase, 6PGD; Δ -pyrroline-5-carboxylate
dehydrogenase,
methyltransferase,
phosphoglycerate dehydrogenase, PHGDH) were
studied in vitro for inhibitory effects by S-
nitrosation. S-nitrosoglutathione (GSNO) was
selected as the nitrosothiol donor because of its
putative physiological role in transnitrosation
ALDH4A1;
COMT;
catechol
and
O-
D-3-
-
/-
wild-type and eNOS knockout (eNOS ) mice. In
this study, unmodified cysteine residues were first
blocked with methyl methanethiosulfonate
(
MMTS), then S-nitrosated proteins were enriched
with an organomercury resin. Bound proteins were
2

S-nitrosation Modifies Metabolic Enzyme Function
signaling (18, 25, 26), but it is important to note that
GSNO may not be the relevant nitrosating agent
that led to the observed S-nitrosation of these
enzymes in mice. All four of the tested enzymes
were inhibited upon GSNO treatment. Through
site-directed mutagenesis, the cysteine residues
responsible for modulating the activity were
identified. Finally, after measuring the fraction of
individual cysteine S-nitrosation, the degree of
enzyme inhibition was confirmed to correlate with
the amount of cysteine S-nitrosation. In summary,
this study integrates comparative proteomics
published previously with metabolomics performed
in the current study and presents quantitative and
functional analysis of metabolic enzymes with the
primary goal of identifying physiologically relevant
protein targets of S-nitrosation.
consistent retention times across all samples) of
eluents from a C18 HPLC column analyzed
separately by positive and negative mode mass
spectrometry as well as a hydrophilic interaction
chromatography (HILIC) column analyzed by
negative
mode
mass
spectrometry,
the
chromatograms of six biological replicates of each
-
/-
WT and eNOS organ were analyzed and mass
feature variations were compared using XCMS
online (27-29). It is important to note that many of
the observed mass features could match multiple
metabolites, isotopes, or salt forms. As a result,
mass features that shared no more than six potential
metabolites were examined, as matched to
metabolites in the METLIN database (30). These
putative metabolites were then mapped to 71
KEGG metabolic pathway maps utilizing Pathos
online (31, 32). S-nitrosated enzymes from the
Ischiropoulos proteomic study (23) were then
annotated with the metabolomics data. Forty-four
S-nitrosated metabolic enzymes with more than
Results
1
.4-fold modulated product and substrate levels
were identified (Table 1). Mass features with p <
.1 (n = 6) when comparing differences in wild-
Metabolomics complements previously published
proteomics to identify functional sites of metabolic
enzyme S-nitrosation–A recent proteomics study by
Ischiropoulos and coworkers of wild-type and
eNOS knockout mice identified 942 S-nitrosated
cysteine residues that are eNOS-dependent (23).
Over half of the cysteine residues identified were in
metabolic enzymes (313 enzymes, some containing
multiple S-nitrosated cysteine residues); however,
the functional consequences of most of these S-
nitrosation sites are unknown. To narrow the list of
target enzymes to those whose activities are
0
-
/-
type versus eNOS mouse organ (note that each
organ and type of LC-MS run were statistically
compared separately) were then identified to
narrow the list to 21 metabolic enzymes (Table S1;
Figures 2B, 3B, 4B, and 5B).
Metabolic enzyme target list–To investigate the
effect of S-nitrosation on metabolic activity, a
subset of the identified enzyme targets was selected
for further characterization. These enzymes were
selected based on the following criteria: (i)
confidence in metabolite identification as outlined
above; (ii) conservation of S-nitrosated cysteine
residue in vertebrates or species known to contain
an eNOS isoform (Figure S1); (iii) established
protocols for enzyme expression, purification, and
activity determination; (iv) availability of enzyme
crystal structure to map the site of S-nitrosation
relative to the enzyme active site (Figures 2A, 3A,
inhibited or activated by S-nitrosation,
a
metabolomics comparison of wild-type and eNOS
knockout mice was carried out and putative
metabolite changes were cross-referenced with the
eNOS-dependent metabolic enzyme S-nitrosation
sites identified by the Ischiropoulos proteomics
study (23).
The approach to identify metabolic enzyme
targets of S-nitrosation that are functionally-
relevant is outlined in Figure 1. To parallel the
proteomics study, we analyzed the brain, heart,
kidney, liver, lung and pancreas (instead of the
thymus). The combined data narrows down the
metabolic enzyme targets of S-nitrosation to only
those S-nitrosation sites that impact enzymatic
activity. After obtaining a list of 176,035 mass
features (observed monoisotopic m/z values with
4
A, and 5A); (v) disease relevance of the metabolic
enzyme; and (vi) evidence in the literature implying
NO involvement in the metabolic pathway. To
•
relate findings to human health, human instead of
mouse protein sequences were used for this study.
Based on these criteria, the following four enzymes
3

S-nitrosation Modifies Metabolic Enzyme Function
–
1 –1
were selected for in vitro determination of the
effects of S-nitrosation: 6PGD, ALDH4A1,
COMT, and PHGDH. Detailed descriptions of each
enzyme can be found in the Supporting
Information. Two control enzymes, aldolase A
µM s while 6PGD activity when incubated with
2 mM GSNO was two orders of magnitude lower at
0.0057 µM s (Figure 2D, Table 2). The kcat/K
value of ALDH4A1 treated with 1 mM GSH was
–
1 –1
M
–
1 –1
0.29 µM s while S-nitrosation from 1 mM GSNO
(
(
ALDOA) and triose phosphate isomerase 1 (TPI1)
Figure 6), were selected for in vitro
treatment lowered activity to 7% that of GSH-
–
1 –1
treated ALDH4A1 at 0.021 µM s (Figure 3D,
Table 2). Treatment of COMT with 150 µM GSNO
resulted in 26% of the 150 µM GSH-treated activity
characterization. These control enzymes were S-
nitrosated in the Ischiropoulos study (23), but did
not fit our metabolomics criteria. For these two
glycolytic enzymes, we anticipated that S-
nitrosation would not modulate activity but could
have another function in signaling (33, 34).
with kcat/K
M
values decreasing from 0.55 to 0.14
–
1
–1
µM min (Figure 4D, Table 2). It should be noted
that higher concentrations of GSNO incubated with
COMT resulted in complete inhibition of enzyme
activity. Finally, S-nitrosation of PHGDH resulted
in 29% of the GSH-treated activity, decreasing
–
1 –1
–1 -1
from 0.070 mM s to 0.021 mM s with 2 mM
GSNO (Figure 5D, Table 2). The control enzymes
ALDOA and TPI1 were unaffected, giving the
S-nitrosation-dependent inhibition of target
enzymes–The activity of each enzyme was assayed
as described in the Experimental Procedures
section. Since specific physiological mechanisms
of S-nitrosation for these target metabolic enzymes
are unknown, the small-molecule transnitrosation
donor GSNO was used. In in vitro studies, any
accessible cysteine may be S-nitrosated, which may
differ from specific in vivo targeted S-nitrosation
reactions (35, 36). All enzymes were purified
following recombinant overexpression in E. coli for
in vitro characterization. To confirm each enzyme
same kcat/K
M
values for both GSNO and GSH
–1 –1
treatments within experimental error: 0.41 µM s
for ALDOA and 2.0 µM s for TPI1 (Figure 6B,
–1 –1
Table 2). In summary, the metabolic enzyme targets
identified through the metabolomics study (6PGD,
ALDH4A1, COMT, and PHGDH) were all
inhibited when incubated with GSNO, while
neither control enzyme (ALDOA and TPI1)
showed significant inhibition with similar
concentrations of GSNO as the target enzymes.
was S-nitrosated upon GSNO treatment,
a
TAMRA-maleimide switch assay was used, which
is a variant of the biotin switch assay, where
tetramethylrhodamine fluorescent imaging was
substituted for a biotin immunoblot (37, 38). The
enzymes 6PGD and PHGDH (at 20 and 150 µM,
respectively), were incubated with 2 mM GSH or
GSNO for 1 h at 37 ˚C; 20 µM ALDH4A1,
ALDOA, and TPI1 were incubated with 1 mM
GSH or GSNO for 1 h at 37˚C; and 20 µM COMT
was incubated for 150 µM GSH or GSNO for 1 h at
Identification of cysteine residues responsible for
metabolic enzyme inhibition–The four enzyme
targets (6PGD, ALDH4A1, COMT, and PHGDH)
contain multiple cysteine residues. To determine
the cysteine residue(s) responsible for the observed
enzyme inhibition, a series of cysteine to serine
variants were prepared to compare the kcat/K
M
values of the GSH versus GSNO treated samples.
We anticipated that the activity of the cysteine to
serine variant enzyme when treated with GSNO
would be comparable to the GSH-treated enzyme.
For 6PGD, residue C289 was identified in the
Ischiropoulos study as S-nitrosated (23) and the
3
7 ˚C. All enzymes were S-nitrosated after
incubation with GSNO followed by buffer
exchange to remove residual GSNO (Figures 2C,
3
C, 4C, 5C, and 6A). To determine the effects of S-
nitrosation on each enzyme, activity assays were
performed after exposure to GSNO (or reduced
glutathione, GSH, followed by buffer exchange to
remove residual GSH) and data was analyzed under
steady-state kinetic conditions (Table 2, Figure S2).
variant C289S basal kcat/K
M
is 22% of wild type
activity, which is indicative of the importance of
this cysteine residue for maximal activity. It should
be noted that nearly all the cysteine residues
mutated are highly conserved in vertebrates (Figure
S1), indicating these residues are important for
function. Therefore, it is not surprising that the
The specificity constant (kcat/K
M
) of 6PGD
negative control (2 mM GSH-treated) was 0.68
4

