Mechanistic studies on transcriptional coactivator protein arginine methyltransferase 1

Article abstract of DOI:10.1021/bi102022e

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family, and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH-rate studies, and the determination of solvent isotope effects (SIEs) indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer.

Full text of DOI:10.1021/bi102022e

ARTICLE
pubs.acs.org/biochemistry
Mechanistic Studies on Transcriptional Coactivator Protein Arginine
Methyltransferase 1
†
,‡
§
§
,†,‡
Heather L. Rust, Cecilia I. Zurita-Lopez, Steven Clarke, and Paul R. Thompson*
†
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States
‡
Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, United
States
§
Department of Chemistry and Biochemistry, University of California, 607 Charles E. Young Drive East, Los Angeles, California 90095,
United States
S Supporting Information
b
ABSTRACT: Protein arginine methyltransferases (PRMTs)
catalyze the transfer of methyl groups from S-adenosylmethio-
nine (SAM) to the guanidinium group of arginine residues in a
number of important cell signaling proteins. PRMT1 is the
founding member of this family, and its activity appears to be
dysregulated in heart disease and cancer. To begin to character-
ize the catalytic mechanism of this isozyme, we assessed the
effects of mutating a number of highly conserved active site
residues (i.e., Y39, R54, E100, E144, E153, M155, and H293),
which are believed to play key roles in SAM recognition,
substrate binding, and catalysis. The results of these studies,
as well as pHꢀrate studies, and the determination of solvent
isotope effects (SIEs) indicate that M155 plays a critical role in
both SAM binding and the processivity of the reaction but is not
responsible for the regiospecific formation of asymmetrically
dimethylated arginine (ADMA). Additionally, mutagenesis
studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general
base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/
base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double
mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily
driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of
the substrate guanidinium is not required for methyl transfer.
ost-translational modifications of proteins are well-known
for the variety of roles they play in controlling cellular
PRMTs that produce ADMA, whereas PRMT5 is a definitive
6
P
type II PRMT and produces SDMA. Note that the modified
6
function. One specific modification, the methylation of arginine
residues, which occurs on numerous proteins (e.g., histones H3
and H4, p53, and p300), is known to modulate a number of cell
signaling pathways, including gene transcription, RNA splicing,
signal transduction, cell growth, and proliferation (reviewed in
refs 1ꢀ6). Arginine methylation is catalyzed by the protein
arginine methyltransferases (PRMTs), a family of enzymes that
catalyze the transfer of a methyl group from S-adenosylmethio-
nine (SAM) to the guanidinium moiety of arginine residues in
proteins, but not free arginine. This reaction first produces an
ω-monomethylarginine residue (ω-MMA), which can then be
further methylated to produce either an asymmetrically dimethy-
lated arginine residue (ADMA) or a symmetrically dimethylated
arginine (SDMA) residue (Figure 1). In humans, there are nine
PRMT family members; PRMT1, -2, -3, -4, -6, and -8 are type I
arginine products of PRMT7 remain to be clearly established
and that enzymatic activity has yet to be demonstrated for
6
PRMT9 (4q31).
All PRMTs possess a highly conserved ∼310-amino acid
catalytic core that is responsible for methyltransferase activity.
This core consists of a SAM binding domain that contains a
Rossmann-type fold typical of class I methyltransferases, a unique
β-barrel domain, and a dimerization arm. All family members
also possess an N-terminal extension, and several also contain
1
ꢀ6
C-terminal extensions.
PRMT1 is an ∼42 kDa, 353-residue
5,7
protein that is responsible for ∼85% of in vivo PRMT activity.
Received: December 20, 2010
Revised:
March 18, 2011
Published: March 21, 2011
r 2011 American Chemical Society
3332
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Figure 1. PRMT-catalyzed reactions. PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the guanidinium group of
an arginine residue. Type I PRMTs produce asymmetric dimethylarginine (ADMA), and type II PRMTs produce symmetric dimethylarginine (SDMA)
via an ω-monomethylated (ω-MMA) intermediate.
8
This isozyme is located in both the nucleus and the cytoplasm
and is active as a head-to-tail dimer, which is formed by the
interaction of the dimerization arm of one monomer with the
group of SAM. The γ-carboxylate of E153 also likely contributes
to the alignment of the substrate guanidinium via electrostatic
9,18
interactions and two hydrogen bonds with N and N , although
η
δ
1
9
SAM binding domain of another monomer.
it should be noted that, in structures of PRMT1, the position of
this residue does not appear to be catalytically competent as it is
Given the links between dysregulated PRMT1 activity and
10ꢀ12
9
cancer and heart disease,
we initiated kinetic and mechan-
“flipped” out of the active site.
istic studies on PRMT1 to aid our efforts to develop inhibitors
Examination of the crystal structure of CARM1 also identified
Y154, a conserved tyrosine residue that corresponds to Y39 in
PRMT1, as potentially playing a role in PRMT catalysis. Although
Y39 is not visible in the crystal structure of PRMT1, the side
chain phenol of this residue forms the top of the SAM binding
pocket and is likely important for cofactor binding. Additionally,
on the basis of the CARM1 structure, the phenol appears to
interact with E153 (PRMT1 numbering) and helps orient this re-
sidue and, as a consequence, the substrate guanidinium to promote
13ꢀ16
targeting this isozyme.
Previously, we demonstrated that
PRMT1 preferentially methylates a 21-residue peptide based on
the N-terminus of histone H4 with kinetics comparable to those
13
of the parent protein. Additionally, these studies demonstrated
that positively charged residues present in the C-terminus of this
peptide, which is denoted AcH4ꢀ21, are critical for the high rates
of catalysis observed with this substrate. We further demon-
strated that PRMT1 catalyzes the methylation of the AcH4ꢀ21
substrate in a partially processive manner; i.e., PRMT1 can
rebind SAM and subsequently produce ADMA before the first
19,20
catalysis.
The backbone carbonyl oxygen of Y154 in CARM1
also appears to be important for CARM1 catalysis via the for-
mation of a hydrogen bond with the hydroxyl group of a con-
served serine residue, S217 in CARM1 (S102 in PRMT1); this
latter interaction likely helps to orient the Y154 phenol for
interaction with E267. It has been hypothesized that S217 and
13
methylation product, ω-MMA, is released. Because ADMA
formation is not obligatory, we have suggested that PRMT1
displays partial processivity. The partially processive nature of
this reaction is entirely consistent with the fact that PRMT1 uses
a rapid equilibrium random kinetic mechanism with “dead-end”
E SAM ω-MMA and E AcH4ꢀ21 SAH complexes, where
2
1
Y154 play an important role in regulating CARM1 activity. This
is the case because S217 can be phosphorylated in vivo, and this
modification is associated with a loss of CARM1 activity; phosphor-
ylation presumably disrupts the hydrogen bond between the S217
hydroxyl and the backbone carbonyl of Y154, leading to a loss of
affinity for SAM and an inability to properly position the Y154
3
3
3
3
the E SAM ω-MMA complex can undergo a second methyl
3
3
17
transfer reaction to produce ADMA.
To follow up on these studies and provide a mechanistic
basis for the methylation of an arginine residue, which is arguably
a weak nucleophile, we examined the structure of PRMT1 bound
21
phenol. Because residues from the N-terminal tail are absent in the
holo structure of PRMT1 and an apo structure does not exist, it is
unclear whether this interaction occurs in PRMT1.
9
to SAH. On the basis of this structure, there are a number of
highly conserved active site residues that likely play key roles in
SAM recognition, substrate binding, and catalysis (Figure 2A).
For example, in PRMT1, it has been suggested that R54 and
E100 are involved in SAM binding by forming hydrogen bonds
and electrostatic interactions with the carboxylate group and ribose
Also present in the active site is M155. Although this residue is
not thought to play a direct role in rate acceleration, it has been
22
suggested that M155 is responsible for the formation of ADMA
as the end product of dimethylation, as opposed to SDMA,
because of steric hindrance that would prevent the transfer of a
9,18
moiety of SAM, respectively.
The R54 residue also likely
18,22
forms a hydrogen bond with the side chain of E144 to orient the
methyl group to N after methylation of N .
This hypoth-
η
η
2
1
γ-carboxylate of this residue for optimal electrostatic and hydro-
esis is supported by the fact that PRMT5, a type II PRMT, has a
serine residue at this position that presumably creates a more
open pocket that allows symmetric dimethylation.
gen bonding interactions with N of a substrate arginine residue.
η
2
22
This interaction likely helps position N for attack on the methyl
η
2
3
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Figure 2. Active site and proposed catalytic mechanism of PRMT1. (A) Structure of PRMT1 (white) highlighting key residues in the active site believed
to play roles in substrate binding and/or catalysis. Note that the PRMT1 structure is overlaid with PRMT3 (green) because the electron density of Y39 is
not present in the crystal structure of PRMT1 and the positioning of E153 in PRMT1 is different from that in PRMT3, which is likely due to the
crystallization conditions. This figure was prepared with UCSF Chimera using the coordinates from PRMT1 (PDB entry 1ORI) and PRMT3 (PDB
entry 1F3L). (B) Proposed catalytic mechanism of PRMT1. The proposed mechanism of catalysis involves three conserved active site residues. It has
η
been suggested that R54 and E144 help position N 2 of a substrate Arg residue for attack on the methyl group of SAM. E153 plays a role in positioning the
Arg residue as well as initiating an electron rearrangement that results in the formation of a more nucleophilic guanidinium moiety. Methyl transfer
results in the formation of a dication intermediate that then undergoes the loss of a proton to form the first methylation product, ω-MMA. A second
round of methylation occurs via the same mechanism to form the final product, ADMA.
