Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3

Article abstract of DOI:10.1021/bi100363t

Protein citrullination has been shown to regulate numerous physiological pathways (e.g., the innate immune response and gene transcription) and is, when dysregulated, known to be associated with numerous human diseases, including cancer, rheumatoid arthri

Full text of DOI:10.1021/bi100363t

4852 Biochemistry 2010, 49, 4852–4863
DOI: 10.1021/bi100363t
Substrate Specificity and Kinetic Studies of PADs 1, 3, and 4 Identify Potent and Selective
Inhibitors of Protein Arginine Deiminase 3†
Bryan Knuckley,‡ Corey P. Causey,‡ Justin E. Jones,‡ Monica Bhatia,‡ Christina J. Dreyton,‡ Tanesha C. Osborne,‡
Hidenari Takahara,§ and Paul R. Thompson*,‡
‡Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, and
§Department of Applied Bioresource Sciences, School of Agriculture, Ibaraki University, 21-1 Chuou 3, Ami, Inashiki,
Ibaraki 3000393, Japan
Received March 9, 2010; Revised Manuscript Received May 13, 2010
ABSTRACT: Protein citrullination has been shown to regulate numerous physiological pathways (e.g., the
innate immune response and gene transcription) and is, when dysregulated, known to be associated with
numerous human diseases, including cancer, rheumatoid arthritis, and multiple sclerosis. This modification,
also termed deimination, is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). In
mammals, there are five PAD family members (i.e., PADs 1, 2, 3, 4, and 6) that exhibit tissue-specific
expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was
recently reported, and these efforts guided the development of the two most potent PAD4 inhibitors (i.e.,
F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors, we show here that Cl-amidine
also exhibits a strong inhibitory effect against PADs 1 and 3, thus indicating its utility as a pan PAD inhibitor.
Given the increasing number of diseases in which dysregulated PAD activity has been implicated, the
development of PAD-selective inhibitors is of paramount importance. To aid that goal, we characterized the
catalytic mechanism and substrate specificity of PADs 1 and 3. Herein, we report the results of these studies,
which suggest that, like PAD4, PADs 1 and 3 employ a reverse protonation mechanism. Additionally, the
substrate specificity studies provided critical information that aided the identification of PAD3-selective
inhibitors. These compounds, denoted F4- and Cl4-amidine, are the most potent PAD3 inhibitors ever
described.
Aberrantly increased protein arginine deiminase (PAD)1 activity
has been associated with numerous human diseases, including
rheumatoid arthritis (1), multiple sclerosis (2), Alzheimer’s dis-
ease (3), ulcerative colitis (4), and numerous cancers (5, 6), thereby
suggesting these enzymes as novel therapeutic targets (7). Five
PAD isozymes (i.e., PADs 1, 2, 3, 4, and 6) exist in humans, and
these enzymes catalyze the posttranslational conversion of pepti-
dylarginine to peptidylcitrulline in numerous protein substrates,
e.g., histones H2A, H3, and H4, fibrinogen, keratin, and tricho-
hyalin (Figure 1A). While there is significant evidence to suggest
that PAD4 plays a causative role in the aforementioned diseases,
there is also emerging evidence suggesting that other PADs, in
particular PAD2, may also play a role (8). Significant questions
regarding the contributions of PADs 1, 3, and 6 to human disease
also remain unanswered; thus a greater understanding of the
physiological and pathophysiological roles of these enzymes is
necessary in order to define the individual contributions of these
enzymes to human disease and normal human physiology.
In humans, the genes encoding the five PAD isozymes are all
clustered on chromosome 1 (1p35-36), and this genomic orga-
nization is conserved among mammals. Each isozyme is also
highly conserved at the amino acid sequence level (70-95%
homology retained in mammals), with PADs 1 and 3 being the
most closely related isozymes (68% AA sequence identity) (9, 10).
Deiminated proteins have been identified throughout mamma-
lian tissues, and the resulting reduction in net positive charge can
potentially have dramatic effects on protein folding and protein-
protein interactions (11, 12).
To date, PAD4 has been the focus of many biochemical studies
(1, 10, 13-15), thus making it the best characterized PAD. For
example, this isozyme has been shown to regulate chromatin
structure and function through its ability to deiminate histones
H2A, H3, and H4, as well as p300 (12, 14-18). Putative roles for
PAD4 in apoptosis, and in the formation of neutrophil extra-
cellular traps (NETs), have also been described (19-24). The
prominent role of PAD4 in transcriptional regulation, as well as
its role in RA, prompted our efforts to design and synthesize
two highly potent mechanism-based inhibitors that are denoted
F- and Cl-amidine (Figure 1B) (7, 19, 25). These compounds
irreversibly inactivated PAD4 by covalently modifying an active
site cysteine (Cys645) that is critical for catalysis. Inactivation likely
proceeds via an initial attack of the Cys645 thiolate on the iminium
carbon of the haloacetamidine warhead, resulting in the formation
of a tetrahedral intermediate. His471 then likely donates a proton
to stabilize this intermediate, thereby promoting halide displace-
ment by the sulfur atom. The resultant three-membered sulfonium
†This work was supported in part by funds from the University of
South Carolina (P.R.T.) and by National Institutes of Health Grant
GM079357 to P.R.T.
*To whom correspondence should be addressed at the Department of
Chemistry, The Scripps Research Institute, Scripps Florida, 130 Scripps
Way, Jupiter, FL 33458. Tel: (561) 228-2860. Fax: (561) 228-3050.
E-mail: Pthompso@scripps.edu.