S-nitrosation Modifies Metabolic Enzyme Function
untreated activity of these variants is lower that the
activity of the wild type enzyme. However, the
from the active site. Treatment of COMT C241S
with 150 µM GSNO resulted in inhibition similar
to the inhibition observed with wild-type COMT
(Figure 4D, Table 2), suggesting that C241 is not
the residue responsible for GSNO-mediated
inhibition. After surveying the COMT structure
(PDB 3A7E), C223 was identified as a surface-
exposed cysteine at the entrance to the active site: a
potential location that could influence activity upon
S-nitrosation (Figure 4A). The C223S variant was
6
PGD variant C289S is still sensitive to GSNO
treatment and therefore is likely not the S-nitrosated
cysteine residue responsible for the activity
decrease (Figure 2D). A complete series of variants
was generated for the nine 6PGD cysteine residues
(
Figure S1A), where C289S was used as a
background mutation so all other variants are
double mutants (along with one triple mutant).
–
1
–1
found to have kcat/K
M
values of 0.21 µM min
–
1
–
The 6PGD variants C199S/C289S and
C289S/C422S could not be expressed as soluble
protein. Cys170 and Cys171 are adjacent to each
other in a buried loop, so the triple mutant of
C170S/C171S/C289S was expressed and purified;
however, this triple mutant was inactive. Of the
remaining five 6PGD variant constructs
upon GSNO treatment compared to 0.36 µM min
1
for GSH-treated enzyme, corresponding to 59%
activity upon GSNO treatment (Figure 4D, Table
2
). The C223S variant was much less inhibited by
GSNO treatment compared to wild-type COMT,
which exhibited a 26% decrease in activity upon
GSNO treatment.
(
C30S/C289S,
C113S/C289S,
C289S,
C289S/C366S and C289S/C402S), only 6PGD
C289S/C366S activity was not significantly
An initial activity assay of the C281S
variant in PHGDH, the residue identified in the
Ischiropoulos proteomics study (23), exhibited
22% activity upon GSNO treatment, which is even
more inhibited than wild-type PHGDH (Figure 5D,
Table 2). A survey of the PHGDH crystal structure
(PDB 2G76) identified four additional PHGDH
cysteine residues that could affect enzymatic
activity by either disrupting the active site or overall
structure. Serine variants of each cysteine were
generated and C116 was identified as the cysteine
residue most affecting activity from GSNO
inhibited by S-nitrosation with a kcat/K
0
M
value of
.037 µM s for the GSH-treated negative control
compared to the GSNO-treated kcat/K value of
.024 µM s (Figure 2D). This represents 67% of
–
1 –1
M
–
1 –1
0
GSH-treated activity for C289S/C366S compared
to 0.8% activity for wild-type 6PGD. The activity
of GSH-treated 6PGD C289S/C366S is low (6%
compared to wild type), which can be attributed to
the importance of C366 for activity; however, the
k
cat/K
M
value of the GSNO-treated 6PGD
C289S/C366S is 650% that of the GSNO-treated
WT enzyme.
M
treatment (Figure 5A). The kcat/K value for
GSNO-treated PHGDH C116S was 83% of GSH-
treated enzyme, which is a small change compared
to the 29% activity observed for GSNO-treated
wild-type PHGDH (Figure 5D). C116 is found at
the homodimer interface of PHGDH in a potential
location to form a disulfide bond with C116 of the
neighboring monomer. To the best of our
knowledge, there is no evidence in vivo to suggest
that PHGDH forms a disulfide, although S-
nitrosation has been known to induce disulfide
formation (39, 40). Therefore, it is possible that S-
nitrosation of C116 induces disulfide formation,
leading to a structural change that lowers the
activity of PHGDH.
Both C95 and C315 of ALDH4A1
displayed eNOS-dependent S-nitrosation in the
Ischiropoulos study (23). We determined that the
k
M
cat/K values for ALDH4A1 C95S were 0.26
–
1 –1
(
GSH treatment) and 0.017 µM s (GSNO
treatment), while the C315S variant exhibited
–
1 –1
k
cat/K
M
values 0.037 and 0.0091 µM s for GSH-
and GSNO-treated samples, respectively (Figure
D, Table 2). These results suggest that C315 has a
3
greater effect on the inhibition of ALDH4A1 by S-
nitrosation: GSNO-treated C315S exhibited 25%
the activity of GSH-treated, whereas the C95S
variant exhibited 6% activity of GSH-treated.
The Ischiropoulos study identified C241 in
COMT as an S-nitrosated residue (23). This
cysteine is located on the protein surface and distant
Percentage of S-nitrosation correlates with
inhibition–To further evaluate the effect of S-
5

S-nitrosation Modifies Metabolic Enzyme Function
nitrosation on the identified cysteine residues and
enzyme inhibition, 6PGD and ALDH4A1 were
incubated with increasing GSNO concentrations
Discussion
S-nitrosation is difficult to study under
physiological conditions due to the cysteine
reactivity of •NO-derived nitrosating agents, low
cellular •NO concentrations, and the indirect
detection methods used for this posttranslational
modification. In addition, varying levels of cysteine
S-nitrosation will occur based on the relative
(
20–2000 µM) and the extent of S-nitrosation was
analyzed with a D-switch assay (41). This LC-MS-
based assay provides relative abundances of S-
nitrosated and unmodified cysteine residues.
COMT and PHGDH were not included because
peptides containing essential cysteine residues were
not detected during LC-MS analysis. It is expected
that some free thiols are more reactive towards
GSNO in vitro and the results here show that. The
cysteine residues C366 in 6PGD and C315 in
ALDH4A1, identified through mutagenesis as the
likely sites of S-nitrosation, exhibited increasing
reactivity
of
specific
cysteine
residues.
Additionally, it remains unclear how S-nitrosation
occurs enzymatically, as there are only a small
number of known transnitrosating enzymes (17).
Since it is known that S-nitrosation occurs in vivo
(
42), we focused on metabolic enzymes that are
targets of S-nitrosation in which S-nitrosation
modulates activity. To complement the
Ischiropoulos S-nitrosation proteomics study (23),
we performed metabolomics with wild-type and
eNOS knockout mice to search for S-nitrosated
metabolic enzymes with eNOS-dependent
fluctuations in their substrate and product levels.
This provided a list of 21 enzymes that are S-
nitrosated and exhibited modulated substrate and
product levels in an eNOS-dependent manner
levels
of
S-nitrosation
with
increasing
concentrations of GSNO (Figures 2E and 3E).
Other cysteine residues quantified in the D-switch
assays, including C422 in 6PGD and C66 in
ALDH4A1, were found mostly unmodified
compared to other quantified cysteine residues. Still
other cysteine residues, such as C113 in 6PGD,
exhibited similar S-nitrosation efficiency as the
targeted cysteine residues, but the activity
differences from mutagenesis clearly demonstrated
that these cysteine residues were not responsible for
the observed enzyme inhibition upon GSNO
treatment.
(
Table S1). As both studies indicate, S-nitrosation
abundance and metabolic effects are likely tissue-
specific and this specificity needs to be further
addressed in future studies. Of these 21 enzymes,
we further characterized the effects of S-nitrosation
on 6PGD, ALDH4A1, COMT, and PHGDH
(Figures 2, 3, 4, and 5).
For comparison, the same GSNO
treatments were used to test enzyme activity.
Increasing GSNO concentrations correlated with
decreasing activity for each enzyme, which
corresponds to approximate in vitro GSNO IC50
values of 556 µM for 6PGD and 78 µM for
ALDH4A1 (Figures 2F and 3F). These values are
too large to be physiologically relevant compared
to in vivo concentrations of GSNO (predicted to be
in the low µM range). Since the exact physiological
mechanism of S-nitrosation is unknown for these
proteins, it is more relevant to correlate levels of S-
nitrosation with activity. When comparing these
IC50 values to the D-switch results, increasing S-
nitrosation of C366 in 6PGD and C315 in
ALDH4A1 clearly correlates with decreasing
The transnitrosating agent GSNO was used
to S-nitrosate each enzyme, but increasing
concentrations of GSNO will eventually S-nitrosate
all the accessible cysteine residues of a protein. In
addition, inhibition of each enzyme was not
necessarily due to the specific cysteine residue
identified in the Ischiropoulos study (23). To relate
enzyme inhibition to a specific cysteine residue,
mutagenesis was performed and a range of GSNO
concentrations were used to compare enzyme
activity with the percent modification of each
cysteine residue (23). Based on our experiments,
C366 in 6PGD, C315 in ALDH4A1, C223 in
COMT, and C116 in PHGDH appear to be the
cysteine residues most responsible for the observed
decrease in activity upon GSNO treatment. Apart
from a study by Fox and coworkers, where an
iNOS/S100A8/A9 complex was shown to S-
k
M
cat/K values (Figures 2E/F and 3E/F). This result,
in conjunction with the mutagenesis data, indicates
that these cysteine residues are responsible for the
activity decreases observed upon GSNO treatment.
6