Given that the guanidinium group is a relatively weak nucleo-
phile, it has been suggested that its interaction with E153 causes a
This attack potentially results in the formation of a dication
intermediate that undergoes the loss of a proton to possibly E144
or via a proton wire to H293. However, because the formation of
a dication intermediate is somewhat unfavorable, it has been
redistribution of electrons that activates Nη2 for an S 2-type
N
18
nucleophilic attack on the methyl group of SAM (Figure 2B).
3
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suggested that PRMT1 uses a stepwise or concerted mechanism
in which the proton is removed prior to or simultaneously with
methyl transfer.
Table 1. Peptide Sequences
1
8
peptide
sequence
To confirm the assumed roles of the aforementioned resi-
dues and to begin to validate the proposed mechanism, we initiated
studies to characterize the catalytic mechanism of PRMT1. Herein
we describe those efforts. Specifically, we report the results of
site-directed mutagenesis of a number of highly conserved active
site residues (i.e., Y39, R54, E100, E144, E153, M155, and
H293), which are believed to play key roles in SAM recognition,
substrate binding, and catalysis, as well as pHꢀrate profiles,
processivity studies, and the determination of solvent isotope
effects (SIEs). In particular, we demonstrate that while E100 is
not important for SAM binding, M155 plays a critical role in this
process, as well as the processivity of the enzyme; however, M155
is not responsible for the regiospecific formation of ADMA.
Additionally, while R54 is not important for SAM binding, this
residue likely forms a hydrogen bond with E144, and this inter-
action appears to be important for orienting the substrate
guanidinium for nucleophilic attack on the S-methyl group of
SAM. While our data suggest that the charge of E144 is not im-
portant for catalysis, the charge and position of E153 are critical.
The Y39 phenol also plays an important role in rate enhance-
ment, likely via its ability to form a hydrogen bond with the E153
carboxylate or the substrate guanidinium. Additionally, mutagen-
esis studies on H293, combined with the lack of a normal SIE, do
not support a role for this residue as a general base. Furthermore,
the lack of a normal SIE with either the wild type (WT) or
catalytically impaired mutants suggests that general acid/base
catalysis is not important for promoting methyl transfer. This
result, combined with the fact that the E144A/E153A double
mutant retains considerably more activity then the single mutants
alone, suggests that the PRMT1-catalyzed reaction is primarily
driven by proximity effects and that the prior deprotonation of
the substrate guanidinium is not required for methyl transfer.
AcH4ꢀ21
1-Ac-SGRGKGGKGLGKGGAKRHRKV
GGRGGFGGRGGFGGRGGFG
RGG3
Synthesis of Peptides. AcH4ꢀ21 and RGG3 peptides were
13
synthesized as previously described on a Rainin PS3 automatic
peptide synthesizer using Fmoc chemistry on a Wang resin.
The sequences of these peptides can be found in Table 1. The
peptides were cleaved from the resin with 95% TFA, 2.5%
triisopropylsilane, and 2.5% water and then precipitated with
diethyl ether. Peptides were purified by reverse phase HPLC with
a mobile phase of water and 0.05% TFA and eluted with
acetonitrile and 0.05% TFA. The masses were determined using
a Bruker Ultraflex II MALDI-TOF mass spectrometer.
Gel-Based Activity Assay. A previously described gel-based
assay was used to determine the steady state kinetic parameters of
WT and PRMT1 mutants. Assays were performed in a reaction
mixture of 50 mM HEPES (pH 8.0), 1 mM EDTA, 50 mM NaCl,
13
14
0
.5 mM dithiothreitol, 15 μM C-labeled SAM, and a varying
final concentration of AcH4ꢀ21 (0ꢀ1000 μM). Reaction mix-
tures were preincubated at 37 °C for 10 min. WT PRMT1, or a
PRMT1 mutant, was then added, and the reaction was quenched
after 15 min. For the assays with a varying final SAM concentra-
tion (0ꢀ39.7 μM), the same reaction mixture was used except
the concentration of AcH4ꢀ21 was held constant at 100 μM.
Each assay was conducted in duplicate, and the standard devia-
tion of the duplicate raw data values agreed within e20%. GraFit
23
version 5.0.11 was used to fit the data to eq 1 or 2 if substrate
inhibition was observed
υ ¼ Vmax½Sꢁ=ðKm þ ½SꢁÞ
ð1Þ
ð2Þ
υ ¼ Vmax½Sꢁ=½ðK þ ½SꢁÞð1 þ ½Sꢁ=K Þꢁ
m
i
’
MATERIALS AND METHODS
MALDI-MS-Based Activity Assay. A previously described
matrix-assisted laser desorption ionization (MALDI) MS-based
assay was used to determine the processivity of WT and select
PRMT1 mutants. Briefly, assays were performed in a reaction
mixture of 50 mM HEPES (pH 8.0), 1 mM EDTA, 50 mM NaCl,
500 μM SAM, and 20 μM AcH4ꢀ21. Reaction mixtures were
then preincubated at 37 °C for 10 min. WT PRMT1 or a PRMT1
mutant was then added, and the reaction was quenched with 3 μL
Chemicals. Sodium dodecyl sulfate (SDS), tris(hydroxymethyl)
aminomethane (TRIS), tetramethylethylenediamine, acryla-
mide, and ammonium persulfate were purchased from Bio-Rad
13
(
Hercules, CA). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), Tricine, and dithiothreitol (DTT) were pur-
chased from RPI (Mt. Prospect, IL). Acetonitrile and methanol
were purchased from Fisher Scientific (Pittsburgh, PA). Sodium
chloride and dimethylformamide (DMF) were purchased from
Alfa Aesar (Ward Hill, MA). Piperidine was purchased from
Sigma-Aldrich (St. Louis, MO). Fmoc-protected amino acids,
of 50% TFA in ddH O after the appropriate time period. Spectra
2
were recorded on a Bruker Ultraflex II MALDI-TOF MS or Applied
Biosystems 4800 Plus MALDI TOF/TOF MS instrument and
analyzed using Flex Analysis. The percent turnover was determined
by dividing the intensity of the modified peptide by the sum of the
intensities of the unmodified and modified substrates times 100%.
Chemical Analysis of Methylation Products. A reaction
(
ethylenedinitrilo)tetraacetic acid (EDTA), and trifluoroacetic
14
acid (TFA) were purchased from EMD (Gibbstown, NJ). C-
14
labeled SAM was purchased from Perkin-Elmer and C-labeled
BSA from Sigma-Aldrich. Mutagenic primers were purchased
from IDT Inc. (Coralville, IA). The purification of PRMT1 has
3
mixture of 10 μg of GST-GAR and 1.4 μM [ H]SAM in
13
been described previously.
50 mM HEPES (pH 7.5) was incubated with 2 μg of WT
PRMT1 or a PRMT1 mutant for 2 h at 37 °C. The products were
then precipitated with an equal volume of 50% trichloroacetic
acid, washed with acetone, and hydrolyzed for 20 h at 110 °C in 6
M HCl. The hydrolysate was dried and mixed with standards of
ADMA, SDMA, and ω-MMA before being fractionated on a
Site-Directed Mutagenesis. PRMT1 mutants were generated
using the QuikChange site-directed mutagenesis kit (Stratagene).
The sequences of the mutagenic primers can be found in Table
S1 of the Supporting Information. The full open reading frame
was sequenced for each mutant to ensure that only the desired
mutation had been incorporated. DNA that contained the desired
mutation was then transformed into Escherichia coli BL21(DE3)
24
high-resolution cation exchange column as described previously.
One-tenth of the fractions were used for ninhydrin analysis of
the standards, and nine-tenths were counted.
13
cells and purified using our established protocol for WT PRMT1.
3
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Partial Proteolysis. Partial proteolysis assays were performed
in a reaction mixture of 50 mM HEPES (pH 8.0), 1 mM EDTA,
atomic radii were assigned on the basis of the Amber99 force field
25
using AMBER. pK values were computed taking into account
a
5
0 mM NaCl, and 0.5 mM dithiothreitol in the absence or
desolvation effects and intraprotein interactions, including the
proximity of neighboring functional groups.
26
presence of 0.5 mM SAH and 0.75 μg/mL subtilisin on ice. WT
PRMT1, or a PRMT1 mutant, was then added, and the reaction
was quenched after 60 min with 5 mM phenylmethanesulfonyl
fluoride. Protein fragments were separated by 12% sodium
dodecyl sulfateꢀpolyacrylamide gel electrophoresis and visua-
lized with Coomassie Brilliant Blue.
’
RESULTS AND DISCUSSION
Mutagenesis Studies of Proposed Catalytic Residues. To
begin to investigate the catalytic mechanism of PRMT1, we used
site-directed mutagenesis to probe the roles of Y39, R54, E100,
S102, E144, E153, M155, and H293. The mutant enzymes were
pH Profile. The steady state kinetic parameters for the WT
enzyme as well as the Y39F and H293A mutants were deter-
mined over a pH range of 6.0ꢀ9.25 using the gel-based activity
assay described above. Assays were performed in a reaction mix-
ture of 50 mM Bis-Tris (6.0ꢀ7.0), 50 mM HEPES (7.0ꢀ8.5),
1
3
purified and characterized according to described procedures.