1Abbreviations: PAD, protein arginine deiminase; Cit, citrulline; RA,
rheumatoid arthritis; BAEE, benzoyl-
L
-arginine ethyl ester; BAA,
-arginine methyl ester;
benzoyl- -arginine amide; BAME, benzoyl-
L
L
DTT, dithiothreitol; TCEP, tris(2-carboxyethyl)phosphine; EDTA,
ethylenediaminetetraacetic acid.
r
pubs.acs.org/Biochemistry
Published on Web 05/14/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 23, 2010 4853
FIGURE 1: (A) PADs catalyze the posttranslational conversion of peptidylarginine to peptidylcitrulline. (B) Structures of PAD inhibitors
including F- and Cl-amidine, F4- and Cl4-amidine, and F- and Cl-ethylamidine. (C) Proposed mechanism of PAD inactivation.
ring then collapses to form a thioether adduct, rendering the
enzyme inactive (Figure 1C) (19, 25, 26).
hair follicle differentiation. Additionally, their potential roles in these
processes suggest that inhibition of these enzymes could lead to a
number of undesired side effects. Therefore, in an effort to guide the
design of isozyme-specific PAD inhibitors, we initiated studies to
both characterize the catalytic mechanism of PADs 1 and 3 and
identify important substrate recognition elements. Herein, we de-
scribe the results of these studies. Specifically, we demonstrate that
Cl-amidine is a pan PAD inhibitor. Additionally, the results of pH
profiles and pKa measurements on the active site Cys demonstrate
that PADs 1 and 3, like PAD4, utilize a reverse protonation mecha-
nism. Furthermore, the substrate specificities of PADs 1, 3, and 4
were probed by determining steady-state kinetic parameters for both
small synthetic substrates and various peptides. The results of these
studies identified isozyme-specific differences that led to the dis-
covery of the PAD3-specific inhibitors F4- and Cl4-amidine; these
compounds are the most potent PAD3 inhibitors described to date.
Unlike PAD4, little is known about the physiological roles of the
remaining PADs, although it has been speculated that PADs 1, 2,
and 3 play an important role in skin homeostasis and moisturiza-
tion. Consistent with this hypothesis is the fact that PAD1 is
expressed throughout the epidermis and that PADs 2 and 3 are
expressed in the spinous and granular layer, respectively (27, 28). In
addition to these roles, PAD3 is also believed to play an important
role in hair follicle formation due to its ability to deiminate
trichohyalin, an abundant, arginine-rich (22.7%), structural pro-
tein present in the inner root sheath and medulla of hair follicles
(28, 29). Given the presumed roles of PAD activity in normal skin
moisturization, dysregulation of these enzymes has been suggested
to play a causative role in psoriasis, an autoimmune disorder that
causes red, scaly patches to form on the skin (27, 29, 30). The fact
that vitamin D derivatives are known to induce the expression of
PADs 1, 2, and 3 in keratinocytes, and are used to treat this disease
(29, 31), suggests that decreased PAD activity is associated with
psoriasis.
MATERIALS AND METHODS
Chemicals. Dithiothreitol (DTT), N-R-benzoyl
L-arginine
ethyl ester (BAEE), citrulline, β-mercaptoethanol, Triton X-100,
imidazole, protease inhibitor cocktail (catalog no. P8465), and
tris(2-carboxyethyl)phosphine (TCEP) were acquired from Sigma
These and other studies demonstrate the importance of under-
standing the roles that PADs 1 and 3 play in skin moisturization and

4854 Biochemistry, Vol. 49, No. 23, 2010
Knuckley et al.
hygroscopic solid (162 mg, 94%). 1H NMR (400 MHz, D2O) δ
(ppm): 7.51 (d, J=8 Hz, 2H), 7.37-7.22 (m, 3H), 4.27 (dd, J=2, 8
Hz, 1H), 2.97 (q, J=7.2 Hz, 2H), 2.78 (t, J=7.6 Hz, 2H), 1.75-
1.45 (m, 4H), 0.84 (t, J=7.2 Hz, 3H). 13C NMR (100 MHz, D2O)
δ (ppm): 172.90, 170.77, 132.58, 132.30, 128.56, 127.08, 53.99,
38.69, 34.35, 27.85, 23.28, 13.32. HRMS (C14H22N3O2þ): calcd
264.1712, observed 264.1711.
Table 1: Histone Peptide Derivatives
peptide
sequence
AcH4-21
AcH4-18
AcH4-16
AcH4-15
1-Ac-SGRGKGGKGLGKGGAKRHRKV
1-Ac-SGRGKGGKGLGKGGAKRH
1-Ac-SGRGKGGKGLGKGGAK
1-Ac-SGRGKGGKGLGKGGA
1-Ac-SGRGKGGKGLGKG
AcH4-13
Synthesis of Cl-ethylamidine. N-R-Benzoyl-L-ornithine ethyl
AcH4-5
1-Ac-SGRGK
amide (38 mg, 0.1 mmol) and ethyl chloroacetimidate (HCl) (28.2
mg, 0.2 mmol) were dissolved in dry methanol (3 mL). Cs2CO3
(48.8 mg, 0.15 mmol) was added, and the reaction was stirred
at room temperature. After 16 h, the reaction was quenched
with TFA and purified by RP-HPLC to yield the product as a
AcH4-5 S1A
AcH4-5 G2A
AcH4-5 G4A
AcH4-5 K5A
AcH4-21 N(1)
AcH4-21 N(2)
AcH4-21 K16A
AcH4-21 K20A
AcH4-21 R3A
AcH4-21 R17A
AcH4-21 R19A
AcH4-21 R17,19K
AcH4-21 all Ac-K
1-Ac-AGRGK
1-Ac-SARGK
1-Ac-SGRAK
1-Ac-SGRGA
2-Ac-GRGKGGKGLGKGGAK
3-Ac-RGKGGKGLGKGGAK
1-Ac-SGRGKGGKGLGKGGAARHRKV
1-Ac-SGRGKGGKGLGKGGAKRHRAV
1-Ac-SGAGKGGKGLGKGGAKRHRAV
1-Ac-SGRGKGGKGLGKGGAKAHRAV
1-Ac-SGRGKGGKGLGKGGAKRHAAV
1-Ac-SGRGKGGKGLGKGGAKKHKAV
1-Ac-SGRGKAcGGKAcGLGKAcGGAKAcRHRKAc
1
white powder. H NMR (400 MHz, D2O) δ (ppm): 7.60-7.57
(m, 2H), 7.45-7.39 (m, 1H), 7.35-7.29 (m, 2H), 4.22 (dd, J = 2,
8 Hz, 1H), 4.19 (s, 2H), 3.18 (t, J = 6.8 Hz, 2H), 3.03 (q, J =
7.2 Hz, 2H), 1.82-1.45 (m, 4H), 0.90 (t, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, D2O) δ (ppm): 173.07, 170.92, 162.68, 132.68,
132.36, 128.63, 127.13, 54.12, 41.82, 38.94, 34.38, 28.08, 23.03,
13.40. HRMS (C16H24ClN4O2þ): calcd 339.1588, observed
339.1593.