S-nitrosation Modifies Metabolic Enzyme Function
nitrosate cysteine residues in a [I/L]-X-C-X
motif (16), no consensus sequence has been
2
-[E/D]
account for the GSNO-mediated decrease in
activity.
reported that predicts sites of cysteine S-nitrosation
While both C95 and C315 of ALDH4A1
were found to be S-nitrosated in the Ischiropoulos
study (23), we found C315 was more responsible
for inhibition, with C315S decreasing to 25%
activity, upon S-nitrosation (Figure 3D). This
change in activity is not as great as the 7% activity
of wild-type and 6% activity of the C95S variant
upon GSNO treatment. However, the 25% activity
in C315S is still significant, suggesting additional
cysteine residues contribute to S-nitrosation-
derived inhibition. The residue C315 is highly
conserved in animals, but is a threonine in most
single-celled organisms, suggesting that C315 is
important for proper protein function in
multicellular organisms and may have evolved to
mediate ALDH4A1 activity via S-nitrosation
(Figure S1B). Similar to C366 of 6PGD, C315 of
ALDH4A1 is located between two half domains at
the periphery of the active site and borders the
catalytic residue E314. S-Nitrosation of C315 could
disrupt substrate binding to inhibit the enzyme
(Figure 3A). Unlike 6PGD, the C315 side chain in
ALDH4A1 borders a hydrophobic cleft and resides
within a Van der Waals distance of 4–5 Å from
F284, V288 and F291. S-nitrosation of this residue
would likely disrupt a portion of the protein
hydrophobic core as well as misalign the catalytic
proton acceptor E314 and disrupt catalytic
efficiency.
(
35, 43). The cysteine residues identified in this
study are also not part of any discernable consensus
sequence. This is in contrast to other post-
translational
modifications
such
as
phosphorylation, glycosylation or protease
cleavage sites where conserved consensus
sequences exist (44). While each cysteine identified
here is conserved to varying degrees, these cysteine
residues are mostly invariant in vertebrates and
more variant in invertebrates (Figure S1). Since all
vertebrates but only a subset of invertebrates
encodes NOS isoforms, cysteine residues would be
more likely to be targeted by S-nitrosation when the
organism or host possesses a NOS homolog.
Of the four enzymes tested, 6PGD was
most highly affected by S-nitrosation, exhibiting a
decrease in kcat/K
M
by two orders of magnitude,
while also requiring a higher GSNO concentration
for inhibition (Figure 2D/F). Based on mutagenesis
experiments and D-switch assays, C366 was
identified as the residue most responsible for this
significant activity change (Figure 2D/E). The
C366S variant was the only variant tested in this
study that lost sensitivity to GSNO (i.e. the activity
was not significantly inhibited upon GSNO
treatment). Additionally, S-nitrosation at C366 of
the wild-type enzyme, as measured by a D-switch
assay, correlated with a decrease in enzyme activity
(
Figure 2F). This strongly indicates that S-
COMT treated with 150 µM GSNO
exhibited 26% of the activity of GSH-treated
COMT (Figure 4D). COMT activity was not
detectable at higher GSNO concentrations. The
C223 residue is highly conserved among
vertebrates with a notable exception being it is a
valine in mice (Figure S1C). Therefore, C223 could
not be identified as a S-nitrosation target in the
Ischiropoulos study (23), although it may be
important for COMT regulation and activity in
humans and other vertebrates. C223 is located on
the protein surface, near the entrance to the active
site pocket (Figure 4A). S-nitrosation could reduce
ligand access to the active site due to increased
steric bulk and modulated electrostatic potential on
the surface. Since C223 is surface exposed, the
solvent accessibility probably contributes to the
increased sensitivity of C223 to GSNO treatment.
nitrosation of C366 is responsible for the observed
inhibition of 6PGD upon GSNO treatment. Of all
the residues identified in this study, C366 is the
only cysteine residue that is highly conserved from
mammals to bacteria, demonstrating the functional
importance of this residue (Figure S1A). C366 is
centered between the two half-domains of 6PGD
and borders the active site pocket 6–8 Å away from
the active-site catalytic and ligand-binding residues
(
Figure 2A). The hydroxyl side-chain of residues
S129 and S140 are within hydrogen-bonding
distance of the C366 thiol at 3.3 Å and 3.6 Å,
respectively (Figure 2A), and their interactions may
play a role in the structural orientation and rigidity
of each half-domain. The changes in steric bulk and
electrostatic potential that would occur upon S-
nitrosation of C366 may disrupt this orientation and
critical active site interactions, which would
7

S-nitrosation Modifies Metabolic Enzyme Function
Of the four enzyme targets, PHGDH is the
least sensitive to S-nitrosation, exhibiting 29%
activity upon treatment with 2 mM GSNO. The
C116S variant is largely unaffected by GSNO
treatment and displays 83% of the GSH-treated
activity (Figure 5D). While C116 is variable in
invertebrates, C116 is highly conserved in
vertebrates (Figure S1D) and is located at the
surface, but on the opposite side from the PHGDH
active site. Since the sequence of the C-terminal
regulatory domain of human PHGDH indicates it is
a type I PHGDH enzyme, it is predicted to form a
tetramer in vivo (45). Although the construct used
in this study does not include the C-terminal
domain, it could likely dimerize; the crystal
structure of human PHGDH, which is also lacking
the C-terminal domain, also forms a dimer (PDB
Since the physiological mechanism of S-nitrosation
is unclear for the metabolic enzymes studied herein,
varying GSNO concentrations were used to S-
nitrosate the cysteine residues that affect activity. In
this case, modifying the correct cysteine residues to
observe inhibition was more important than the
non-specific modifications of cysteine residues at
higher concentrations of GSNO that had less of an
inhibitory affect. This study highlights a novel
strategy to identify physiologically relevant targets
of S-nitrosation with an emphasis on functional
effects of S-nitrosation of metabolic enzymes. The
synergistic
analysis
of
proteomics
and
metabolomics data was successful in identifying
four functionally modulated S-nitrosated enzymes.
The remaining 17 metabolic enzymes identified in
this study as potentially regulated by S-nitrosation
await further characterization.
2
G76). Residue C116 is located at the putative
dimer interface and the thiol side chain is oriented
3
5
.3 Å away from C116 of the dimer partner (Figure
A). Therefore, S-nitrosation of C116 may either
Experimental procedures
disrupt dimerization of PHGDH or induce an S-
nitrosation-derived disulfide. Disulfide formation
and generation of nitroxide (NO ) as a consequence
tm1Unc
Metabolomics.
(
Adult
B6.129P2-Nos3
/J
–
−/−
eNOS ) C57BL/6J mice (Jax cat# 002684) were
of S-nitrosation has been demonstrated in a few
cases (39, 40, 46). A disulfide bond could force the
enzyme to adopt a more rigid conformation,
resulting in GSNO-mediated inhibition. However,
the exact mechanism requires further investigation.
purchased from Jackson Laboratories (Bar Harbor,
ME). Age-matched wild-type C57BL/6J mice were
bred in an AAALAC-approved facility at the
Scripps Research Institute. All procedures followed
the guidelines of the Institutional Animal Care and
Use Committee (IACUC) of the Scripps Research
Institute. Animal housing followed a typical 6
AM/6 PM light/dark phase with free access to water
and food. Six sets of both wild-type and eNOS
mouse brain, heart, kidney, liver, lung and pancreas
were harvested, flash frozen in liquid N , and stored
at –80 °C until further processing. The organs were
extracted by the Scripps Center for Metabolomics
and Mass Spectrometry, as previously described
Using
a
metabolomics approach to
prioritize metabolic enzymes for further study, we
demonstrated that four important metabolic
enzymes (6PGD, ALDH4A1, COMT, and
PHGDH) are inhibited by GSNO transnitrosation in
vitro. These results demonstrate that S-nitrosation
of C366 of 6PGD, C315 of ALDH4A1, C223 of
COMT, and C116 of PHGDH inhibit enzyme
activity. Although the enzymes ALDOA and TPI-1
were identified to be S-nitrosated in the proteomics
study (23) as well as the TAMRA-maleimide-
switch assay (Figure 6A), the metabolomics data
did not suggest that ALDOA or TPI-1 would be
inhibited by S-nitrosation and were unaffected by
GSNO treatment as anticipated. While the
concentrations of GSNO required for enzyme
inhibition are higher than physiological levels (low
µM range in vivo), •NO and GSNO may reach
higher local concentrations proximal to NOS.
Additionally, other •NO-derived agents and
proteins may convey S-nitrosation specificity.
-
/-
2
(
47, 48). LC-MS analysis of each sample was
performed at the Scripps Center for Metabolomics
and Mass Spectrometry using negative and positive
mode MS for the C18 column and negative mode
MS for the HILIC column. Resulting MS spectra
and LC peak integrations were collected and
analyzed using XCMS Online (27-29).
Proteomics and metabolomics analysis. Proteomic
data was taken from the supplemental materials of
Doulias et al. (23) and rearranged to identify each
enzyme with eNOS dependent S-nitrosation. From
this list, less than 10% of identified cysteine
8