The kinetic parameters of each mutant were determined for the
AcH4ꢀ21 peptide as well as SAM (Tables 2 and 3); AcH4ꢀ21 is
a 21-amino acid peptide substrate whose sequence is based on
5
5
0 mM Tricine (8.5ꢀ9.0), or CHES (8.75ꢀ9.25), 1 mM EDTA,
0 mM NaCl, and 0.5 mM dithiothreitol, with constant and
the N-terminus of histone H4 (Table 1). Note that, like other
14
27,28
varying final concentrations of [ C]SAM (0ꢀ41 μM) and
RGG3 (0ꢀ1000 μM). Each assay was conducted in duplicate,
and the standard deviation of the duplicate measurements agreed
within e20%. Note that, to make certain that the variances in
kinetic parameters were not the result of a buffer effect, we
utilized an overlapping buffer method. The kinetic parameters
for the overlapping buffers were similar, and thus, the average was
used. In addition, time course assays were performed at each pH
to demonstrate that activity was not lost over time. GraFit
systems,
partial proteolysis studies were performed to ensure
that the loss of activity associated with a particular mutation was
not due to a gross structural perturbation (Figure S1 of the
Supporting Information).
SAM Binding Mutants. R54 Mutants. R54 forms hydrogen
bonds and/or electrostatic interactions with E144, one of the two
key glutamate residues, and presumably orients this residue for
the productive recognition of the substrate guanidinium. Addi-
tionally, both Nη1 and Nη2 of this residue interact with the car-
boxylate group on the methionine portion of SAM, which
suggests that this residue could play a key role in SAM binding
2
3
version 5.0.11 was used to fit the data to eq 3 or 4
y ¼ ðLim1 þ Lim2
(
Figure 2B). Consistent with this prediction is the 42-fold
pH ꢀ pKa1
pH ꢀ pKa2
pH ꢀ pKa1
pH ꢀ pKa2
ꢂ 10
ꢂ 10
Þ=ð10
þ 1Þ ꢀ ½ðLim2 ꢀ Lim3Þ
þ 1Þ
decrease in the k /K value observed for SAM with the R54K
cat
m
ꢁ=ð10
ð3Þ
mutant. However, the effect on k /K is primarily driven by a
cat
m
decrease in kcat and not K , suggesting that the lack of a more
m
y ¼ ðLim1 þ Lim2 þ 10pH ꢀ pKa1 Þ=ð10pH ꢀ pKa1 þ 1Þ ð4Þ
dramatic effect on the SAM K reflects the multistep nature of
m
the reaction, where K is not equal to K ; k /K represents
d
m
cat
m
For eqs 3 and 4, Lim1 corresponds to the activity measured at low
pH and Lim2 corresponds to the maximal activity measured at
the optimal pH, and for eq 3, Lim3 is equal to the activity
measured at high pH.
all steps up to and including the first irreversible step of the
reaction. To evaluate whether this was indeed the case, we
determined the dissociation constants, i.e., K , for binding of
i
SAH to both the WT and R54A enzymes; SAH was used for
these experiments as a proxy for SAM to more accurately gauge
the effects of a particular mutation on SAM binding. The results
of these studies confirm that R54 is not critical for SAM binding,
Solvent Isotope Effect. SIEs were investigated by determin-
ing the steady state kinetic parameters for the WT enzyme using
the gel-based activity assay described above. The reaction
mixture consisted of 50 mM HEPES (pH 8.0), 1 mM EDTA,
as evidenced by the fact that the K for SAH is similar to that
i
14
5
0 mM NaCl, 0.5 mM dithiothreitol, 15 μM C-labeled SAM,
and a varying final concentration of AcH4ꢀ21 (0ꢀ1000 μM) in
92% D O. The assay was conducted in duplicate, and the
obtained for the WT enzyme (Table 4).
With respect to the peptide substrate, large changes in both
>
2
kcat(app) (42- and 7.7-fold) and kcat(app)/K (32- and 32-fold)
m
standard deviation of the duplicate measurements agreed within
were observed for both the R54K and R54A mutants, respec-
tively. Although these effects are at least partially related to our
inability to completely saturate the enzyme with SAM in our
radioactive methyltransferase assay, which is why the term
kcat(app) is used, their magnitude, particularly on kcat(app), is
consistent with a role for this residue in orienting the substrate
guanidinium via E144 for nucleophilic attack on the methyl
group of SAM. Consistent with this notion is the fact that the kcat
values obtained with SAM, where the peptide substrate is
saturating, are decreased by a similar order of magnitude.
23
e20%. GraFit version 5.0.11 was used to fit the data to eq 1.
SAH Inhibition Studies. The inhibition constants for SAH
were determined for the WT and mutant enzymes using the gel-
based activity assay described above. The reaction mixture
consisted of 50 mM HEPES (pH 8.0), 1 mM EDTA, 50 mM
14
NaCl, 0.5 mM dithiothreitol, 15 μM C-labeled SAM, 100 μM
AcH4ꢀ21, and a varying final concentration of SAH (0ꢀ500 μM).
The assay was conducted in duplicate, and the standard deviation
of the duplicate measurements agreed within e20%. GraFit
2
3
version 5.0.11 was used to fit the data to a Dixon plot of 1/υ
E100 Mutants. E100 forms hydrogen bonds with the ribose
moiety of SAM and thus would be expected to play an impor-
tant role in SAM binding. Three mutants, i.e., the E100D,
E100Q, and E100A mutants, were made to confirm this hypoth-
esis. With respect to the kinetic parameters determined for SAM,
there is an only very small effect on k /K . For example, the
versus SAH concentration. K was determined using eq 5
i
slope ¼ Km=ðVmaxKi½SꢁÞ
ð5Þ
pKa Calculations. The structure of PRMT1 (PDB entry
cat
m
1
ORI) was rebuilt using Amber topology parameters and hydro-
complete removal of the E100 carboxylate, which occurs in the
gen atoms added to the structure. Atom partial charges and
E100A mutant, decreases k /K by only 3.2-fold. The K for
cat
m
i
3
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Table 2. Kinetic Parameters of PRMT1 Mutants for the AcH4ꢀ21 Peptide Substrate
ꢀ1
ꢀ1
ꢀ1
K
m
(μM)
x-fold
kcat(app) (min
)
x-fold
k
cat(app)/K
m
(M min
)
x-fold
a
ꢀ1
ꢀ2
ꢀ3
5
WT
1.1 ( 0.5
2.0 ( 0.7
0.8 ( 0.6
5 ( 1
ꢀ
4.6 ꢂ 10 ( 2 ꢂ 10
ꢀ
11
4.1 ꢂ 10
2.1 ꢂ 10
ꢀ
20
a
ꢀ2
4
Y39F
1.8
0.7
4.5
4.5
5.5
2.4
4.5
5.5
1.5
1.8
0.3
2.8
1.8
1.6
2.7
0.5
1.6
1.8
10
4.2 ꢂ 10 ( 2 ꢂ 10
a
ꢀ2
ꢀ4
4
R54K
1.09 ꢂ 10 ( 4 ꢂ 10
42
1.3 ꢂ 10
32
a
ꢀ2
ꢀ3
4
R54A
6.0 ꢂ 10 ( 3 ꢂ 10
7.7
1.3
1.8
2.5
0.8
0.7
2.0
3.5
50
1.3 ꢂ 10
7.6 ꢂ 10
4.5 ꢂ 10
32
a
a
ꢀ1
ꢀ2
4
E100D
5 ( 1
3.6 ꢂ 10 ( 1 ꢂ 10
5.4
9.1
5.7
3.7
3.7
3.2
6.1
14
ꢀ1
ꢀ2
4
E100Q
6 ( 2
2.5 ꢂ 10 ( 1 ꢂ 10
a
ꢀ1
ꢀ3
4
E100A
2.6 ( 0.9
5 ( 1
1.85 ꢂ 10 ( 8 ꢂ 10
7.2 ꢂ 10
a
ꢀ1
ꢀ2
5
S102E
S102A
5.7 ꢂ 10 ( 2 ꢂ 10
1.1 ꢂ 10
1.1 ꢂ 10
1.3 ꢂ 10
a
ꢀ1
ꢀ2
5
6 ( 1
6.3 ꢂ 10 ( 2 ꢂ 10
a
a
ꢀ1
ꢀ2
ꢀ3
ꢀ4
5
E144D
1.7 ( 0.4
2.0 ( 0.6
0.3 ( 0.2
3.1 ( 0.5
2.0 ( 0.8
1.8 ( 0.6
3.0 ( 0.7
0.6 ( 0.4
1.8 ( 0.5
2 ( 2
2.2 ꢂ 10 ( 1 ꢂ 10
ꢀ1
4
E144Q
1.3 ꢂ 10 ( 4 ꢂ 10
6.7 ꢂ 10
a
ꢀ3
4
E144A
E153D
9.5 ꢂ 10 ( 2 ꢂ 10
3 ꢂ 10
a
a
ꢀ2
ꢀ4
3
2.65 ꢂ 10 ( 5 ꢂ 10
17
8.6 ꢂ 10
3.8 ꢂ 10
2.2 ꢂ 10
50
ꢀ3
ꢀ4
3
E153Q
7.7 ꢂ 10 ( 3 ꢂ 10
60
110
190
9.3
1.4
16
a
ꢀ3
ꢀ4
3
E153A
3.8 ꢂ 10 ( 1 ꢂ 10
121
3.5
2.7
10
a
ꢀ1
ꢀ3
ꢀ3
4
E144A/E153A
1.32 ꢂ 10 ( 4 ꢂ 10
4.4 ꢂ 10
a
ꢀ1
5
M155L
1.73 ꢂ 10 ( 5 ꢂ 10
3 ꢂ 10
b
ꢀ2
ꢀ3
4
M155A
4.6 ꢂ 10 ( 1 ꢂ 10
2.6 ꢂ 10
3.7 ꢂ 10
3
a
ꢀ3
ꢀ4
3
H293Q
7.6 ꢂ 10 ( 6 ꢂ 10
61
110
256
a
ꢀ2
ꢀ4
H293A
11.1 ( 0.6
1.81 ꢂ 10 ( 2 ꢂ 10
25
1.6 ꢂ 10
a
b
At 15 μM SAM. At 30 μM SAM.