V
Aldrich (St. Louis, MO). N-R-Benzoyl-
(BAME) and N-G-methyl- -arginine acetate (MMA) were ob-
tained from MP Biomedicals (Irvine, CA). N-R-Benzoyl- -argi-
nine (BA) and N-R-benzoyl- -arginine amide (BAA) were obtained
L-arginine methyl ester
Synthesis of F-ethylamidine. N-R-Benzoyl-L-ornithine ethyl
L
amide (30 mg, 0.08 mmol) and ethyl fluoroacetimidate (HCl) (25
mg, 0.16 mmol) were dissolved in dry methanol (3 mL). Cs2CO3
(39 mg, 0.12 mmol) was added, and the reaction was stirred at
room temperature. After 16 h, the reaction was quenched with
TFA and purified by RP-HPLC to yield the product as white
powder. 1H NMR (400 MHz, D2O) δ (ppm): 7.57-7.55 (m, 2H),
7.44-7.38 (m, 1H), 7.33-7.27 (m, 2H), 5.05 (d, JH-F = 44.8 Hz,
2H), 4.21 (dd, J = 2.4, 8 Hz, 1H), 3.18 (t, J = 6.8 Hz, 2H), 3.01
(q, J = 7.2 Hz, 2H), 1.79-1.48 (m, 4H), 0.88 (t, J = 7.2 Hz, 3H).
13C NMR (100 MHz, D2O) δ (ppm): 173.10, 170.92, 162.47 (d,
L
L
from Acros (Hampton, NH). The synthesis of a subset of the
peptides listed in Table 1 has been previously described (32). The
AcH4-21 R3A, AcH4-21 R17A, and AcH4-21 R19A peptides
were synthesized on the solid phase using the Fmoc strategy and
purified by reverse-phase liquid chromatography. The synthesis of
F- and Cl-amidine, as well as F4- and Cl4-amidine, has previously
been described (19, 25, 33).
Synthesis of N-R-Benzoyl-N5-(tert-butoxycarbonyl)-L-or-
JC-F=19.9 Hz), 132.66, 132.34, 128.61, 127.11, 77.43 (d, JC-F
=
nithine Ethyl Amide. N-R-Benzoyl-N5-(tert-butoxycarbonyl)-
L-
178.29 Hz), 54.13, 41.29, 34.37, 27.83, 23.17, 13.37. HRMS
(C16H24FN4O2þ): calcd 323.1883, observed 323.1884.
ornithine (200 mg, 0.59 mmol), S-(1-oxido-2-pyridyl)-N,N,N0,N0-
tetramethylthiouronium hexafluorophosphate (HOTT) (330 mg,
0.89 mmol), and triethylamine (0.17 mL, 1.2 mmol) were dissolved
in dry DMF (2 mL) and allowed to stir at room temperature. After
10 min, ethylamine (0.6 mL of a 2 M solution in THF, 1.2 mmol)
was added, and stirring was allowed to continue at room tempera-
ture. After 45 min, the reaction was partitioned between EtOAc (15
mL) and brine (30 mL). The organics were separated, and the
aqueous layer was extracted twice more with EtOAc. The organics
were combined, washed with 2 M HCl (2 ꢀ 15 mL), H2O (2 ꢀ 15
mL), saturated NaHCO3 (2 ꢀ 15 mL), H2O (3 ꢀ 15 mL), and brine
(15 mL), dried over MgSO4, and concentrated under vacuum to
yield the product as a white powder (167 mg, 78%). 1H NMR (400
MHz, CDCl3) δ (ppm): 7.76 (d, J=7.2 Hz, 2H), 7.42-7.3 (m, 4 H),
7.05 (br, 1H), 4.86 (t, J=6 Hz, 1H), 4.8-4.73 (m, 1H), 3.33-3.14
(m, 3H), 2.98-3.08 (m, 1H), 1.8-1.76 (m, 1H), 1.76-1.65 (m, 1H),
1.56-1.47 (m, 2H), 1.35 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
(ppm): 171.84, 167.48, 156.58, 133.84, 131.70, 128.49, 127.19, 79.18,
52.33, 39.29, 34.40, 30.38, 28.40, 26.53, 14.66. HRMS (C19H30-
N3O4þ): calcd 364.2236, observed 364.2240.
Cloning, Expression, and Purification of PADs 1 and 3.
For protein expression, the genes encoding PADs 1 and 3 were
cloned from the original pGEX-6P1 vector (34) into the pET16B
vector, which contains a 6ꢀ-His tag followed by a factor Xa
cleavage site. PAD1 was cloned using the following primers:
forward 50-AAAAAACATATGGCCCCAAAGAGAG-30 and
reverse 50-AAAAAACTCGAGGGGCACCATGTTC-30. PAD3
was cloned using the following primers: forward 50-AAAAAA-
CATATGTCGCTGCAGAGAATC-30 and reverse 50-AAAAAA-
CTCGAGGGGCACCATGTTC-30. The forward primers contain
an NdeI restriction site (underlined) and either 13 base pairs or 15
base pairs that correspond to the 50-coding region of the PAD 1 and
3 genes, respectively. The reverse primers contain an XhoI restriction
site (underlined) followed by 13 base pairs that correspond to the 30-
coding region of the PAD 1 and 3 genes. The resulting pET16b-
PAD1 and pET16b-PAD3 constructs were sequenced to ensure that
no mutations were incorporated during PCR amplification. PADs
1 and 3 were purified analogously to previously described meth-
ods (35). Although the His-tagged proteins were recovered in
modest yield (1.0-1.5 mg/L), this procedure, in one step, afforded
PADs 1 and 3 in greater than 95% purity (Supporting Information
Figure S1). A detailed description of the purification protocol can
be found in the Supporting Information.