S-nitrosation Modifies Metabolic Enzyme Function
residues had an annotated function as either a
catalytic, ligand/metal-binding, post-translationally
modified, disulfide or protein-protein interaction
residue. For metabolomics analysis, XCMS Online
and METLIN analysis for each organ (n = 6) only
utilized MS peaks with p < 0.1 when comparing
Two methods of subcloning were utilized for the
remaining enzyme targets, separated by method:
Gateway cloning. COMT (pDONR221 to
pDEST42, C-His), PHGDH (pDONR221 to
pDEST527, N-His), and TPI-1 (pDONR221 to
pDEST42, C-His) were cloned utilizing Gateway
cloning methods (51). Both constructs were
initially cloned into pDONR221 with BP Clonase
II. After purification, transformation and mini-
prepping pDONR221 constructs, each construct
was transferred to their respective destination
vectors utilizing LR Clonase II reaction.
-
/-
differences between wild-type and eNOS data (27,
0, 48). The average integrated peak area for each
3
mass feature was compiled into tables, with
separate tables for negative (C18 and HILIC) and
positive (C18 only) mode MS analysis for each
organ. Worksheets for each organ and ionization
mode were uploaded to Pathos online to compare
-
/-
wild-type and eNOS metabolomes (32). The
combination of differences from all organs for each
metabolite was plotted onto KEGG metabolic
pathway maps with minimal difference in mean
mass feature integrations between wild-type and
The soluble isoform of COMT was used for domain
boundaries, excluding the first 50 amino acids that
are present in membrane-bound COMT. The final
construct ranged from M51 to P271 and was cloned
into pDEST42 with a C-terminal V5 epitope and 6×
His tag. PHGDH domain boundaries were chosen
based on the published crystal structure (PDB ID:
-
/-
eNOS organs of 1.4-fold used in the analysis (31,
9, 50). Metabolic enzymes showing eNOS-
4
dependent S-nitrosation from the Ischiropoulos
proteomics study (23) were then added to the
KEGG maps. S-nitrosated enzymes with
metabolomic mass features matching increased
product and decreased substrate (or decreased
2
G76), which lacks the regulatory C-terminal
domain and ranges from M1 to V315, and was
cloned into pDEST527 with a N-terminal 6× His
tag. These domain boundaries were chosen because
full-length PHGDH was found to be largely
insoluble.
-
/-
product and increased substrate) levels in eNOS
compared to WT mice were compiled to generate
an initial enzyme targets list (Table 1). Targets were
further narrowed based on criteria outlined in the
Results section to identify 21 enzymes (Table S1),
including four for in vitro testing: 6PGD,
ALDH4A1, COMT and PHGDH.
Golden Gate cloning. 6PGD and ALDH4A1 were
cloned utilizing a Golden Gate cloning method into
a pET28-GG vector (kindly provided by the
Tullman-Ercek lab) using constructs with N-
terminal 6× His tags and TEV protease cleavage
sites for each (52). Since the 6PGD N-terminal
methionine is removed in vivo, the domain
boundaries for this construct were from A2 to
A483. ALDH4A1 contains a signal sequence on the
N-terminus that is removed in vivo and this signal
sequence was not included in the construct to give
domain boundaries from T18 to Q563.
Subcloning. Human ALDOA (Uniprot ID P04075)
in a pMCSG7 expression vector (AmpR) with a N-
terminal 6× His tag and Tobacco Etch Virus (TEV)
cleavage site was ordered from Harvard PlasmID
repository (Clone ID HsCD00286766). An intron
was removed from the construct using a standard
site-directed mutagenesis loop excision protocol
with primers in Table S2. DNA encoding human
genes of 6PGD (Uniprot ID P52209), ALDH4A1
Site-directed mutagenesis. Sequence and cysteine
numbering is based on the human enzyme. Primers
were obtained from Sigma-Aldrich or Integrated
DNA Technologies (Table S2). Mutagenesis was
accomplished through PCR and sequences were
confirmed using standard Sanger sequencing by
GeneWiz or the UC-Berkeley sequencing facility.
Site-directed mutants were transformed into
BL21(DE3) E. coli and purified similarly to their
respective wild-type constructs as detailed below.
(
Uniprot ID P30038), COMT (Uniprot ID P21964),
PHGDH (Uniprot ID O43175), and TPI1 (Uniprot
ID P60174) were obtained from Harvard PlasmID
repository
with
ID’s
HSCD00438431,
HSCD00324442,
HSCD00042264,
HSCD00331467,
HSCD00322318,
respectively.
and
9

S-nitrosation Modifies Metabolic Enzyme Function
Enzyme expression and purification. Each
construct was transformed into BL21(DE3) E. coli
and inoculated with 2 mL of a 6 mL overnight
culture into 1 L terrific broth plus appropriate
antibiotic. Cells were grown to OD600 nm = 0.4-0.6.
ALDOA, TPI1, and COMT (and cysteine variants)
were induced with 1 mM IPTG at 37 °C for 4 h. The
enzymes 6PGD, ALDH4A1, and PHGDH along
with their variants were incubated on ice for 15 min
and induced with 0.7 mM IPTG overnight at 18 °C.
Cell pellets were obtained by centrifugation at 4000
rpm for 15 min at 4 °C. Pellets were washed with
5% v/v glycerol, 1 mM EDTA and 1 mM DTT) and
COMT was further purified with a GE HiLoad
16/600 Superdex 75 column pre-equilibrated in
freezing buffer. The purest fractions, as assessed by
SDS-PAGE, were combined. ALDOA, PHGDH
and TPI1 were buffer exchanged into freezing
buffer with a PD-10 column. All proteins were
concentrated to 10-20 mg/mL, flash frozen in liquid
nitrogen, and stored at –80 °C.
S-nitrosation of purified enzymes. Frozen protein
aliquots were thawed on ice. Proteins were diluted
to 150 µL in the appropriate activity buffer (see
2
0 mM Tris, pH 8.0, pelleted once more, flash
“
Enzyme activity assays” subsection), reduced with
mM TCEP for 20-30 min and then buffer
exchanged into appropriate activity buffers using
Bio-Rad Bio-Spin6 columns. Protein
frozen in liquid nitrogen and stored at –80 °C until
purification. Frozen cell pellets were thawed
quickly in room temperature water, resuspended in
ice cold lysis buffer (50 mM Tris-HCl at pH 8.0,
2
concentrations were determined by Bradford assay
or the absorbance at 280 nm (using molar extinction
3
00 mM NaCl, 10% v/v glycerol and 10 mM
imidazole) supplemented with 0.25 mg/mL
lysozyme, 20 µg/mL DNaseI and 20 µg/mL
benzamidine. Resuspended cells were stirred at 4
–
1
–1
–
coefficients of 63.4 mM cm for 6PGD, 74.3 mM
1
–1
–1
–1
cm for ALDH4A1, 22.9 mM cm for COMT
–
1
–1
and 13.9 mM cm for PHGDH). GSNO stock
concentrations were measured before use (ε335 nm
0
°
C for 30 min and sonicated to break cells (5 min
=
with 2 s on, 4 s off). Cell debris was removed by
centrifugation (35,000 rpm for 35 min or 15,000
rpm for 1 h) and the supernatant was mixed with
ClonTech His60 nickel (Ni) resin pre-equilibrated
in lysis buffer. The Ni resin slurry was stirred for
–
1
–1
.992 mM cm ). All proteins, except PHGDH,
were incubated separately at 20 µM with varying
GSNO or GSH concentrations (20, 50, 100, 150,
2
00, 500, 1000 or 2000 µM) at 37 °C for 1 h.
PHGDH was incubated at 150 µM with GSNO or
GSH concentrations of 150, 500, 1000 or 2000 µM
for 1 h. For kinetics experiments, 6PGD and
PHGDH were incubated with 2 mM GSH or GSNO
for 1 h at 37 ˚C, ALDH4A1, ALDOA, and TPI1
were incubated with 1 mM GSH or GSNO for 1 h
at 37˚C and COMT was incubated for 150 µM GSH
or GSNO for 1 h at 37 ˚C. Proteins were spun at
4
5 min to 2 h. Unbound proteins were removed by
application of the resin to a gravity column and the
flow-through was reapplied to the Ni. The Ni resin
was then washed with ten column volumes of lysis
buffer. Bound protein was eluted with increasing
concentrations of imidazole in lysis buffer (40 to
4
50 mM). Most protein eluted between 100 and 250
mM imidazole. Purity of the eluted fractions was
confirmed by SDS-PAGE using Bio-Rad Stain-
Free methods; fractions containing the desired
protein were combined and concentrated with 10
kDa molecular weight cut-off (MWCO) spin
concentrators in a Beckman Coulter Allegra X-14R
centrifuge at 4 °C at 3,000-4,000 rcf. ALDH4A1
was further purified with anion-exchange
chromatography with a POROS HQ20 column
using a 0-100% buffer B in buffer A gradient
~
21,000 rcf for 1 min to remove precipitation and
the supernatant was buffer exchanged into the
appropriate activity buffer using Bio-Spin6
columns. Protein concentrations were measured
and diluted with activity buffer to stock solutions of
1
µM for 6PGD, ALDH4A1, TPI1 and ALDOA; 10
µM for COMT; or 50 µM for PHGDH.
TAMRA-maleimide switch assay. Each enzyme was
diluted to 100-400 µM in HEN buffer (250 mM
HEPES at pH 7.7, 1 mM EDTA, and 0.1 mM
neocuproine) and reduced with 3 mM TCEP for 20
min at room temperature. Enzymes were then
exchanged into HEN buffer with pre-equilibrated
Bio-Rad Spin6 columns to remove excess TCEP. A
(
Buffer A: 100 mM Tris-HCl at pH 8.0, 10% v/v
glycerol, 2 mM DTT, 5 mM EDTA. Buffer B:
Buffer A plus 1 M NaCl). The enzymes 6PGD and
ALDH4A1 were further purified by size exclusion
chromatography with a GE HiLoad 26/600
Superdex 200 column pre-equilibrated in freezing
buffer (50 mM Tris-HCl at pH 8.0, 50 mM NaCl,
5
0 µL aliquot of 20 µM enzyme and 100-2000 µM
1
0