Table 3. Kinetic Parameters of PRMT1 Mutants for SAM
ꢀ1
ꢀ1
ꢀ1
K
m
(μM)
x-fold
kcat (min
)
x-fold
kcat/K
m
(M min
)
x-fold
a
ꢀ1
ꢀ2
ꢀ3
ꢀ3
ꢀ3
ꢀ2
ꢀ2
ꢀ2
ꢀ2
ꢀ2
ꢀ2
5
WT
6 ( 1
ꢀ
5.8 ꢂ 10 ( 3 ꢂ 10
ꢀ
1 ꢂ 10
ꢀ
20
a
ꢀ2
3
3
Y39F
14 ( 3
14 ( 1
10 ( 1
6 ( 2
2.3
2.3
1.7
1.0
1.2
0.8
0.9
0.9
0.7
1.4
0.5
0.8
2.8
1.5
1.7
1.7
18
7.0 ꢂ 10 ( 5 ꢂ 10
8.3
18
6.1
5.1 ꢂ 10
2.4 ꢂ 10
9.1 ꢂ 10
a
ꢀ2
R54K
3.3 ꢂ 10 ( 1 ꢂ 10
42
a
ꢀ2
3
R54A
9.5 ꢂ 10 ( 4 ꢂ 10
11
a
a
ꢀ1
4
E100D
4.8 ꢂ 10 ( 5 ꢂ 10
1.2
2.1
3.6
1.1
1.1
2.1
3.6
7.8 ꢂ 10
3.8 ꢂ 10
3.1 ꢂ 10
1.3
2.6
3.2
1.0
1.1
1.5
5.3
29
ꢀ1
4
E100Q
7 ( 2
2.8 ꢂ 10 ( 2 ꢂ 10
a
ꢀ1
4
E100A
5 ( 2
1.6 ꢂ 10 ( 2 ꢂ 10
a
ꢀ1
4
S102E
S102A
5.1 ( 0.7
5.6 ( 0.6
4.0 ( 0.9
8.3 ( 0.9
3.1 ( 0.6
5 ( 1
5.1 ꢂ 10 ( 2 ꢂ 10
9.9 ꢂ 10
a
ꢀ1
4
5.2 ꢂ 10 ( 2 ꢂ 10
8.8 ꢂ 10
6.8 ꢂ 10
1.9 ꢂ 10
3.4 ꢂ 10
5.8 ꢂ 10
1.1 ꢂ 10
a
a
ꢀ1
4
E144D
2.7 ꢂ 10 ( 2 ꢂ 10
ꢀ1
ꢀ3
ꢀ4
4
3
3
3
E144Q
1.60 ꢂ 10 ( 7 ꢂ 10
a
ꢀ2
E144A
E153D
1.05 ꢂ 10 ( 4 ꢂ 10
55
a
a
ꢀ2
ꢀ3
2.8 ꢂ 10 ( 2 ꢂ 10
21
31
17
ꢀ2
ꢀ3
E153Q
17 ( 4
9 ( 3
1.9 ꢂ 10 ( 2 ꢂ 10
91
a
ꢀ3
ꢀ4
2
E153A
4.1 ꢂ 10 ( 4 ꢂ 10
141
4 ꢂ 10
250
9.1
10
a
ꢀ1
ꢀ3
ꢀ3
4
3
E144A/E153A
10 ( 2
10 ( 2
110 ( 21
17 ( 6
37 ( 2
1.06 ꢂ 10 ( 6 ꢂ 10
5.5
1.1 ꢂ 10
a
ꢀ1
4
M155L
1.00 ꢂ 10 ( 8 ꢂ 10
5.8
2.0
1 ꢂ 10
a
ꢀ1
ꢀ2
M155A
2.9 ꢂ 10 ( 4 ꢂ 10
2.8 ꢂ 10
36
a
ꢀ2
ꢀ3
2
H293Q
2.8
6.2
1.4 ꢂ 10 ( 2 ꢂ 10
41
10
8 ꢂ 10
125
50
a
ꢀ2
ꢀ3
3
H293A
5.6 ꢂ 10 ( 1 ꢂ 10
2 ꢂ 10
a
At 100 μM AcH4ꢀ21.
SAH with this mutant is similarly unaffected, quite clearly de-
monstrating that this residue is not important for SAM binding
or catalysis. This result is especially surprising when one con-
siders that the distances between the ribose hydroxyls and the R-
be an artifact of the crystallization conditions (the enzyme was
crystallized at pH ∼4.7, which would favor protonation of E100
and potentially promote hydrogen bond formation), similar
distances and orientations are observed in the crystal structures
of the PRMT3 SAH and CARM1 SAH complexes that were
carboxylate of E100 in the PRMT1 SAH complex are only
3
3
3
18,29
2
.6ꢀ2.7 Å, which are distances typically associated with relatively
crystallized at pH 6.3 and 7, respectively.
explanation is intellectually unsatisfying. Nevertheless, these results
Thus, such an
strong hydrogen bonds. Although the observed interactions may
3
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Table 4. SAH Inhibition Studies
formation of ω-MMA and ADMA. Thus, despite the fact that
this mutation relieves the steric constraint thought to prevent
SDMA formation, our results indicate M155 is not responsible
for the formation of ADMA over SDMA.
Nevertheless, given that the mutation of this residue strongly
impacts the kinetics of the PRMT1-catalyzed reaction, particu-
i
K (μM)
WT
1.28 ( 0.06
26 ( 1
Y39F
R54A
E100A
M155A
1.8 ( 0.2
1.9 ( 0.1
34 ( 6
larly with respect to the K for SAM, we reasoned that it may
m
play a role in the processivity of ADMA formation. To investigate
this possibility, we utilized a previously established MALDI-MS
13
assay. Consistent with previous results with the WT enzyme,
ω-MMA- and ADMA-containing peptides were initially pro-
duced in equimolar amounts, followed by a decrease in the levels
of ω-MMA (Figure 4A). As described previously, these results
are characteristic of an enzyme that has the ability to rebind SAM
and subsequently produce ADMA before the first methylation
are consistent with the fact that methylthioadenosine is a re-
latively poor PRMT1 inhibitor.
With respect to the kinetic parameters determined for the
AcH4ꢀ21 peptide, the effects, while still small, are significantly
larger than those observed with SAM. For example, the decreases
13
13
in kcat(app)/K for the E100D, E100Q, and E100A mutants are
product, ω-MMA, is released. Because ADMA formation is not
obligatory, we have suggested that PRMT1 displays partial pro-
cessivity. With respect to the M155L mutant, similar results were
obtained; however, the level of ADMA formed is significantly
lower at the early time points (Figure 4B). This result indicates
that the M155L mutant is significantly less processive than the
WT enzyme. For the M155A mutant, little to no ADMA is
formed until the 20 min time point (Figure 4C), which suggests
that ω-MMA is released prior to the rebinding of SAM and that
this mutant utilizes a distributive mechanism. Note that the
concentration of SAM used in these assays was 500 μM; thus,
the loss of processivity is not due to a failure to saturate the
enzyme with SAM. In total, these results are consistent with
M155 playing a key role in SAM binding, as reduced affinity for
SAM, which occurs with the M155L and M155A mutants, would
be expected to decrease the processivity of the enzyme because,
for these mutants, the off rate for the monomethylated peptide is
larger than the rate constant for SAM binding.
m
5
.4-, 9.1-, and 5.7-fold, respectively. As the effects are largely
driven by an increase in K , these results suggest that an interplay
m
exists between the binding of both substrates or, alternatively, a
change in the kinetic mechanism.
M155 Mutants. To probe the importance of M155, we created
the M155L and M155A mutants. With respect to the peptide
substrate, the kinetic parameters for the M155L mutant were
similar to those obtained for the WT enzyme; the kcat(app) and
kcat(app)/Km values were only decreased by 2.7- and 1.4-fold,
respectively. In contrast to these relatively minor effects, 5.8- and
1
0-fold decreases in kcat and k /K , respectively, were observed
cat m
when SAM was tested as the varied substrate. These effects
represent an inability to properly position the S-methyl group of
SAM or, alternatively, an effect on SAM binding. Given that
M155 forms the bottom of the adenine portion of the SAM
binding pocket, these results are most consistent with the latter
possibility because the structural differences between a leucine
and a methionine would be expected to alter SAM binding.