Synthesis of N-R-Benzoyl-L-ornithine Ethyl Amide. Cold
TFA (10 mL) was added to N-R-benzoyl-N5-(tert-butoxycar-
bonyl)-L-ornithine ethyl amide (166 mg, 0.46 mmol), and the
reaction was stirred at 0 °C. After 30 min, the reaction was
allowed to warm to room temperature and stir for an additional
30 min. The TFA was removed under a stream of nitrogen. The
remaining residue was dissolved in H2O (20 mL), extracted with
ether (2 ꢀ 5 mL), and lyophilized to yield the product as a clear
Citrulline Production Assay. After a 10 min preincubation
period at 37 °C, either PAD 1 or 3 was added to assay buffer (60
μL total volume; 10 mM CaCl2, 50 mM NaCl, 100 mM Tris-HCl,
pH 7.6, 2 mM DTT) plus 10 mM BAEE to initiate the reaction.

Article
Biochemistry, Vol. 49, No. 23, 2010 4855
Following the addition of enzyme, the reaction was allowed to
proceed for 10 min and then flash frozen in liquid nitrogen.
Citrulline production was then quantified using previously
established methods (32, 36, 37). PAD activity was linear with
respect to time and enzyme concentration.
either iodoacetamide or 2-chloroacetamidine (dissolved in 50
mM buffer) to initiate the reaction (60 μL final volume). At
various time points (0-30 min), an aliquot (6 μL) was removed
and added to assay buffer, which was preincubated for 10 min at
37 °C to measure residual PAD1 activity (60 μL total volume).
Reactions were allowed to proceed for 15 min before being flash
frozen in liquid nitrogen. Citrulline production was measured
according to the methodology described above, and the residual
activity data, thus obtained, were fit to eq 4
Ammonia Production Assay. The amount of ammonia
produced as a function of time was determined by preincubating
assay buffer containing 10 mM BAEE at 37 °C for 10 min before
adding 0.2 μM enzyme (PAD 1 or 3) to start the reaction. At
specific time points (0, 2, 4, 6, 10, 15 min), 60 μL of the reaction
was removed and quenched by flash freezing. In order to quantify
ammonia production, 180 μL of 50 mM EDTA was added to the
quenched reaction, and the method of Sugawara and Oyama was
used to measure the amount of ammonia produced (38).
Calcium Dependence. Varying concentrations of calcium
(0-10 mM) were incubated in 50 mM NaCl, 2 mM DTT, 10 mM
BAEE, and 100 mM Tris-HCl, pH 7.6. Reactions were preincu-
bated at 37 °C for 10 min before the addition of 0.2 μM enzyme
(PAD 1 or 3). The reactions were allowed to proceed for 10 min
and then flash frozen in liquid nitrogen. Citrulline production
was determined as described above, and the data were fit to eq 1
v ¼ v0e- kt
ð4Þ
using Grafit 5.0.1.1 (39). v is the velocity, v0 is the initial velocity, k
is the pseudo-first-order rate constant for inactivation, and t is
time. Due to a lack of inactivator saturation, second-order rate
constants for enzyme inactivation, kinact/KI, were determined by
plotting the observed inactivation rates (kobs) versus inactivator
concentration and fitting the data to eq 5
kobs ¼ ðkinact=KIÞ½Iꢁ
ð5Þ
using Grafit 5.0.1.1 (39). kinact is the maximal rate of inactivation,
KI is the concentration of inactivator that yields half-maximal
inactivation, and [I] is the concentration of inactivator. The
slopes thus obtained, i.e., kinact/KI, were plotted versus pH and
subsequently fit to eq 6
n
n
v=Vmax ¼ ½Ca2þꢁ =ðKD þ ½Ca2þꢁ Þ
ð1Þ
using GraFit version 5.0.11 (39). KD is the dissociation constant
and n is the Hill coefficient. Note that for these assays PADs 1
and 3 were dialyzed into EDTA-free long-term storage buffer (20
mM Tris-HCl, pH 8.0, 2 mM DTT or 250 μM TCEP, 500 mM
NaCl, and 10% glycerol).
pH - pKa
y ¼ ððymin þ ymaxÞ ꢀ 10pH - pK Þ=ð10
þ 1Þ
ð6Þ
a
using GraFit version 5.0.11 (39). Ymin is the minimum rate, and
ymax is the maximum rate.
Substrate Specificity Studies. The steady-state kinetic
parameters were determined for peptide substrates and synthetic
arginine substrates using the methodology described above.
Briefly, assay buffer containing various concentrations of sub-
strate was preincubated at 37 °C for 10 min prior to the addition
of either PAD 1, 3, or 4 (0.2 μM). The reaction was allowed to
proceed for 15 min (peptides) or 10 min (arginine derivatives)
before freezing in liquid nitrogen. Citrulline formation was
measured using the methodology described above. Peptide sub-
strates were dissolved in 50 mM HEPES, pH 8.0. The initial rates
obtained from these experiments were fit to eq 2
IC50 Values. IC50 values were measured as previously
described for PAD4 (19, 33). The data were fit to eq 7
fractional activity of PAD ¼ 1=ð1 þ ½Iꢁ=IC50Þ
using Grafit 5.0.1.1 (39).
ð7Þ
RESULTS AND DISCUSSION
PADs 1 and 3 Produce Ammonia and Citrulline in Equi-
molar Amounts. Previous studies have established that PAD4
uses a hydrolytic mechanism to produce ammonia and peptidyl-
citrulline in equimolar amounts (40). To confirm that citrulline
and ammonia are also the major products of the PAD 1 and 3
catalyzed reactions, experiments to measure the formation of
these products were undertaken. As described previously (32),
these experiments were performed because the central thiour-
onium intermediate can be hydrolyzed to form one of several
different products, including citrulline plus ammonia or urea and
ornithine. The results of these experiments demonstrated that,
like PAD4, PADs 1 and 3 produce equimolar amounts of citrulline
and ammonia (Supporting Information Figure S2). Thus, PADs 1
and 3 are bona fide PADs.