S-nitrosation Modifies Metabolic Enzyme Function
GSNO or GSH were incubated at 37 °C for 1 h.
Reactions were quenched with an equal volume of
cysteine blocking buffer (6 M Urea, 200 mM
iodoacetamide, and 1% w/v SDS in HEN) and
incubated for 1 h at 37 °C. Protein was precipitated
with 1 mL of cold acetone (stored at –20 °C),
thoroughly mixed and pelleted at 21,000 rcf for 10
min at 4 °C and supernatant was removed and
discarded. This acetone precipitation was repeated
ALDH4A1 assay: This assay first required the
synthesis of the substrate L-Δ -pyrroline-5-
carboxylate:
1
1
1
Synthesis of L-Δ -pyrroline-5-carboxylate: L-Δ -
pyrroline-5-carboxylate was synthesized as
previously described (53, 54). All steps were
performed on ice. Briefly, 3.1 mL of 50 mM sodium
periodate (pH to 7.0 with 1 M NaOH) was added to
2
mL of 70 mM DL-5-hydroxylysine hydrochloride
1
-2 more times to remove all the blocking buffer
and incubated for 8 min. Glycerol (50 µL of 1 M)
was added and incubated for 2 min to quench the
periodate. Then, 45 µL of 6 M HCl was added to
acidify the reaction. L-Δ -pyrroline-5-carboxylate
was purified using Bio-Rad AG1-X8 resin in a
and then incubated at 37 ˚C for 10-20 min after the
final supernatant removal to evaporate any residual
acetone. Pellets were resuspended in labeling buffer
1
(
6 M urea, 60 mM ascorbate, and 200 µM
tetramethylrhodamine (TAMRA) maleimide
2
×30 cm column treated with 1.0 M HCl before
diluted in PBS) and incubated at 37 °C for 1 h.
Samples were analyzed by SDS-PAGE and washed
with destain buffer (40% v/v methanol and 10% v/v
acetic acid) 3 times for at least 20 min each
washing with 4 column volumes of MilliQ water.
The reaction mixture was added to the column,
washed with 1 column volume of 50 mM HCl and
eluted with 1 M HCl, collecting 1 mL fractions.
2
followed by rehydration in MilliQ H O before
1
Fractions containing L-Δ -pyrroline-5-carboxylate
TAMRA imaging on a Bio-Rad ChemiDoc to
confirm that the enzymes were modified, followed
by Coomassie staining to demonstrate equal protein
loading.
were identified by mixing aliquots with 0.5% o-
aminobenzaldehyde in ethanol and monitoring the
1
absorbance at 444 nm. Fractions containing L-Δ -
pyrroline-5-carboxylate were combined and the
concentrations were noted before storage at –80 °C.
Enzyme activity assays. To determine kcat and K
M
values, eight substrate concentrations were used for
each protein and variant. All substrates, cofactors
and enzymes were dissolved or exchanged into
buffers specific for each assay, as described below.
Product formation for all proteins (except COMT)
was continuously monitored by absorbance
changes at 340 nm over a period of 5 to 10 min.
Initial rates were determined from the least-square
fit of the linear portion of substrate turnover.
COMT was assayed using a discontinuous HPLC-
based end-point assay as described below. Initial
rates were plotted versus substrate concentration
using GraphPad Prism and fitted using non-linear
regression to the Michaelis-Menten equation to
ALDH4A1 assay: The activity assay buffer was
comprised of 100 mM HEPES at pH 8.1, 50 mM
KCl and 1 mM EDTA. The assay mixture contained
+
3
00 µM NAD with varying concentrations of L-
1
Δ -pyrroline-5-carboxylate (5, 10, 15, 25, 50, 75,
00 or 200 µM); the assay was initiated by addition
1
of 20 nM ALDH4A1.
COMT assay: The activity assay buffer was 100
mM sodium phosphate at pH 7.6 and 1.2 mM
MgCl . The assay mixture contained 1 mM SAM
2
with varying concentrations 3,4-dihydroxybenzoic
acid (DHBA) (5, 10, 15, 20, 40, 60 or 100 µM) and
was pre-incubated for 2-3 min at 37 °C before
enzyme addition. The assay was initiated by the
addition of 200 nM COMT in a 450 µL Eppendorf
tube and incubated at 37 °C. For each time point (0,
determine kcat and K
M
values.
6
PGD assay: The activity assay buffer was
comprised of 100 mM HEPES at pH 7.5, 50 mM
2
, 4 and 6 min for 5, 15 and 20 µM DHBA; 0, 3, 6
KCl and 1 mM EDTA. The assay mixture contained
+
and 9 min for 40, 60 and 100 µM DHBA), 100 µL
aliquots were removed and quickly mixed with 15
µL 60% w/v perchloric acid to quench the reaction.
Quenched reaction aliquots were stored at –80 °C
until HPLC analysis. Aliquots were thawed and
centrifuged at 21,000 rcf for 5 min at 4 °C. The
2
00 µM NADP and varying concentrations of 6-
phosphogluconate (5, 10, 25, 50, 75, 100, 250 or
5
2
00 µM); the assay was initiated by the addition of
0 nM 6PGD. For 6PGD GSNO-treated samples,
the assay was initiated with 600 nM enzyme.
1
1

S-nitrosation Modifies Metabolic Enzyme Function
supernatant was transferred to an HPLC vial and
Aldrich
with
varying
concentrations
of
1
00 µL of the sample was injected onto a C18
glyceraldehyde-3-phosphate (0.05, 0.1, 0.2, 0.5, 1,
2, 4 or 8 mM); the assay was initiated by the
addition of 0.2 nM TPI1.
reverse phase column (Agilent Eclipse Plus XDB
C18 4.6×100 mm 3.5 µm particle size, 80 Å pore
size) with an Agilent 1260 Infinity HPLC system.
A gradient consisting of two buffers (buffer A:
D-switch assay. 6PGD and ALDH4A1 were
analyzed by a D-switch assay (41). After 20 µM
enzyme was incubated with various GSNO
concentrations for 1-2 h, as described above for the
TAMRA-switch assay, an equal volume of
blocking buffer [50 mM N-ethylmaleimide (NEM),
0
.1% v/v formic acid in MilliQ filtered water and
buffer B: 99.9% v/v acetonitrile with 0.1% v/v
formic acid) was used with the following
conditions: 5% B for 0-5 min, 5-17% B for 5-20
min, 17-100% B for 20-21 min, 100% B for 21-26
min, 100-5% B for 26-27 min and 5% B for 27-34
min. The absorbance of the substrate (DHBA) and
product (vanillic and isovanillic acid) peaks was
monitored at 260 nm. Concentrations of vanillic
and isovanillic acid were determined by plotting a
standard curve using standards obtained from
Sigma-Aldrich. Rates were determined from
vanillic acid production as it is produced in
approximately 5:1 ratio compared to isovanillic
acid.
7
M urea and 1% w/v SDS dissolved in HEN Buffer
(
100 mM HEPES at pH 7.5, 1 mM EDTA and 0.1
mM neocuproine)] was added to each reaction and
sonicated in a water bath for 5 min. Samples were
then incubated for 1 h at 55 °C. Protein was
precipitated by the addition of 1 mL of cold acetone
(
–
stored at –20 °C) followed by 10 min incubation at
80 °C and centrifugation at 21,000 rcf for 10 min
at 4 °C. The supernatant was gently removed and
the pellet was resuspended in 1 mL of cold acetone
one or two more times, until the pellets were white
and all of the buffer was exchanged. Residual
acetone was removed by incubating samples for a
PHGDH assay: The activity assay buffer was
comprised of 200 mM HEPES at pH 7.5, 50 mM
KCl and 1 mM EDTA. The assay mixture contained
5
few minutes at 37 °C before adding 5 mM d -NEM
1
µM PHGDH with varying concentrations of
dissolved in HBS (100 mM HEPES at pH 7.2 and
150 mM NaCl) with 7 M urea and 30 mM sodium
ascorbate and incubating 1 h at 37 °C. Samples
oxaloacetate (OAA) (0.2, 0.5, 1, 5, 10, 15, 20 or 40
mM); the assay was initiated by addition of 200 µM
NADH. Since higher concentrations of OAA (> 20
mM) absorb at 340 nm and slowly oxidize NADH,
a control without enzyme was used to account for
the background reactivity of the substrate. To
minimize decomposition, OAA was dissolved in
the activity assay buffer immediately before use.
2
were diluted 14× in HBS plus 5 mM CaCl and
protein was digested overnight while shaking at 37
°C with chymotrypsin for 6PGD or trypsin/LysC
for ALDH4A1. Samples were pelleted at 21,000 rcf
for 5 min at room temperature to remove any
precipitate and the supernatant was transferred to a
new Eppendorf tube. Samples were analyzed by
LC-MS and peaks of peptides containing cysteine
residues were integrated and compared to
ALDOA assay: The activity assay buffer was
comprised of 100 mM HEPES at pH 7.5, 50 mM
KCl and 1 mM EDTA. The assay mixture contained
5
determine the relative amounts of NEM and d -
NEM labeled peptides.
2
00 µM NADH, 2 mU/µL GPDH/TPI (α-
Glycerophosphate dehydrogenase/Triosephosphate
isomerase) enzyme mixture from Sigma-Aldrich
with varying concentrations of fructose-1,6-
bisphosphate (2, 5, 10, 20, 50, 100, 250 or 500 µM);
the assay was initiated by the addition of 50 nM
ALDOA.
Acknowledgements
This work was funded in part by the George E.
Hewitt Foundation for Medical Research (J. J. B.),
a National Institutes of Health National Institute of
General Medical Sciences Postdoctoral Fellowship
TPI1 assay: The activity assay buffer was
comprised of 100 mM HEPES at pH 7.5, 50 mM
KCl and 1 mM EDTA. The assay mixture contained
5
F32GM095023 (B. C. S.), and an American Heart
Association Scientist Development Grant
5SDG25830057 (B. C. S.). We thank Bill Webb at
the Scripps Center for Metabolomics and Mass
2
00 µM NADH,
2
mU/µL GPDH (α-
1
Glycerophosphate dehydrogenase) from Sigma-
1
2