Similar effects on SAM binding are observed with the M155A
Catalytic Mutants. E144 Mutants. E144 appears to orient Nη2
of the substrate guanidinium group to facilitate nucleophilic
attack on the S-methyl group of SAM. To investigate this role,
mutant, where the K for SAM was increased by 18-fold and the
m
k /K was decreased by 36-fold. Further confirming that this
we created the E144D, E144Q, and E144A mutants. The k /Km
cat
m
cat
residue is important for SAM binding is the fact that the K for
values for the E144D mutant were decreased by 3.2- and 1.5-fold
i
SAH is increased by 27-fold relative to that of WT. The large
for AcH4ꢀ21 and SAM, respectively. The ability of aspartate to
substitute for the glutamate indicates flexibility within the active
site and reveals that positioning of this residue is not critical for
changes in K , K , and k /K likely reflect a loss of steric
i
m
cat
m
constraint within the SAM binding pocket, which disfavors the
binding of SAM in an orientation that is productive for catalysis.
With respect to the peptide substrate, 10- and 16-fold reductions
catalysis. For the E144Q mutant, the effects on k /Km are
cat
slightly larger, with these values decreased 6.1- and 5.3-fold for
AcH4ꢀ21 and SAM, respectively. These data suggest that the
charge of this residue is significantly less important than its ability
to hydrogen bond with both the substrate guanidinium and R54,
as opposed to modulating the nucleophilicity of that group. Note
in kcat(app) and kcat(app)/K , respectively, were observed. These
m
effects are most likely due to our inability to completely saturate
the enzyme with SAM in our radioactive methyltransferase assay.
Role of M155 in ADMA Formation. Given the postulated role
of M155 in directing the formation of ADMA, as opposed to
that the calculated pK of the E144 carboxylate is ∼2.7; thus, this
a
1
8,22
SDMA,
we also investigated the contribution of this residue
residue is likely deprotonated in the PRMT1 active site. Never-
theless, this residue is important for catalysis, as illustrated by the
14- and 29-fold decreases in the k /K values for AcH4ꢀ21 and
to the regiospecific dimethylation of a substrate arginine residue.
For these studies, the M155A mutant was utilized because we
hypothesized that this mutation would relieve the steric con-
straint imposed by the methionine and thereby open up the
pocket and allow for SDMA formation. To investigate this
possibility, the M155A mutation, along with the WT control,
cat
m
SAM, respectively, for the E144A mutant. Note the effects on
k /K are dominated by a decreased k , suggesting that this
cat
m
cat
residue is relatively unimportant for substrate binding. These
results are consistent with previous findings from mutagenesis
3
9
was used to catalyze the [ H]SAM-dependent methylation of
studies that only measured relative rates. In total, the data suggest
GST-GAR, a fusion protein that links GST to the N-terminus of
human fibrilarin (Figure 3). Subsequently, the reaction mixture
was hydrolyzed in 6 M HCl at 110 °C, and the levels of ω-MMA,
SDMA, and ADMA formed were quantified by high-resolution
cation exchange chromatography. The results of these experi-
ments indicate that the M155A mutant catalyzes the exclusive
that the hydrogen bonding characteristics, and to a lesser extent,
the charge of E144 are important for orienting the substrate
guanidinium for nucleophilic attack on the S-methyl group of SAM.
E153 Mutants. The carboxylate group of E153 is thought to
play a crucial role in catalysis through its electrostatic and
hydrogen bonding interactions with N and N of the substrate
η
δ
1
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Figure 3. Amino acid analysis of methylation products. (A) WT PRMT1 produces a mixture of ω-MMA and ADMA as products of methylation. (B)
The M155A mutant also produces a mixture of ω-MMA and ADMA as products of methylation. In each case, the position of the standards was
3
determined with a ninhydrin assay (dotted lines); the position of the H-radiolabeled derivatives by counting (solid lines). The slightly earlier elution
3
3
a
position of the [ H]ADMA and [ H]-ω-MMA products compared to the standards is due to the mass and pK differences of amino acids with tritium vs
24
hydrogen-containing methyl groups.
guanidinium. To investigate the importance of both the size and
charge of this residue, we created the E153D, E153Q, and E153A
mutants and determined the kinetic parameters. The results are
consistent with this residue playing a key role in catalysis. For
example, the kcat values for the E153D, E153Q, and E153A
mutants are decreased by 21-, 31-, and 140-fold, respectively.
Given that the effects on k /K mirror the effects on k , these
double mutant would likely yield negligible, if any, activity.
However, this was not the case, as kcat is decreased by only 5.5-
fold and the k /K values are decreased by only 9.3- and 9.1-
cat
m
fold for AcH4ꢀ21 and SAM, respectively. The more significant
effect on the single mutants is most easily explained by the for-
mation of a strong salt bridge to the remaining glutamate. Such
an interaction would be expected to decrease activity by altering
both the nucleophilicity of the guanidinium and its position such
that methyl transfer is suboptimal. The fact that the double
mutant retains considerable activity is more difficult to rationa-
lize. However, it is possible that the removal of the two glutamate
residues increases the hydrophobicity of the active site, which
cat
m
cat
data indicate that both the charge and the position of E153 are
more important for catalysis than substrate binding. These
results are also consistent with previous findings from mutagen-
9
esis studies that measured only relative rates. The fact that
mutating E153 has a more dramatic effect than mutating E144 is
consistent with the notion that this residue plays an important
role in redistributing electron density within the guanidinium
group to enhance its nucleophilicity.
E144A/E153A Mutant. Given the results obtained for the E144
and E153 single mutants, we expected that the E144A/E153A
would be expected to depress the pK of the guanidinium and
a
thereby increase its nucleophilicity. Although we cannot rule out
such a possibility, the active site possesses a number of other hy-
drophilic residues in the proximity (e.g., Y39, R54, and H293) that
should minimize any change in hydrophobicity. An alternative
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Figure 4. Processivity of WT PRMT1 and M155 mutants. (A) WT PRMT1 uses a partially processive mechanism to catalyze the formation of ADMA.
B) The M155L mutant uses a partially processive mechanism; however, formation of ADMA is slower than that with WT PRMT1. (C) The M155A
(
mutant uses a distributive mechanism to catalyze the formation of ADMA. The large percent turnover of ω-MMA suggests that ω-MMA is released prior
to the rebinding in preparation for the second round of methylation.
explanation, which is equally supported by the data, involves the
PRMT1-catalyzed reaction being primarily driven by bringing
the substrate guanidinium closer to the S-methyl group of SAM.
H293 Mutants. H293 has been thought to play the role of a
could be interpreted as being consistent with a role for H293 as
the general base, alternative explanations are also possible. For
example, in the structure of the PRMT1 SAH complex, H293
3
appears to form a salt bridge with D51, a conserved residue that is
present on RY. Given the short distance between the side chains
of H293 and D51 (i.e., 2.6 Å), this interaction likely plays a
critical role in forming the two-helix boundary that separates the
SAM and peptide binding portions of the active site (Figure 5).
As such, one would expect that disruption of this interaction
would lead to decreased activity via the inability to properly form
the substrate and cofactor binding pockets. This is especially
likely when one considers that Y39, H45, M48, and R54 are
present on helix RY and RZ and likely play key roles in both
PRMT1 catalysis (e.g., Y39 and R54) and formation of the active
site cleft (e.g., H45 and M48). Consistent with this possible role
general base by accepting a proton from N ; however, this
η
2
residue is >6 Å from the approximate position of the substrate
guanidinium, a distance that is too far to directly remove the
proton. Although a water-mediated proton transfer mechanism is
possible, the high basicity of the substrate guanidinium makes
this mechanism intellectually unsatisfying, and we have suggested
(
see above) that proton loss could occur prior to, concomitant
with, or even after methyl transfer, and that H293 may not act as a
general base. To probe this hypothesis, we created the H293Q
and H293A mutants. For the H293Q mutant, the kcat is de-
creased by 41-fold, whereas negligible changes in the K values
m
were observed, indicating that the 110- and 125-fold decreases in
for H293 is the fact that the K for SAH is increased by 11-fold
i
the k /K values for AcH4ꢀ21 and SAM, respectively, are
(Table 4). This is the case because either the alanine or glutamine
mutations would not be expected to affect SAM binding, only kcat.
Given that R54 is also present on helix RY, some of the effects of
mutating this residue may also be due to destabilizing the formation
of the PRMT1 substrate and cofactor binding pockets.
cat
m
driven by k . For the H293A mutant, the effects on k /K are
cat
cat
m
similar in magnitude, with the k /K values for AcH4ꢀ21 and
cat
m
SAM decreased by 256- and 50-fold, respectively. Although these
results indicate that H293 plays a critical role in catalysis and
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Figure 5. Interaction of D51 and H293. The kinetic effects of the H293
mutations can be explained by the fact that a salt bridge likely forms
between D51 and H293, which are separated by only 2.6 Å. A disruption
of this interaction could possibly prevent the proper formation of the
binding pockets. This figure was prepared with UCSF Chimera using the
coordinates for PRMT1 (PDB entry 1ORI).