Calcium Dependence. PADs are known to be calcium-
dependent enzymes; therefore, the concentration dependence of
calcium activation for PADs 1 and 3 was determined using BAEE
as the substrate. The sigmoidal nature of the curve indicates that
calcium activates these enzymes in a cooperative fashion. For
both enzymes, the concentration of calcium required for half-
maximal activity, that is, the K0.5, is in the mid to high micro-
molar range (Table 2). The fact that the Hill coefficients are
greater than 1 for both enzymes at the pH optimum indicates that
multiple calcium ions are required to efficiently activate these
enzymes. These data are consistent with previously reported
v ¼ Vmax½Sꢁ=ðKm þ ½SꢁÞ
using Grafit 5.0.1.1 (39).
ð2Þ
pH Profiles. pH profiles for PADs 1 and 3 were generated by
measuring the steady-state kinetic parameters for the deimination
of BAEE over the pH range 6.0-9.5. Stock concentrations of
BAEE were prepared in 50 mM buffer at the desired pH. Reac-
tion mixtures containing 50 mM NaCl, 2 mM DTT, 100 mM
buffer (Bis-tris, pH 6.5-7, or Tris-HCl, pH 7-9.5), 10 mM CaCl2,
and BAEE at various concentrations (0-10 mM in a final volume
of 60 μL) were preincubated for 10 min prior to the addition of
either PAD 1 or 3. The initial rates obtained from these experiments
were fit to eq 2 using GraFit version 5.0.11 (39). The kcat and kcat
/
Km values obtained from this analysis were plotted as a function of
pH and fit to eq 3
a1 - pH
log½yobsꢁ ¼ logðymax=ð1 þ 10pK
þ 10pH - pK ÞÞ
ð3Þ
a2
using GraFit version 5.0.11 (39). ymax is the amount of activity at
the pH optimum.
Inactivation Studies. Inactivation reactions containing 10
mM CaCl2 and 100 mM of buffer (pH 6-8.5 Tris-HCl) were
incubated with 2.0 μM PAD1 at 37 °C for 10 min before adding

4856 Biochemistry, Vol. 49, No. 23, 2010
Knuckley et al.
Table 2: Calcium Dependence of PADs 1 and 3 with BAEEa
Table 3: Steady-State Kinetic Parameters for Benzoylated Arg Substratesa
pH
K0.5 (μM)b
nc
substrate
PAD1
PAD3
PAD4c
PAD1
PAD3
6.0
7.6
9.0
6.0
7.6
8.5
550 ( 40
140 ( 90
400 ( 50
950 ( 110
550 ( 80
1500 ( 1100
5.0 ( 3.6
1.4 ( 0.6
3.2 ( 1.1
3.7 ( 1.1
2.2 ( 0.8
1.0 ( 0.4
Km (mM)
BAA
0.16 ( 0.05
0.19 ( 0.07
13.9 ( 1.8 0.25 ( 0.06
NDb
1.36 ( 0.19
10.8 ( 2.7 1.66 ( 0.26
1.85 ( 0.15 2.76 ( 0.16
BAEE
BAME 0.37 ( 0.07
BAA
k
cat (s-1
)
3.57 ( 0.18
0.27 ( 0.02
BAEE
ND
5.94 ( 0.26
BAME 3.85 ( 0.16
BAA
1.26 ( 0.19 5.57 ( 0.28
kcat/Km (s-1 M-1
)
22000 ( 3600 130 ( 80 11000 ( 2700
1500 ( 300
aValues were determined in duplicate by incubating the enzyme with
b
BAEE
25 ( 6.0
4400 ( 1400
3300 ( 1100
BAEE at 37 °C. K0.5 is the concentration of calcium that yields half-
maximal activity. cn is the Hill coefficient.
BAME 10400 ( 2300 120 ( 70
aKinetic parameters were measured by incubating the enzyme at 37 °C.
bND = not determined. cValues taken from Kearney et al. (32).
values for these isozymes (41-43) as well as PAD4 (32). The
requirement for multiple calcium ions is also consistent with the
PAD4 calcium BAA complex revealed that PAD3 lacks a residue
structure of the PAD4 calcium complex, which shows that this
3
3
3
corresponding to Arg374 in PAD4; this residue is a glycine in
PAD3. As this residue has previously been shown to be important
for PAD4 substrate recognition (19, 45, 46), by forming two key
hydrogen bonds to the backbone carbonyls surrounding the site of
deimination, we generated the PAD3G374R mutant to evaluate
whether the lack of this residue causes the comparatively low
activity of PAD3. Surprisingly, the kinetic parameters obtained
with the PAD3G374R mutant, for both BAA (Km=17.5 ( 6.4
enzyme binds to up to five calcium ions at sites distal from the active
site. Additional studies (not shown) demonstrated that calcium
activates these enzymes by g10000-fold, similarly to PAD4.
We also determined whether or not other cations (i.e., Mg2þ
,
Mn2þ, Zn2þ, and Ba2þ) could substitute for calcium. With the
exception of barium, little to no activity was observed with most of
these cations, even after long incubation times (up to 2 h)
(Supporting Information Table S1); barium, another group II
metal ion, activated PADs 1 and 3 to ∼15% and 2.5%, respec-
tively, of the level of calcium. These results indicate that PADs 1
and 3, like PAD4, are highly specific for calcium. Given that
supraphysiological levels of calcium are required for activity in
vitro, typical intracellular calcium concentrations range from 100
nM to 1 μM, and given that other metal ions could not substitute
for calcium, our results suggest that the PADs must be subject to
an additional layer of regulation, e.g., a chaperone or posttransla-
tional modification that decreases the amount of calcium required
to activate these enzymes in vivo.
mM, kcat=2.1 ( 0.6 s-1, kcat/Km=120 M-1 s-1) and BAEE (kcat
/
Km=13.8 M-1 s-1), are nearly identical to those obtained with the
wild-type enzyme. The fact that the PAD3G374R mutant does not
display PAD4-like kinetics with benzoylated arginine derivatives
suggests either that this enzyme has a very different substrate
specificity profile than PAD4, that the inserted Arg residue cannot
be properly positioned to interact with the substrate, or, alter-
natively, that, in addition to its requirement for calcium, PAD3
may be subject to additional levels of regulation (e.g., interacting
proteins or posttranslational modifications) that activate it toward
physiologically relevant substrates.