S-nitrosation Modifies Metabolic Enzyme Function
Spectrometry for assistance with metabolomics
sample preparation and LC-MS analysis. We thank
Kim Masuda for assistance with harvesting mouse
organs and the Cravatt laboratory for generously
donating the wild-type mice. We thank the QB3 MS
facility at UC Berkeley, especially Anthony T.
Iavarone. We thank the Marletta laboratory for
helpful input.
localization, regulation, and clinical
implications. Nitric Oxide. 20, 223–230
Förstermann, U., and Sessa, W. C. (2012)
Nitric oxide synthases: regulation and
function. European Heart Journal. 33,
829–837
Serafim, RAM, Primi, M., Trossini, G., and
Ferreira, E. I. (2012) Nitric oxide: state of
the art in drug design. Curr. Med. Chem.
4.
5.
1
9, 386–405
Derbyshire, E. R., and Marletta, M. A.
2012) Structure and regulation of soluble
guanylate cyclase. Annu. Rev. Biochem. 81,
33–559
6
7
.
.
Conflict of interest
(
The authors declare that they have no conflicts of
interest with the contents of this article.
5
Hess, D. T., Matsumoto, A., Kim, S.O.,
Marshall, H. E., and Stamler, J. S. (2005)
Protein S-nitrosylation: purview and
parameters. Nat. Rev. Mol. Cell Biol. 6,
Author contributions
1
50–166
J. J. B. analyzed and combined proteomics and
metabolomics data, subcloned all enzymes except
ALDOA and TPI1, expressed and purified all
enzymes, performed site-directed mutagenesis on
all constructs, conducted S-nitrosation reactions
and performed TAMRA-maleimide switch on all
enzymes, performed and analyzed all enzyme
activity assays, and performed and analyzed D-
switch assays. S. L. W.-S. subcloned ALDOA and
TPI1. S. L. W.-S and B. C. S. initiated and prepared
mouse organs for metabolomics. J. J. B., S. L. W.-
S., B. C. S., and M. A. M. conceived and designed
the experiments, prepared figures and wrote the
manuscript. M. A. M. acquired funding and
coordinated the study. All authors reviewed the
results and approved the final version of the
manuscript.
8
9
.
.
Anand, P., and Stamler, J. S. (2012)
Enzymatic mechanisms regulating protein
S-nitrosylation: implications in health and
disease. J. Mol. Med. 90, 233–244
Yang, L., Calay, E. S., Fan, J., Arduini, A.,
Kunz, R. C., Gygi, S. P., Yalcin, A., Fu, S.,
and Hotamisligil, G. S. (2015) Metabolism.
S-nitrosylation links obesity-associated
inflammation to endoplasmic reticulum
dysfunction. Science. 349, 500–506
1
1
1
0.
1.
2.
Nakamura, T., and Lipton, S. A. (2016)
Protein S-nitrosylation as a therapeutic
target for neurodegenerative diseases.
Trends Pharmacol. Sci. 37, 73–84
Huang, B., Chen, S. C., and Wang, D. L.
(
2009) Shear flow increases S-nitrosylation
of proteins in endothelial cells.
Cardiovascular Research. 83, 536–546
Wynia-Smith, S. L., and Smith, B. C.
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Footnotes:
Current address:
B. C. S. and S. L. W.-S. current address is the
Department of Biochemistry, Medical College of
Wisconsin, 8701 Watertown Plank Rd.,
Milwaukee, Wisconsin 53226.
The abbreviations used are: NO, nitric oxide;
iNOS, inducible nitric oxide synthase; eNOS,
endothelial nitric oxide synthase; nNOS, neuronal
nitric oxide synthase; sGC, soluble guanylate
cyclase; 6PGD, 6-phosphogluconate
1
dehydrogenase; ALDH4A1, Δ -pyrroline-5-
carboxylate dehydrogenase; COMT, catechol-O-
methyltransferase; PHGDH, D-3-
phosphoglycerate dehydrogenase; ALDOA,
Aldolase A; TPI1, triose phosphate isomerase 1;
GSNO, S-nitrosoglutathione.
1
6

S-nitrosation Modifies Metabolic Enzyme Function
Table 1: Initial enzyme target list. Initial list of enzyme targets of S-nitrosation, obtained through the combined proteomics and metabolomics
approach. Each enzyme on the list is S-nitrosated based on the Ishiropoulos proteomics study; the listed substrate is found in overall increased levels
and product at decreased levels (or vice versa) of at least 1.4-fold in eNOS knockout mice compared to wild-type mice from the metabolomics.
Metabolite p-values were not used to generate in this initial list and the most relevant enzyme targets are found in Table S1.
Uniprot
Accession
Number
Enzyme Commission
Number
Primary
Substrate
2-Amino-3-carboxymuconate
semialdehyde
Primary
Product
Enzyme Name
-amino-3-carboxymuconate-6-
semialdehyde
2
1
2
3
4
Q8R519
Q60597
P50136
Q61694
4.1.1.45
1.2.4.2
2-Aminomuconate semialdehyde
3
-Carboxy-1-hydroxyproyl-
ThPP
2-Methyl-1-hydroxybutyl-ThPP,
1 more
2-oxoglutarate dehydrogenase
2-oxoisovalerate dehydrogenase
Thiamin Diphosphate (ThPP)
Thiamine Diphosphate
1.2.4.4
(
ThPP)
3
beta-hydroxysteroid
3β,17β-Dihydroxy-androst-5-
1.1.1.145/5.3.3.1
Testosterone
dehydrogenase type 5
ene
3
-Oxohexanoyl-CoA, or 3-
Oxodecanoyl-CoA
5
3-ketoacyl-CoA thiolase
Q8BWT1
2.3.1.16
Acetyl CoA, 1 more
6
-phosphogluconate
dehydrogenase
6
7
8
9
Q9DCD0
O55137
1.1.1.44
3.1.2.2
3.1.2.-
D-Gluconate-6-phosphate
D-Ribulose-5-phosphate
Palmitic acid
Acyl-coenzyme A thioesterase 1
Acyl-coenzyme A thioesterase
C16:0-CoA
(7Z,10Z,13Z,16Z)-
Docosatetraenoyl-CoA
4-Amino-butanal, 1 more
Galactitol, 1 more
Phenyl-ethylamine, 1 more
L-Argino succinate
L-Aspartate
Q32MW3
Adrenic Acid
1
0
Aldehyde dehydrogenase
Aldose reductase
1.2.1.3
1.1.1.21
1.4.3.4
4-Amino-butanoate, 1 more
D-Galactose, 1 more
Phenyl-acetaldehyde, 1 more
Arginine
1
0
1
2
3
4
P45376
Q8BW75
Q91YI0
P05202
1
1
1
1
Amine oxidase B
Argininosuccinate lyase
Aspartate aminotransferase
Aspartoacylase-2
4.3.2.1
2.6.1.1/2.6.1.7
3.5.1.15
Oxaloacetate
Aspartate
Q91XE4
N-Formyl-L-aspartate
Bifunctional purine biosynthesis
protein PURH
Carnitine O-palmitoyltransferase
1
Carnitine O-palmitoyltransferase
2
1
1
1
5
6
7
Q9CWJ9
Q924X2
P52825
2.1.2.3/3.5.4.10
2.3.1.21
AICAR
FAICAR
Hexadecanoyl-CoA
L-Palmitoyl-carnitine
L-Palmitoyl- carnitine
Hexadecanoyl-CoA
2.3.1.21
1
7

S-nitrosation Modifies Metabolic Enzyme Function
1
8
Catechol O-methyltransferase
O88587
2.1.1.6
L-Noradrenaline, 1 more
L-Normetanephrine, 1 more
Several
Possible
1
9
Cytochrome P450
1.14.13.-
Several possible
Several Possible
3
α,7α,12α-Trihydroxy-5β-
cholestane
L-Cysteinyl-glycine
3α,7α,12α-Trihydroxy-5β-
cholestan-26-al
2
2
2
0
1
2
Cytochrome P450 27
Q9DBG1
Q9CPY7
Q61753
1.14.13.15
3.4.11.5/3.4.11.1
1.1.1.95
Cytosol aminopeptidase
D-3-phosphoglycerate
dehydrogenase
L-Cysteine
3-Phospho-D-glycerate
3-Phospho-hydroxypyruvate
Delta-1-pyrroline-5-
carboxylate dehydrogenase
L-1-Pyrroline-3-hydroxy-5-
carboxylate
L-Erythro-4-
hydroxyglutamate
2
3
Q8CHT0
1.2.1.88
(
S)-3-Hydroxybutanoyl-CoA, 1
2
4
Enoyl-CoA hydratase
Q8BH95
4.2.1.17
Crotonoyl-CoA, 1 more
more
Tetradecanoic acid, 2 more
LTD4
2
2
5
6
Fatty acid synthase
Gamma-glutamyltranspeptidase1
GDH/6PGL endoplasmic
bifunctional protein
P19096
Q60928
2.3.1.85
2.3.2.2/3.4.19.13/3.4.19.14
Malonyl CoA
LTC4
2
2
2
3
3
7
8
9
0
1
Q8CFX1
P48318
P13707
Q9D964
Q61425
1.1.1.47/3.1.1.31
4.1.1.15
β-D-Glucose-6-phosphate
L-Aspartate
D-Gluconate-6-phosphate
β-Alanine
Glutamate decarboxylase 1
Glycerol-3-phosphate
dehydrogenase
Glycine amidinotransferase
Hydroxyacyl-coenzyme A
dehydrogenase
1.1.1.8
sn-Glycerol-3-phosphate
4-Amino-butanoate
Glycerone-phosphate
4-Guanidino-butanoate
Acetoacetyl-CoA
2.1.4.1
1.1.1.35
(S)-3-Hydroxybutanoyl-CoA
Hydroxymethylglutaryl-CoA
lyase
(S)-3-Hydroxy-3-
methylglutaryl-CoA
3
2
P38060
4.1.3.4
Acetoacetate
Indolethylamine N-
methyltransferase
L-lactate dehydrogenase
3
3
3
4
P40936
P06151
2.1.1.49/2.1.1.96
1.1.1.27
Tryptamine
N-Methyl tryptamine
3-Mercaptolactate
3-Mercaptopyruvate
3
5
Lambda-crystallin
Q99KP3
1.1.1.45
L-Gulonate
3-Dehydro-L-gulonate
1
8