Y39 Mutant. In CARM1, Y154 appears to be important for
both cofactor binding and orienting E267 (Y154 and E267
19ꢀ21
correspond to Y39 and E153 in PRMT1, respectively
).
Figure 6. pH profiles of WT PRMT1 with SAM. (A) The log kcat/K
m
vs
of
Although the Y154F mutant appears to abolish CARM1 activity,
this was a single-point assay and only relative rates were measured.
pH plot is used to determine the pK of ionizable groups on the enzyme
21
a
or substrate. (B) The log kcat vs pH plot is used to determine the pK
a
Thus, to establish the role of the corresponding residue in
PRMT1, we created the Y39F mutant and determined the kinetic
parameters. Although this mutation has only small, ∼2-fold
effects, on the K values for AcH4ꢀ21 and SAM, the K for
ionizable groups in the ES complex.
m
i
residues that typically ionize within the pH range under study.
Note that at all pH values enzyme activity was linear with respect
to time, indicating that the loss of activity at the pH extremes was
not due to a nonspecific effect on enzyme structure. Also note
that k /K is the apparent second-order rate constant for the
SAH is increased by ∼20-fold, thereby confirming that this
residue is important for cofactor binding; the lack of a more
dramatic effect on the SAM K reflects the multistep nature of
m
the reaction, where K is not equal to K . The importance of this
d
m
cat
m
residue was further illustrated by the 20-fold decrease in the
reaction of free substrate and free enzyme [or when one substrate
(A) is saturating, the EA complex], and therefore, the pH
dependence of k /K monitors the ionization state of these
k /K value observed for AcH4ꢀ21 and the 20-fold decrease
cat
m
seen with SAM. The position of this residue within the PRMT1
active site and the fact that kcat is decreased by 8.3-fold suggest
that this residue may also play an important role in rate
enhancement; the specific role of this residue in catalysis is
described below (see pH Studies).
cat
m
entities. In contrast, effects on kcat are interpreted as being due to
the presence of important ionizable groups in the enzyme sub-
3
strate complex.
With SAM as the varied substrate, the plot of log k /Km
cat
S102 Mutants. Given that disruption of the interaction between
versus pH is bell-shaped and is consistent with the presence of
two ionizable groups that are important for substrate capture;
the S102 hydroxyl and the Y39 backbone carbonyl appears to
21
negatively regulate CARM1, we also investigated the role of
S102. For these studies, we generated the S102A and S102E
mutants; the latter mutant was generated to mimic constitutively
phosphorylated PRMT1. Somewhat surprisingly, the results of
these studies indicate that neither mutant negatively impacts the
PRMT1 kinetic parameters. The k , K , and k /K values are
pK values of 6.2 ( 0.3 and 10.5 ( 0.4 were assigned to the
a
ascending and descending limbs, respectively (Figure 6A). With
respect to k , the rate of the reaction increases with increasing
cat
pH until a limiting value is reached (Figure 6B). Fitting the data
to eq 4 yields a pK value of 5.1 ( 0.8. Although it is difficult to
a
cat
m
cat
m
definitively assign an observed pK to a particular residue, or
a
virtually identical to those obtained for the WT enzyme. These
results suggest that in contrast to the situation with CARM1,
even if this residue is phosphorylated, no effect on PRMT1
activity is likely.
functional group on a substrate, the structures of PRMT1, SAM,
and the RGG3 peptide suggest several possible residues and/or
functional groups whose ionization could alter substrate capture
or k . With respect to the ascending limb, protonation of the
cat
pH Studies. To further improve our understanding of PRMT1
catalysis, pHꢀrate profiles were generated for the WT enzyme
by determining k and k /K values for both SAM and the
SAM carboxylate (pK ∼ 2 in solution), D51, E100, E144, and
a
E153 could explain the loss in activity as the pH decreases.
However, most of these groups are readily ruled out. For
cat
cat
m
RGG3 peptide over a pH range of 6.0ꢀ9.25. The RGG3 peptide
was used in place of the AcH4ꢀ21 peptide to simplify the
interpretation of the pHꢀrate profiles. This is the case because
this peptide has kinetic parameters comparable to those of the
AcH4ꢀ21 peptide and, more importantly, because, with the
example, the pK of the SAM carboxylate is significantly lower
a
than the pK for the ascending limb of the k /K versus
a
cat
m
pHꢀrate profiles, effectively ruling this group out. Consistent
with this notion is the fact that the R54A mutation does not affect
the K for SAM. Although protonation of D51, which is the
i
exception of the N-terminus (pK ∼ 8.0), this peptide lacks
residue that interacts with H293, could also explain the loss of
a
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activity at low pH, the fact that the pHꢀrate profiles obtained
for the H293A mutant show a similar loss of activity at low pH
(
vide infra) argues against this possibility. Protonation of E100
could also potentially explain the loss of activity at low pH.
However, the fact that little to no effect on the kinetic parameters
was observed when this residue was mutated to alanine makes
this suggestion unlikely. Of the two remaining residues, i.e., E144
and E153, the residue most likely responsible for the loss of
activity at low pH is E153. We surmise that this is the case
because the E144Q mutant retains considerable activity; thus,
the ionization of this residue would also be expected to minimally
impact the reaction rate. In contrast, the activity of the E153Q
mutant is significantly decreased and similar to that obtained for
the E153A mutant, thereby suggesting that protonation of the
E153 carboxylate would have a profound negative impact on rate
acceleration. Also consistent with the ionization of this residue
corresponding to the ascending limb is the fact that the pK of a
a
glutamate residue in solution is typically in the range of 4ꢀ5. In
total, these data suggest that E153 must be deprotonated for
optimal PRMT1 activity.
With respect to the descending limb, deprotonation of the
amino group on SAM (pK ∼ 9.5 in solution), the N-terminal
a
amino group on the RGG3 peptide (pK ∼ 8 in solution), Y39,
a
and H293 could explain the loss in activity as the pH increases.
Given the similarities in the pK values of these functional groups,
a
it is more difficult to definitively assign the pK of the descend-
a
Figure 7. pH profiles of WT PRMT1 and mutants with the RGG3
peptide. (A) The log kcat/K vs pH plot is used to determine the pK of
m a
ionizable groups on the enzyme or substrate. (B) The log kcat vs pH plot
ing limb. Nevertheless, the fact that the pK of the N-terminal
a
amine on the RGG3 peptide is significantly lower than the pK of
a
the descending limb argues against the notion that the ionization
of this group is responsible for the decreased activity at high pH.
is used to determine the pK of ionizable groups in the ES complex.
a
Note also that the pK of an arginine residue is 12.5 and is sig-
a
nificantly higher than the pK observed for the descending limb,
difference most likely reflects a change in the rate-determining
step for the reaction. Given that PRMT1 methylates its substrates
in a partially processive fashion, these data are most consistent
with product release being rate-limiting when the peptide is the
varied substrate, as opposed to the case when the concentration
of SAM is varied and chemistry, a conformational change, or
SAM binding is potentially rate-limiting.
pHꢀrate profiles were also generated for the H293A and
Y39F mutants, using the RGG3 peptide as the varied substrate.
Note that we focused on these mutants because of their putative
roles as the general base and because the tyrosine residue possesses
an ionizable group that potentially corresponds to the basic limb
of the WT pHꢀrate profile. Also note that we generated profiles
for only the RGG3 peptide because we envisioned that these data
would provide greater insights into the factors that are important
for promoting the transfer of methyl groups to the peptide
substrate. For the H293A and Y39F mutants, the plots of log kcat
versus pH are sigmoidal, with the rates increasing as a function of
pH to a limiting value (Figure 7B). Fitting the data to eq 4
a
arguing against the idea that ionization of these residues in the
RGG3 peptide is responsible for the loss of activity. The fact that
the concentration of the RGG3 peptide is fixed in these experi-
ments further argues against these possibilities because, here,
k /K is the second-order rate constant for the reaction of SAM
cat
m
with the E RGG3 complex, and thus, k /K monitors the
3
cat
m
ionization state of these entities, and not the RGG3 peptide. The
fact that the calculated pK of H293 (7.9) is significantly lower
a
than the pK observed for the descending limb suggests that this
a
residue is also not responsible for the loss of activity at high pH.
Of the two remaining functional groups or residues, i.e., the
amino group on SAM and the Y39 phenol, deprotonation of
either one could be responsible for the loss of activity at high pH,
as the pK values of these groups (i.e., 9.5 and 10.5, respectively)
a
are similar to that obtained for the descending limb.