Steady-State Kinetic Analysis of Synthetic Arginine
Derivatives. The steady-state kinetic parameters were deter-
mined for BAA, BAME, and BAEE (Table 3) (32, 37). BAA was
identified as the best small molecule substrate for PAD1, with a
kcat/Km value of 22000 M-1 s-1, which is similar to the previously
reported value obtained for PAD4 (40). The values obtained for
BAME and BAEE were also comparable to those determined for
PAD4, although we note that the Km value obtained with BAME
was significantly lower for PAD1 (0.37 ( 0.07 mM) than PAD4
(1.66 ( 0.26 mM). In comparison to PADs 1 and 4, all of the
small molecule substrates tested are relatively poor PAD3 sub-
strates. For example, kcat/Km values of 130, 120, and 26 M-1 s-1
were obtained for BAA, BAME, and BAEE, respectively. The
large decrease in kcat/Km is driven by an increase in Km; this is best
exemplified by the fact that saturation kinetics were not observed
for BAEE, and thus only kcat/Km values could be determined
(Table 3). These values are in agreement with previously estab-
Working Model of PAD Catalysis. Over the past few years,
the conclusions of numerous crystallographic and biochemical
studies have aided the construction of a working model for PAD4
catalysis (26, 32, 47) In this model, Cys645 acts as the active site
nucleophile, and His471 is involved in general acid/base catalysis.
The reaction is initiated by the nucleophilic attack of the thiolate
of Cys645 on the substrate guanidinium, which gives rise to the
first tetrahedral intermediate. His471 then acts as a general acid,
donating a proton to the departing amine either during or after
the formation of the first tetrahedral intermediate (26). Collapse
of this intermediate results in the release of ammonia and the
formation of an S-alkylthiouronium intermediate, the existence
of which has been verified in numerous members of the guanidino
group modifying enzyme family. His471 then acts as a general
base to activate a water molecule for nucleophilic attack on the
thiouronium intermediate. This attack results in the formation of
a second tetrahedral intermediate that ultimately collapses to
regenerate the enzyme and release citrulline.
Based on the proposed mechanism of catalysis, one would
expect that the rates of catalysis would increase with increasing pH
as the concentration of the active site thiolate increases and then
fall upon deprotonation of the active site His. However, previous
studies with PAD4, as well as other members of this family of
enzymes (e.g., dimethylarginine diaminohydrolase (DDAH) (48),
a number of bacterial arginine deiminases (ADIs) (48-50), and
agmatine deiminase (J. E. Jones and P. R. Thompson, unpublished
data)), have shown that the pKa of the active site Cys is g8.0 and
lished values (41, 44). For example, Mechin et al. observed a kcat
/
Km for BAEE of 32000 M-1 s-1 (kcat of 11.31 s-1 and Km of
0.35 mM) for PAD1 and 230 M-1 s-1 (kcat of 1.73 s-1 and Km of
7.50 mM) for PAD3. Similar trends have been reported by
Guerrin et al. (44). The slight variations in kcat and Km may be
due to the high temperature (55 °C) and high level of reducing
agent (10 mM DTT) used in previous studies.
To better understand the molecular basis for why these
benzoylated arginine derivatives are such poor PAD3 substrates,
a structural model of this enzyme was generated using Swiss-
Model. A comparison of this model to the previously determined

Article
Biochemistry, Vol. 49, No. 23, 2010 4857
FIGURE 2: Plot of log kcat/Km versus pH for (A) PAD1 and (B)
PAD3. (C) Plot of log kcat versus pH for PAD1.
F
IGURE 3: Time- and concentration-dependent inactivation of
PAD1 by iodoacetamide. (A) Inactivation of PAD1 at pH 7.6 at
various concentrations of iodoacetamide: (ꢀ) 0 μM, (b) 50 μM, (9)
100 μM, and (2) 250 μM. (B) Plot of the pseudo-first-order rate
constants of inactivation, i.e., kobs, versus iodoacetamide concentra-
tion for PAD1 inactivation. (C) Plot of the second-order rate con-
stants of inactivation, i.e., kinact/KI, versus pH to identify the pKa of
the active site cysteine. (D) Substrate protection experiments with
PAD1 demonstrate the substrate can protect against the iodoaceta-
mide-induced inactivation of PAD1.
likely corresponds to the descending limb of the kcat/Km versus pH
rate profile. Given these precedents, we set out to assess whether
PADs 1 and 3 employ a similar catalytic mechanism by determin-
ing pH rate profiles for PADs 1 and 3 and by determining the pKa
of the active site Cys in PAD1 via iodoacetamide and 2-chlor-
oacetamidine inactivation kinetics.
pH Rate Profiles. The steady-state kinetic parameters were
determined for the deimination of BAEE over a range of pH
values (5.5-9.0) for both PADs 1 and 3. These experiments were
performed because such studies can often identify functional
groups in the free enzyme (kcat/Km), ES complex (kcat), and
substrate (kcat/Km) that are critical for catalysis (51). The pH rate
profiles for PADs 1 and 3 showed trends similar to those
previously reported for PAD4 (47). For example, like PAD4,
the plots of log kcat/Km versus pH, for both PADs 1 and 3, are
bell-shaped, suggesting that two ionizable groups must be in the
correct protonation state to facilitate substrate capture. The log
kcat/Km versus pH profile for PAD1 identified a pKa value of
7.5 ( 0.2 for the ascending limb and a pKa of 7.6 ( 0.2 for the
descending limb (Figure 2A). For PAD3 the pKa values are 7.0 (
0.4 and 8.2 ( 0.5 for the ascending and descending limbs, respec-
tively (Figure 2B). Based on the architecture of the PAD active site
and previous studies with PAD4, these pKa values likely correspond
to the protonation states of His471 and Cys645 (PAD4 numbering)
in PADs 1 and 3. Note that for PADs 1 and 3 the pKa values obtained
from this analysis are within 3.5 pH units of one another; therefore,
the actual pKa values were calculated according to the methods of
Segel (52) (Supporting Information Table S2).