S-nitrosation Modifies Metabolic Enzyme Function
Long-chain-fatty-acid--CoA
3
6
P41216
ligase 1
6.2.1.3
Hexadecanoate
Hexadecanoyl-CoA
3
3
7
8
Neutral alpha-glucosidase C
Pyruvate dehydrogenase
Q8BVW0
P35486
3.2.1.20
1.2.4.1
β-D-Fructose, 1 more
Thiamin Diphosphate (ThPP)
L-Gulono-1,4-lactone, 1
more
Sucrose, 1 more
2-Hydroxyethyl-ThPP
3
4
4
9
0
1
Regucalcin
Q64374
Q8K2B3
Q9D0K2
3.1.1.17
1.3.5.1
2.8.3.5
L-Gulonate, 1 more
Fumarate
Succinate dehydrogenase
Succinyl-CoA:3-ketoacid-
coenzyme A transferase 1
Succinate
Acetoacetyl-CoA
Acetoacetate
Testosterone glucuronide, 3
more
4
2
UDP-glucuronosyltransferase 1-1
Q63886
2.4.1.17
Testosterone, 3 more
4
4
3
4
Urocanate hydratase
Xanthine dehydrogenase/oxidase
Q8VC12
Q00519
4.2.1.49
1.17.1.4/1.17.3.2
Urocanate
Paraxanthine, 1 more
4-Imidazolone-5-propanoate
1,7-Dimethyluric acid, 1 more
1
9

S-nitrosation Modifies Metabolic Enzyme Function
Table 2. Steady-state kinetics. Steady-state kinetic parameters of wild-type and cysteine variants treated
with GSNO or GSH. 6PGD and PHGDH at 20 µM and 150 µM, respectively, were treated with 2 mM
GSH or GSNO; 20 µM ALDH4A1, ALDOA and TPI-1 were treated with 1 mM GSH or GSNO and 20
µM COMT was treated with 150 µM GSH or GSNO for 1 h at 37 ˚C. All kinetics were performed with at
least two independent experiments, each performed with 8 distinct substrate concentrations ran in
duplicate, except those denoted with the (*) symbol that indicates one experiment with 8 substrate
concentrations was performed. Standard error was reported for error values.
k
cat
K
(µM)
M
kcat/K
M
(µM s )
0.678 ± 0.121
–
1
–1 –1
(
s )
WT- GSH
18.7 ± 1.2
0.154 ± 0.056
22.1 ± 2.5
0.40 ± 0.1
1.91 ± 0.07
1.05 ± 0.01
28.6 ± 6.1
27.3 ± 1
156 ± 37
128 ± 45
50.9 ± 2.6
43.4 ± 4.4
WT-GSNO
C289S-GSH
C289S-GSNO
C366S-GSH
C366S-GSNO
0.0057 ± 0.0022
0.147 ± 0.019
0.0033 ± 0.0004
0.037 ± 0.001
0.024 ± 0.003
6
PGD
WT-GSH
11.4 ± 0.3
2.39 ± 0.55
9.4 ± 1.0
39 ± 1
0.293 ± 0.006
0.021 ± 0.005
0.257 ± 0.03
WT-GSNO
C95S-GSH
C95S-GSNO
C315S-GSH
C315S-GSNO
121 ± 38
36.6 ± 0.3
187 ± 67
26.9 ± 3.8
206 ± 25
ALDH4A1
2.78 ± 0.23
1.02 ± 0.33
1.87 ± 0.24
0.0165 ± 0.0046
0.0369 ± 0.0072
0.00907 ± 0.00006
–
1
–1
–1
(
min )
(µM)
(µM min )
0.547 ± 0.036
WT-GSH
7.97 ± 0.45
2.27 ± 0.42
9.5 ± 2.1
5.2 ± 1.4
8.3*
14.7 ± 1.7
16 ± 1
26.8 ± 5.7
25 ± 2.7
14*
WT-GSNO
C223S-GSH
C223S-GSNO
C241S-GSH
C241S-GSNO
0.142 ± 0.038
0.356 ± 0.058
0.208 ± 0.049
0.58*
COMT
1.2*
20*
0.063*
–
1
–1 –1
(mM s )
(
s )
(mM)
WT-GSH
0.229 ± 0.011
0.091 ± 0.01
0.215 ± 0.005
0.146 ± 0.001
0.241 ± 0.045
0.066 ± 0.024
3.03 ± 0.3
0.070 ± 0.004
WT-GSNO
C116S-GSH
C116S-GSNO
C281S-GSH
C281S-GSNO
4.41 ± 0.56 0.021 ± 0.001
4.01 ± 0.04 0.0536 ± 0.002
3.24 ± 0.36 0.0455 ± 0.005
4.78 ± 0.08 0.0502 ± 0.009
PHGDH
5.77 ± 1.27 0.0110 ± 0.0018
–
1
–1 –1
(µM s )
(
s )
(µM)
WT-GSH
WT-GSNO
WT-GSH
WT-GSNO
8.4 ± 0.1
8.6 ± 0.6
20.4 ± 1.2
20.9 ± 0.3
901 ± 7
0.413 ± 0.021
ALDOA
TPI1
0.413 ± 0.024
2.09 ± 0.33
1.96 ± 0.09
1880 ± 270
1970 ± 90
1010 ± 10
2
0

S-nitrosation Modifies Metabolic Enzyme Function
Figure Legends
Figure 1. Flow chart used to narrow target enzyme identification for in vitro characterization. eNOS-
dependent S-nitrosated metabolic enzymes were identified in the Ischiropoulos study (23). Metabolites from
this metabolomics study with >1.4-fold difference in relative abundance between wild type and eNOS
knockout mice were also distinguished. These metabolites were plotted on KEGG maps using the program
Pathos (32) along with eNOS-dependent S-nitrosated enzymes. Enzymes that were S-nitrosated and whose
substrate was upregulated and product downregulated (or vice versa) were categorized in Table 1. Enzymes
whose substrates and products were further distinguished based on p-values (p < 0.1, n = 6) and confidence
in metabolite identification (mass features closely match profiles in METLIN metabolic database) were
categorized as enzyme targets that may be functionally affected by S-nitrosation in Table S1.
Figure 2. A. Crystal structure of 6PGD (PDB 4GWK). The approximate location of the active site is
outlined and faded. A black box highlights the zoomed in region. The zoomed in region highlights the
targeted cysteine C366 in red. Annotated active-site catalytic and substrate/cofactor-binding residues are
shown in blue. 6PGD C366 is within potential hydrogen-bonding or Van der Waals interaction distances
of 3.3 Å to S129 and 3.6 Å to S140 (in green). B. 6PGD catalyzes the oxidative decarboxylation of 6-
-
/-
phosphogluconate to ribulose-5-phosphate in the pentose phosphate pathway. In eNOS compared to wild
type mice, there is a lower abundance of a mass feature consistent with the substrate 6-phosphogluconate
in heart and kidney organs as identified with C18 analysis in positive mode and a greater abundance of a
mass feature consistent with the product ribulose-5-phosphate in the liver identified with both C18 using
negative mode and HILIC using negative mode (see Table S1 for more details). C. TAMRA-switch SDS-
PAGE gel indicates GSNO-treated 6PGD is S-nitrosated while GSH-treated samples exhibit only
background fluorescence. Coomassie staining is shown as a loading control. D. The specificity constants
of 6PGD wild type and variants upon S-nitrosation. Comparison of kcat/K
GSNO (as the transnitrosation donor in white) or GSH (as a negative control, in grey) (n = 3 for WT and n
2 for variants with each run including 8 substrate concentrations ran in duplicate). The steady-state kinetic
parameters are summarized in Table 1. The procedure exposed 20 µM 6PGD to 2 mM GSH or GSNO for
h at 37˚C, buffer exchanging out GSH or GSNO and determining the protein concentration as outlined in
M
values in either the presence of
=
1
the Experimental Procedures section. Comparisons of percent activity are used for GSNO treatments in
relation to GSH-treated control. Wild-type 6PGD retains 0.8% activity when treated with GSNO, while
cysteine variant C366S (highlighted in red text) is most responsible for S-nitrosation-derived inhibition and
retains 67% activity when treated with GSNO. Other cysteine mutants were more significantly inhibited by
GSNO-treatment with comparable effects as the wild-type 6PGD, including the cysteine C289S, which was
identified as S-nitrosated in the Ischiropoulos proteomics study (highlighted in blue text). E-F. Increasing
GSNO concentration leads to increased S-nitrosation of cysteine residues responsible for S-nitrosation-
dependent inhibition, where wild-type 6PGD was exposed to varying concentrations of GSNO for 1 h at
3
7˚C. E. D-switch LC-MS comparisons of select cysteine residues, highlighting the labeling efficiency of
S-nitrosation with increasing GSNO concentrations. Increased labeling of the cysteine residues responsible
for S-nitrosation-dependent inhibition, C366, correlates with increased enzymatic inhibition. F. Steady-
state kinetic analyses highlighting decreased specificity constant values with increasing GSNO
concentrations (n = 3). GSH was used as a negative control.
Figure 3. A. Crystal structure of ALDH4A1 (PDB 3V9G). The approximate location of the active site is
outlined and faded. A black box highlights the zoomed in region. The zoomed in region highlights the
targeted cysteine C315 in red. Annotated active-site catalytic and substrate/cofactor-binding residues are
shown in blue. ALDH4A1 C315 is the residue after active-site residue E314 and oriented in a hydrophobic
1
cleft. B. ALDH4A1 catalyzes the conversion of L-1-pyrroline-3-hydroxy-5-carboxylate (or Δ -pyrroline-
-
5
-carboxylate) to 4-hydroxy-L-glutamate (or L-glutamate) in the L-proline degradation pathway. In eNOS
/
-
compared to wild type mice, there is a greater abundance of a mass feature consistent with the substrate
L-1-pyrroline-3-hydroxy-5-carboxylate as well as a lower abundance of a mass feature consistent with the
2
1