When the RGG3 peptide is used as the varied substrate, the
plot of log k /K versus pH is also bell-shaped with an ascending
cat
m
limb pK of 5.2 ( 0.2 and a descending limb pK of 10.0 ( 0.3
a
a
(Figure 7A). For the same reasons described above, these pKa
identified pK values of 4.8 ( 0.4 and 5.1 ( 0.8 for the H293A
a
values likely correspond to the protonation states of E153 and
Y39, respectively. Note that the assignment of the descending
limb to the amino group on SAM can be at least partially ruled
out because the concentration of SAM is fixed in these experi-
ments; thus, k /K is the second-order rate constant for the
and Y39F mutants, respectively. Interestingly, these data differ
substantially from those obtained with the WT enzyme, where
changes in pH did not affect kcat. This difference likely reflects a
change in the rate-limiting step. Consistent with this possibility
is the fact that the mutation of either residue decreases kcat by
g11-fold; thus, chemistry is potentially rate-limiting for both the
H293A and Y39F mutants. In total, these data indicate that both
residues play an important role in rate enhancement.
cat
m
reaction of the RGG3 peptide with the E SAM complex, and as
3
such, k /K monitors the ionization state of these entities, and
cat
m
not SAM. Interestingly, and in contrast to the data presented for
SAM, the plot of the log kcat values versus pH is relatively flat,
thereby indicating that when the peptide is the varied substrate
the turnover number is not influenced by pH (Figure 7B). This
With respect to kcat/K
both WT and H293A are similarly bell-shaped, and the pK
obtained with the H293A mutant (i.e., 5.2 ( 1.6 and 10.1 ( 0.9)
, the plots of log kcat/K
versus pH for
m
m
values
a
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Biochemistry
ARTICLE
Table 5. Solvent Isotope Effects (SIEs) and Solvent Viscosity
Effects for WT and PRMT1 Mutants
Inverse SIEs are often attributed to medium effects, the
dissociation of a metal-chelated water, viscosity effects, or effects
30
on thiol ionization. Although medium effects are difficult to
SIE
SVE
SIE
SVE
k
cat
k
cat
kcat/K
m
kcat/K
m
30
exclude, they are typically small and normally ignored. Addi-
WT
1.0
0.8
0.4
1.2
0.1
0.7
0.7
0.4
1.3
1.2
0.9
0.4
0.3
0.6
0.1
0.9
0.6
1.8
1.7
1.6
tionally, the lack of a requirement for metal ions or the presence
of a thiol within the active site of PRMT1 suggests that the
observed inverse SIE is not due to either of these possibilities. To
E144A/E153A
Y39F
control for the effect of the increased viscosity of D O, the kinetic
2
R54A
parameters were determined in the presence of 10% glycerol, a
concentration of glycerol that closely mimics the viscosity of
H293A
D O (Table 5 and Table S3 of the Supporting Information). The
2
are nearly identical to those obtained for the WT enzyme (Figure 7A).
Conversely, only the ascending limb is evident in the Y39F
k /K versus pH plot, which suggests that the phenolic side
results of these solvent viscosity experiments (SVEs) indicate
that, with the exception of the R54A and H293A mutants, the kcat
values for the WT and mutant enzymes were accelerated in the
presence of 10% glycerol, suggesting that for these mutants the
inverse SIE on kcat can be attributed to a viscosity effect. With
respect to k /K , inverse SVEs were detected for only the WT
cat
m
chain of this residue corresponds to the descending limb of the
k /K versus pHꢀrate profile. These results suggest that Y39
cat
m
must be protonated for optimal activity and that deprotonation
of the phenol leads to a decrease in activity either via the loss of a
key hydrogen bond between Y39 and E153 or via electrostatic
repulsion between these two residues that leads to a decrease in
enzyme activity. In contrast, it is interesting to note that the lack
of an effect of mutating H293 on the shape of the pHꢀrate
profiles suggests that the general acid/base properties of this
residue are not important for rate enhancement. As such, these
data lend support to the idea that H293 does not act as the
general base that deprotonates the substrate guanidinium.
Solvent Isotope Effects. To further probe the catalytic mechan-
ism of PRMT1, we also determined the steady state kinetic
cat
m
and the E144A/E153A double mutant, suggesting again that for
these mutants the inverse SIE on k /K can be attributed to a
cat
m
viscosity effect. However, normal SVEs were observed for the
Y39F, R54A, and H293A mutants, indicating that the inverse
SIEs are due to a direct effect of D O on the PRMT1-catalyzed
2
reaction. Although it is difficult to speculate about the molecular
basis for the inverse SIEs, there are at least two possible explanations.
First, given that deuterium atoms are known to form stronger
hydrogen bonds, D O may stabilize the structure of PRMT1 and
2
thereby enhance the rate of the reaction. This may be particularly
true for the H293A mutant, where a very large, and likely un-
precedented, inverse SIE was observed (SIE = 0.1). As men-
tioned previously, the disruption of the salt bridge between H293
and D51, which would be caused by the alanine mutation, likely
prevents the proper formation of the substrate and cofactor
binding pockets. However, when the H293A mutant is assayed in
parameters for the PRMT1-catalyzed reaction in D O, using the
2
AcH4ꢀ21 peptide as the varied substrate. For these experiments,
the rates of the reaction were measured in g92% D O and
2
compared to those obtained in H O at the corresponding pL. For
2
the WT enzyme, a small inverse SIE (0.9) is apparent on k /K
cat
m
when the peptide is the varied substrate (Table 5 and Table S2 of the
Supporting Information). Although this result could be interpreted
as being consistent with general base catalysis being unimportant for
rate enhancement (a normal SIE would be expected if general base
catalysis plays a prominent role in rate enhancement), the fact that
chemistryisunlikelytoberate-limiting for the peptide substrate with
the wild-type enzyme (see above) could suggest that the lack of an
effect is due to the fact that product release is insensitive to the
identity of the solvent. Given this possibility, we also determined
SIEs for the Y39F, R54A, E144A/E153A, and H293A mutants,
because the large decreases in kcat (∼5ꢀ20-fold) suggest that
chemistry is rate-limiting for these enzymes. To help to confirm
this possibility, we examined the processivity of the mutant enzymes
described above. The results of these studies (Figure S2 of the
Supporting Information) indicated that the Y39F, R54A, E144A/
E153A, and H293A mutants do not methylate the AcH4ꢀ21
peptide in a partially processive fashion, further suggesting that
chemistry is rate-limiting for at least a subset of these mutant
enzymes. The change in the kcat versus pHꢀrate profiles observed
for the H293A and Y39F mutants further supports this notion. Note
that for the majority of the mutants examined, relatively small inverse
D O, this solvent stabilizes the structure of PRMT1 and com-
pensates for the loss of the interaction between H293 and D51.
2
As k /K reports on all steps up to and including the first
cat
m
irreversible step of the reaction, which for PRMT1 is likely methyl
transfer, the observed inverse SIEs for the mutant enzymes may,
alternatively, be reporting on the formation of the dication
intermediate because rehybridization of the ω-nitrogen from
2
3
sp to sp would be expected to yield an inverse isotope effect.
Regardless of the nature of the inverse SIE, the lack of a normal
SIE for WT PRMT1, and all of the catalytically impaired mutants,
suggests that general base catalysis is unimportant for the
PRMT1-catalyzed reaction and, more specifically, suggests that
H293 does not act as a general base.
’
CONCLUSIONS
PRMT1 activity impacts a number of important cell signaling
pathways (e.g., gene transcription), and its activity is dysregu-
lated in heart disease and cancer. As such, PRMT1 represents a
novel therapeutic target, and we have been focused on develop-
13ꢀ16
ing inhibitors targeting this isozyme.
To gain insights that
SIE
SIE
SIEs ( kcat and k /K values of ∼0.6ꢀ0.9) were observed on
could guide the design of inhibitors with increased potency and
selectivity, we used a combination of site-directed mutagenesis,
pHꢀrate profiles, and SIEs to begin to characterize the catalytic
mechanism of PRMT1. For the mutagenesis studies, we focused
our efforts on examining the contribution of eight residues lining
the active site pocket of PRMT1, including Y39, R54, E100,
S102, E144, E153, M155, and H293, which, on the basis of
structures of PRMT family members, have been hypothesized to
cat
m
both kcat and k /K (Table 5). The two exceptions are the R54A
cat
m
SIE
and H293A mutants. For the R54A mutant, the kcat is small and
normal ( kcat = 1.2), whereas for the H293A mutant, the SIE on
SIE
SIE
SIE
both kcat and k /K is large and inverse ( kcat and k /K
cat
m
cat
m
values of 0.1). Note that these experiments were performed in
parallel and that representative data from one of at least two
independent experiments are reported in Table 5.
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ARTICLE
be important for SAM binding (i.e., Y39, R54, and E100), the
regiospecific generation of ADMA (i.e., M155), the regulation of
CARM1 (i.e., S102 and Y39), general base catalysis (i.e., H293),
and modulating the nucleophilicity of the substrate guanidinium
support such a hypothesis. This is the case because effects are
observed on both the kinetic parameters determined for SAM
and the peptide substrate. Additionally, the pHꢀrate profiles
obtained for the H293A mutant are similar to those obtained for
the WT enzyme, which indicates that the ionization of this
residue does not contribute to either the rate-limiting step of the
reaction or substrate capture, which should encompass the
methyl transfer step. Although the contribution of this residue
to rate enhancement may not be apparent in the pHꢀrate
profiles, because this residue is unimportant for substrate capture
or the rate-limiting step of the reaction, we deem this possibility
unlikely and suggest that the decreased activity observed when
this residue is mutated is due to the loss of a critical salt bridge
between this residue and D51. The loss of this salt bridge would
be expected to destabilize the two N-terminal helices and impact
cofactor and peptide binding, both of which occur when this
residue is mutated. Consistent with this notion is the fact that the
Ki for SAH is increased by 11-fold. The notion that H293 is not a
general base is consistent with the fact that this residue is g6 Å
distal from the approximate site of the substrate guanidinium, a
distance that is too great for this residue to play such a role.