Also like PAD4, the log kcat versus pH plot for PAD1 is bell-
shaped, which suggests that two active site residues must be
correctly protonated for substrate turnover after the formation of
the enzyme-substrate complex (ES) (Figure 2C). Note that a log
kcat versus pH profile could not be constructed for PAD3 because
nonsaturating kinetics were observed for the deimination of
BAEE. Also note that large differences in the calcium dependence
of PADs 1 and 3 were not apparent over the range of pH values
tested (Table 2); the observed changes in activity as a function of
pH are therefore not attributable to a lack of calcium saturation.
Finally, note that PAD activity was linear with respect to time at
all pH values evaluated in these studies.
Iodoacetamide Inactivation Kinetics. Iodoacetamide inac-
tivation kinetics were next used to measure the pKa of the active
site Cys and thereby definitively assign the pKa of this residue.
Because of the similarities in the pH rate profiles obtained for
PADs 1 and 3, we focused only on PAD1. For these experiments,
various concentrations of iodoacetamide were incubated with
PAD1, and the residual activity was measured as a function of
time in order to obtain pseudo-first-order rates of inactivation,
i.e., kobs (Figure 3A). Subsequently, second-order inactivation
rate constants, i.e., kinact/KI, were obtained at each pH by plotting
the kobs values versus iodoacetamide concentration (Figure 3B).
The kinact/KI values were then plotted against pH. Using this
analysis, a pKa value of 8.8 ( 0.2 was determined (Figure 3C).
Note that substrate protection experiments were used to show
that enzyme inactivation was due to the preferential modification
of an active site residue (Figure 3D).
The pKa value obtained from this analysis is in reasonable
agreement with the descending limb of the pH rate profile and
likely corresponds to the pKa of the active site cysteine of PAD1.
As described above, similar results have been reported for PAD4
and other members of the guanidino group modifying enzymes
(48-50), suggesting that a high pKa active site Cys is a universal
feature of, at least, the hydrolase branch of this enzyme super-
family. Given that the thiolate is the more reactive species, these
results are counterintuitive. However, there are numerous exam-
ples in the literature of thiol-reactive enzymes that possess high pKa
values and where the pKa assignments are the reverse of the
simplest assumption (47, 53); these enzymes are said to employ a
“reverse protonation mechanism”. We have previously invoked
such a mechanism for PAD4, and our data suggest that PAD1 also
utilizes a reverse protonation mechanism to deiminate peptidyl
arginine. Based on the similarities in the pH rate profiles, PAD3
likely employs a similar mechanism.

4858 Biochemistry, Vol. 49, No. 23, 2010
Knuckley et al.
Scheme 1: Binding of a Positively Charged or Neutral Inacti-
vator (I) to either the E-SH (Thiol Form of the Enzyme) or
E-S- (Thiolate) Form of the Enzyme
Consistent with a reverse protonation mechanism is the fact
that the decrease in the kcat/Km at the pH extremes is primarily
due to an increase in Km. A similar trend was observed for PAD4,
and while changes in Km as a function of pH can be difficult to
interpret, the data imply that the substrate binds preferentially to
a specific form of the enzyme at the pH optimum (47, 52); this
form likely corresponds to a negatively charged thiolate and
positively charged imidazolium ion (i.e., the ES-Hþ form of the
enzyme) (Scheme 1). If we make the reasonable assumption that
the ascending limb of the pH profile corresponds to the pKa of
the active site His, it is apparent that only a fraction of the enzyme
will exist as the thiolate and imidazolium ion, a form of the
enzyme that is uniquely poised to carry out the first step of
the reaction. Although speculative, the small portion of “active”
enzyme may serve as a mechanism to protect the PADs from
nonspecific inactivation by, for example, reactive oxygen species
in vivo.
2-Chloroacetamidine Inactivation Kinetics. Fast and col-
leagues have previously suggested that thiol deprotonation
occurs by a substrate-assisted mechanism; i.e., the positively
charged guanidinium depresses the pKa of the active site thiol by
∼3 pH units resulting in the loss of a proton either to solvent or to
an unknown base (47, 48) (Scheme 1). To determine if this step is
obligatory, i.e., thiol deprotonation must occur after substrate
binding, we determined whether a positively charged inactivator,
in this case, 2-chloroacetamidine, can influence the pKa of the
active site Cys.
The methodology described above for the iodoacetamide
inactivation experiments was used to obtain the pseudo-first-
order and second-order rate constants of 2-chloroacetamidine-
induced PAD1 inactivation (Figure 4). Plots of kobs versus
2-chloroacetamidine concentration were linear over the concen-
tration range tested (Figure 4B), and the overall plots of kinact/KI
versus pH assigned a pKa of 9.5 ( 0.2 for the active site Cys
(Figure 4C). Although slightly higher than the pKa value
obtained from the iodoacetamide inactivation experiments, this
pKa value also corresponds reasonably well to the descending
limb of the pH profile. Note that increasing concentrations of
substrate protected against inactivation, thus indicating that
inactivation by 2-chloroacetamidine was most likely due to the
modification of the active site cysteine (Figure 4D). These results
demonstrated that the positive charge of this inactivator does not
influence kinact/KI and are, therefore, inconsistent with a mechan-
ism that involves the obligatory binding of the inactivator to the
thiol form of the enzyme prior to proton loss and enzyme
inactivation, i.e., a pure substrate-assisted mechanism. Although
our results favor substrate binding to the thiolate/imidazolium
F
IGURE 4: Time- and concentration-dependent inactivation of
PAD1 by 2-chloroacetamidine. (A) Inactivation of PAD1 at pH
7.6 for various concentrations of 2-chloroacetamide: (ꢀ) 0 μM, (b)
500 μM, (9) 1000 μM, and (2) 3000 μM. (B) Plot of the pseudo-first-
order rate constants of inactivation, i.e., kobs, versus 2-chloroaceta-
midine concentration for PAD1 inactivation. (C) Plot of the second-
order rate constants of inactivation, i.e., kinact/KI, versus pH to
identify the pKa of the active site cysteine. (D) Substrate protection
experiments with PAD1 demonstrate substrate can protect against
the 2-chloroacetamidine-induced inactivation of PAD1.
form of the enzyme, they do not rule out the possibility that the
substrate can bind to both the thiol and thiolate forms of the
enzyme (Scheme 1), as we have previously suggested (47).