S-nitrosation Modifies Metabolic Enzyme Function
product 4-hydroxy-L-glutamate in the liver where both were identified with HILIC analysis in negative
mode (see Table S1 for more details). C. TAMRA-switch SDS-PAGE gel indicates GSNO-treated
ALDH4A1 is S-nitrosated while GSH-treated samples exhibit only background fluorescence. Coomassie
staining is shown as a loading control. D. The specificity constants of ALDH4A1 wild type and variants
M
upon S-nitrosation. Comparison of kcat/K values in either the presence of GSNO (as the transnitrosation
donor in white) or GSH (as a negative control, in grey) (n = 3 for WT and n = 2 for variants with each run
including 8 substrate concentrations ran in duplicate). The steady-state kinetic parameters are summarized
in Table 2. The procedure exposed 20 µM ALDH4A1 to 1 mM GSH or GSNO for 1 h at 37˚C, buffer
exchanging out GSH or GSNO and determining the protein concentration as outlined in the Experimental
Procedures section. Comparisons of percent activity are used for GSNO treatments in relation to GSH-
treated control. Wild-type ALDH4A1retains 7% activity when treated with GSNO, while cysteine variant
C315S (highlighted in red text) is most responsible for S-nitrosation-derived inhibition and retains 25%
activity when treated with GSNO. The other cysteine mutant tested, C95S, was more significantly inhibited
by GSNO-treatment with comparable effects as the wild-type ALDH4A1. Both C95 and C315 were
identified as S-nitrosated in the Ischiropoulos proteomics study. E-F. Increasing GSNO concentration leads
to increased S-nitrosation of cysteine residues responsible for S-nitrosation-dependent inhibition, where
wild type ALDH4A1 was exposed to varying concentrations of GSNO for 1 h at 37˚C. E. D-switch LC-
MS comparisons of select cysteine residues, highlighting the labeling efficiency of S-nitrosation with
increasing GSNO concentrations. Increased labeling of the cysteine residues responsible for S-nitrosation-
dependent inhibition, C315, correlates with increased enzymatic inhibition. F. Steady-state kinetic analyses
highlighting decreased specificity constant values with increasing GSNO concentrations (n = 3). GSH was
used as a negative control.
Figure 4. A. Crystal structure of COMT (PDB 3A7E). The approximate location of the active site is
outlined and faded. A black box highlights the zoomed in region. The zoomed in region highlights targeted
cysteine C223 in red. Annotated active-site catalytic and substrate/cofactor-binding residues are shown in
blue. COMT C223 is located on the surface and by the entrance to the active site of COMT. B. COMT O-
-
/-
methylates a catechol hydroxyl and has several substrates. In eNOS compared to wild type mice, there is
a lower abundance of a mass feature consistent with the substrate 2-hydroxy-estradiol-17β in the lung and
kidney as identified with C18 analysis in positive mode and a greater abundance of a mass feature consistent
with the product 2-methoxy-estradiol-17β in the brain as identified with C18 using positive mode (not
shown due to p > 0.05) (see Table S1 for more details). C. TAMRA-switch SDS-PAGE gel indicates
GSNO-treated COMT is S-nitrosated while GSH-treated samples exhibit only background fluorescence.
Coomassie staining is shown as a loading control. D. The specificity constants of COMT wild type and
variants upon S-nitrosation. Comparison of kcat/K
transnitrosation donor in white) or GSH (as a negative control, in grey) (n = 3 except for C241S, where n
1 with each run including 8 substrate concentrations including no substrate). The steady-state kinetic
M
values in either the presence of GSNO (as the
=
parameters are summarized in Table 2. The procedure exposed 20 µM COMT to 150 µM GSH or GSNO
for 1 h at 37˚C, buffer exchanging out GSH or GSNO and determining the protein concentration as outlined
in the Experimental Procedures section. Comparisons of percent activity are used for GSNO treatments in
relation to GSH-treated control. Wild-type COMT retains 26% activity when treated with 150 µM GSNO,
while 1 mM GSNO fully eliminates activity. Cysteine variant C223S (highlighted in red text) is most
responsible for S-nitrosation-derived inhibition and retains 59% activity when treated with GSNO. The
other cysteine mutant tested, C241S, was identified as S-nitrosated in the Ischiropoulos proteomics study
(
highlighted in blue text) and was more significantly inhibited by GSNO-treatment.
Figure 5. A. Crystal structure of PHGDH (PDB 2G76). The approximate location of the active site is
outlined and faded. A black box highlights the zoomed in region. The zoomed in region highlights targeted
cysteine C116 in red. Annotated active-site catalytic and substrate/cofactor-binding residues are shown in
blue. PHGDH C116 is at the putative dimer interface and could potentially form a disulfide bond with C116
of its neighboring monomer in the PHGDH homodimer. B. PHGDH converts D-3-phosphoglycerate to 3-
2
2

S-nitrosation Modifies Metabolic Enzyme Function
phosphohydroxypyruvate in the L-serine biosynthesis pathway and can also convert α-ketoglutarate to the
-
/-
oncometabolite D-2-hydroxyglutarate (not shown). In eNOS compared to wild type mice, there is a lower
abundance of a mass feature consistent with the substrate D-3-phosphoglycerate in the liver as identified
with C18 analysis in negative mode and a greater abundance of a mass feature consistent with the product
3
-phosphohydroxypyruvate in the pancreas as identified with HILIC analysis using negative mode (see
Table S1 for more details). C. TAMRA-switch SDS-PAGE gel indicates GSNO-treated PHGDH is S-
nitrosated while GSH-treated samples exhibit only background fluorescence. Coomassie staining is shown
as a loading control. D. The specificity constants of PHGDH wild type and variants upon S-nitrosation.
M
Comparison of kcat/K values in either the presence of GSNO (as the transnitrosation donor in white) or
GSH (as a negative control, in grey) (n = 3 for WT and n = 2 for variants with each run including 8 substrate
concentrations ran in duplicate). The steady-state kinetic parameters are summarized in Table 2. The
procedure exposed 150 µM PHGDH to 2 mM GSH or GSNO for 1 h at 37˚C, buffer exchanging out GSH
or GSNO and determining the protein concentration as outlined in the Experimental Procedures section.
Comparisons of percent activity are used for GSNO treatments in relation to GSH-treated control. Wild-
type PHGDH retains 29% activity when treated with GSNO, while cysteine variant C116S (highlighted in
red text) is most responsible for S-nitrosation-derived inhibition and retains 83% activity when treated with
GSNO. Other cysteine mutants were more significantly inhibited by GSNO-treatment with comparable
effects as the wild-type PHGDH, including the cysteine C281S, which was identified as S-nitrosated in the
Ischiropoulos proteomics study (highlighted in blue text).
Figure 6. ALDOA and TPI1 were tested as control enzymes that were S-nitrosated in the Ischiropoulos
proteomics study but did not fit the metabolomics criteria. A. TAMRA-switch SDS-PAGE gel indicates
GSNO-treated ALDOA and TPI1 are S-nitrosated while GSH-treated samples exhibit only background
fluorescence. Coomassie staining is shown as a loading control. B. As expected, ALDOA and TPI1 were
not inhibited by GSNO treatment. Comparison of kcat/K values in either the presence of GSNO (as the
M
transnitrosation donor in white) or GSH (as a negative control, in grey) (n = 2). The steady-state kinetic
parameters are summarized in Table 1. The procedure exposed 20 µM enzyme to 1 mM GSH or GSNO for
1
h at 37˚C, buffer exchanging out GSH or GSNO and protein concentration determined as outlined in the
Experimental Procedures section. Comparisons of percent activity are used for GSNO treatments in relation
to GSH-treated control. Wild-type enzymes retain 100% and 91% activity for ALDOA and TPI1,
respectively, when treated with GSNO.
2
3

S-nitrosation Modifies Metabolic Enzyme Function
Figure 1
2
4

S-nitrosation Modifies Metabolic Enzyme Function
Figure 2
2
5

S-nitrosation Modifies Metabolic Enzyme Function
Figure 3
2
6

S-nitrosation Modifies Metabolic Enzyme Function
Figure 4
2
7

S-nitrosation Modifies Metabolic Enzyme Function
Figure 5
2
8

S-nitrosation Modifies Metabolic Enzyme Function
Figure 6
A
5
GSNO
0 kDa
–
+
GSNO
–
+
3
7 kDa
25 kDa
7 kDa
25 kDa
Rhodamine
fluorescence
3
7 kDa
0 kDa
5
3
Coomassie
stain
37 kDa
ALDOA
TPI1
91%
B
0.5
0.4
0.3
0.2
0.1
0
100%
Control
GSNO
2
1
0
ALDOA
TPI1
2
9

S-nitrosation Modifies Metabolic Enzyme Function
3
0