In total, the data described above support a mechanism in which
SAM and a protein, or peptide, substrate bind to the enzyme in a
random fashion to form a ternary complex (Figure 2B). E153 then
likely redistributes the electron density toward either N or N , or
(i.e., E144 and E153).
The results of the mutagenesis studies indicate that while R54
and E100 form hydrogen bonds and electrostatic interactions
with the SAM carboxylate and the ribose moiety, respectively,
neither residue is important for SAM binding. However, the fact
that the R54 mutations negatively impact the kinetic parameters
obtained for the peptide substrate helps to confirm that the
observed hydrogen bond or electrostatic interaction between the
R54 guanidinium and the E144 carboxylate is important for rate
enhancement; this interaction likely orients E144 such that it can
properly position Nη2 for nucleophilic attack on the S-methyl
group of SAM. In contrast to R54 and E100, M155 is important
for SAM binding, as evidenced by the 26-fold increase in the Ki
for SAH when this residue is mutated to alanine. The k /K
cat
m
obtained for SAM is similarly affected. As M155 forms the
bottom of the adenine portion of the SAM binding pocket, the
loss of hydrophobic interactions between M155 and the adenine
ring, which occurs in the M155L and M155A mutants, likely
results in both a loss of affinity and an inability to properly position
the cofactor for methyl transfer. M155 is also important for the
processivity but not the regiospecificity of the PRMT1-catalyzed
reaction; i.e., this residue does not direct the formation of ADMA
over SDMA, which has been suggested previously.
η
δ
1
even both, which enhances the nucleophilicity of N . The methyl
η
2
group of SAM is then transferred to the protonated guanidinium of
the substrate arginine to form a dication intermediate. Although
such an intermediate is to the best of our knowledge unprecedented,
dianionic carboxylate intermediates have been proposed for several
enzymes. Rehybridization of the guanidinium destabilizes the dica-
tion intermediate, thereby facilitating the loss of the extra proton to
water or an unknown general base. While E144 could serve such a
role, this seems unlikely when one considers that the effect of mutat-
ing this residue to glutamine has an only small impact on k /K .
E144 and E153 have previously been suggested to orient the
substrate guanidinium and modulate its nucleophilicity to pro-
18
mote methyl transfer. Consistent with previous mutagenesis
9
studies that measured only relative rates, our results indicate that
both residues are important for PRMT1 catalysis. Interestingly,
however, the charge and position of E144 appear to be relatively
unimportant for rate enhancement as both the E144D and E144Q
mutants retain considerable activity. These results suggest that
the hydrogen bond between E144 and the substrate guanidinium
cat
m
Further support for the notion that methyl transfer precedes
proton transfer comes from the lack of a normal SIE on both the
wild-type enzyme and several catalytically impaired mutants for
which chemistry is most likely rate-limiting; a normal SIE would be
expected if proton abstraction was rate-limiting. Given that Hed-
is most important, and that this interaction likely orients N for
η
2
nucleophilic attack on the S-methyl group of SAM. In contrast,
both the charge and position of E153 are important for rate en-
hancement, and the results are consistent with the previous sugges-
tion that this residue modulates the nucleophilicity of the guanidi-
31
strom and colleagues have noted that arginine residues can act as
nium group by redistributing electron density toward N and N .
η
δ
general bases, and that the pK of those residues is potentially
1
a
With respect to Y39, in addition to being important for SAM
binding (because of its position at the top of the SAM binding
pocket), this residue appears to be important for rate enhance-
modulated by slight structural perturbations to the normally planar
guanidinium, we cannot completely rule out the possibility that
E144 and E153 depress the pK of the substrate arginine and
a
ment. This is apparent from the 20-fold decrease in k /K when
cat
m
thereby enhance its nucleophilicity, via such a mechanism. However,
it is difficult to rationalize such a mechanism with our findings that
the E144A/E153A double mutant possesses considerably more
activity than either of the single mutations alone. Thus, we favor the
mechanism proposed above in which the methyl group is trans-
ferred to the protonated guanidinium. In summary, our results
suggest that the PRMT1-catalyzed reaction is primarily driven by
bringing the substrate and cofactor into the proximity of each other
and that the prior deprotontation of the substrate guanidinium is not
required for methyl transfer.
the peptide is the varied substrate. This result suggests that the
phenolic hydroxyl group enhances the rate of catalysis. On the
basis of structures of PRMT1 family members, this residue likely
forms a hydrogen bond with E153, and this interaction is im-
portant for positioning the E153 carboxylate such that it can
modulate the nucleophilicity of the substrate guanidinium. This
hypothesis is supported by the fact that PRMT1 loses activity at
high pH, where the deprotonated form of Y39 would be expected
to predominate and the resultant electrostatic repulsions be-
tween this residue and the E153 carboxylate would lead to a loss
of activity. This latter observation is further supported by the loss
of the high pK when this residue is mutated to phenylalanine.
a
’ ASSOCIATED CONTENT
The results obtained for the H293A mutant are particularly
interesting. Although this residue has been suggested to act as
general base to deprotonate the substrate guanidinium and thereby
enhance the nucleophilicity of this group, our results do not
S
Supporting Information. Supplementary Tables S1ꢀS3
b
and Figures S1 and S2. This material is available free of charge via
the Internet at http://pubs.acs.org.
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ARTICLE
’
AUTHOR INFORMATION
(13) Osborne, T. C., Obianyo, O., Zhang, X., Cheng, X., andThompson,
P. R. (2007) Protein arginine methyltransferase 1: Positively charged
residues in substrate peptides distal to the site of methylation are important
for substrate binding and catalysis. Biochemistry 46, 13370–13381.
(14) Osborne, T., Roska, R. L., Rajski, S. R., and Thompson, P. R.
(2008) In situ generation of a bisubstrate analogue for protein arginine
methyltransferase 1. J. Am. Chem. Soc. 130, 4574–4575.
Corresponding Author
*
Department of Chemistry, The Scripps Research Institute, 130
Scripps Way, Jupiter, FL 33458. Telephone: (561) 228-2860.
Fax: (561) 228-2918. E-mail: Pthompso@scripps.edu.
Funding Sources
This work was supported in part by the University of South Carolina
Research Foundation and TSRI Scripps Florida (P.R.T.).
(15) Obianyo, O., Causey, C. P., Osborne, T. C., Jones, J. E., Lee, Y. H.,
Stallcup, M. R., and Thompson, P. R. (2010) A chloroacetamidine-based
inactivator of protein arginine methyltransferase 1: Design, synthesis, and in
vitro and in vivo evaluation. ChemBioChem 11, 1219–1223.
(16) Bicker, K. L., Obianyo, O., Rust, H. L., and Thompson, P. R.
(
2011) A combinatorial approach to characterize the substrate specifi-
’
ACKNOWLEDGMENT
city of protein arginine methyltransferase 1. Mol. BioSyst. 7, 48–51.
(17) Obianyo, O., Osborne, T. C., and Thompson, P. R. (2008)
Kinetic mechanism of protein arginine methyltransferase 1. Biochemistry
We thank Raman Parkesh for help with the pK calculations.
a
4
7, 10420–10427.
’
ABBREVIATIONS
(18) Zhang, X., Zhou, L., and Cheng, X. (2000) Crystal structure of
PRMT, protein arginine methyltransferase; SAM, S-adenosyl-
methionine; SAH, S-adenosylhomocysteine; ω-MMA, monomethy-
larginine; ADMA, asymmetrically dimethylated arginine; SDMA,
symmetrically dimethylated arginine; NOS, nitric oxide synthase;
the conserved core of protein arginine methyltransferase PRMT3.
EMBO J. 19, 3509–3519.
(19) Yue, W. W., Hassler, M., Roe, S. M., Thompson-Vale, V., andPearl,
L. H. (2007) Insights into histone code syntax from structural and
biochemical studies of CARM1 methyltransferase. EMBO J. 26, 4402–4412.
NO, nitric oxide; ERR, estrogen receptor R;E , estrogen; FAK, focal
2
(20) Troffer-Charlier, N., Cura, V., Hassenboehler, P., Moras, D., and
adhesion kinase; PI3K, phosphoinositide 3-kinase; NR, nuclear re-
ceptor; AR, androgen receptor; TRIS, tris(hydroxymethylamino-
methane); HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; DTT, dithiothreitol; DMF, dimethylformamide; EDTA, (eth-
ylenedinitrilo)tetraacetic acid; TFA, trifluoroacetic acid; BSA, bovine
serum albumin; PDB, Protein Data Bank.
Cavarelli, J. (2007) Functional insights from structures of coactivator-
associated arginine methyltransferase 1 domains. EMBO J. 26, 4391–4401.
(21) Feng, Q., He, B., Jung, S. Y., Song, Y., Qin, J., Tsai, S. Y., Tsai,
M. J., and O'Malley, B. W. (2009) Biochemical control of CARM1
enzymatic activity by phosphorylation. J. Biol. Chem. 284, 36167–36174.
(22) Branscombe, T. L., Frankel, A., Lee, J. H., Cook, J. R., Yang, Z.,
Pestka, S., and Clarke, S. (2001) PRMT5 (Janus kinase-binding protein
1
) catalyzes the formation of symmetric dimethylarginine residues in
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