Additionally, it is possible that at high concentrations of sub-
strate, and at acidic pH values, binding to the thiol form of the
enzyme would predominate, and under those circumstances the
positive charge of the substrate would undoubtedly depress the
pKa of the active site Cys, thereby favoring its deprotonation.
Substrate Specificity Studies. Previous studies have shown
that the N-termini of histones H2A, H3, and H4 are the major in
vivo, and in vitro, sites of PAD4 modification (40, 54). To
characterize the substrate specificity of PAD4, a series of pep-
tides, based on the N-terminus of histone H4, were synthesized
(Table 1). Histone H4 was chosen for these studies because PAD4
is known to deiminate this protein at arginines 3, 17, and 19 in
vitro and R3 in vivo (15, 17) and because we have previously
shown that H4 tail mimics are deiminated with comparable
efficiency to the benzoylated arginine derivatives (32). It was
expected that, by comparing the steady-state kinetic parameters
obtained for these substrates with PAD4 to those obtained with
PADs 1 and 3, we could identify residues surrounding the site of
modification that play significant roles in substrate binding; any
observed differences could then be exploited for the future design
of PAD-specific inhibitors (Table 4).
To further validate the use of these peptides, the steady-state
kinetic parameters were determined for histone H4 and initially
compared to those obtained for BAEE. The results of these
studies indicate that PADs 1 and 4 deiminate BAEE and histone
H4 with roughly comparable kinetics: the kcat/Km values are only
2-3-fold higher for histone H4. A more dramatic 28-fold
increase in kcat/Km was observed for PAD3, suggesting that
longer range interactions are important for PAD3 substrate
recognition. Notably, all three enzymes deiminate the AcH4-21

Article
Biochemistry, Vol. 49, No. 23, 2010 4859
Table 4: Steady-State Kinetic Parameters for Histone-Based Peptidesa
PAD1
PAD3
PAD4
kcat/Km
(M-1 s-1
kcat/Km
(M-1 s-1
kcat/Km
(M-1 s-1
)
peptide
AcH4-21
AcH4-18
AcH4-16
AcH4-15
AcH4-13
AcH4-5
AcH4-5 S1A
AcH4-5 G2A
AcH4-5 G4A
AcH4-5 K5A
AcH4-21 N(1)
AcH4-21 N(2)
AcH4-21 K16A
AcH4-21 K20A
AcH4-21 R3A
AcH4-21 R17A
AcH4-21 R19A
AcH4-21 R17,19K
AcH4-21 all Ac-K
histone H4
kcat (s-1
)
Km (mM)
)
kcat (s-1
)
Km (mM)
)
kcat (s-1
)
Km (mM)
0.81 ( 0.06
0.64 ( 0.05
0.60 ( 0.07
0.75 ( 0.08
N/Ab
0.21 ( 0.04
0.22 ( 0.05
0.20 ( 0.05
0.16 ( 0.04
N/A
4000 ( 800
3000 ( 600
3100 ( 800
4600 ( 1550
<1
0.31 ( 0.07
4.92 ( 0.87
0.08 ( 0.01
N/A
0.42 ( 0.14
1.95 ( 0.49
0.15 ( 0.05
N/A
700 ( 200
3.39 ( 0.19
2.29 ( 0.99
2.30 ( 0.16
2.53 ( 0.15
2.83 ( 0.15
4.97 ( 0.28
N/A
0.64 ( 0.12
0.52 ( 0.13
0.59 ( 0.12
0.58 ( 0.10
0.86 ( 0.16
0.95 ( 0.17
N/A
5300 ( 1700
4400 ( 2000
3900 ( 900
4400 ( 900
3300 ( 400
5200 ( 900
3500 ( 700
5800 ( 800
6600 ( 1100
16000 ( 7000
5700 ( 3500
1400 ( 550
14000 ( 6500
19000 ( 8400
580 ( 40
2500 ( 800
500 ( 180
2000 ( 100
85 ( 25
1000 ( 100
220 ( 80
900 ( 600
80 ( 40
N/A
N/A
0.90 ( 0.12
0.59 ( 0.09
1.13 ( 0.22
0.98 ( 0.08
0.93 ( 0.16
0.45 ( 0.06
0.48 ( 0.06
0.87 ( 0.23
0.77 ( 0.13
1.46 ( 0.05
N/A
0.33 ( 0.09
0.30 ( 0.11
0.80 ( 0.29
0.58 ( 0.09
0.37 ( 0.13
0.11 ( 0.03
0.36 ( 0.08
0.66 ( 0.28
0.34 ( 0.11
0.44 ( 0.07
N/A
2700 ( 500
2000 ( 650
1400 ( 300
1700 ( 850
2500 ( 750
4200 ( 500
1400 ( 200
1300 ( 400
2300 ( 1900
3300 ( 800
600 ( 150
3700 ( 400
1200 ( 150
3900 ( 150
4300 ( 900
N/A
N/A
0.21 ( 0.08
0.62 ( 0.16
N/A
0.95 ( 0.63
0.69 ( 0.34
N/A
N/A
N/A
N/A
N/A
N/A
N/A
50 ( 30
200 ( 20
<1
N/A
N/A
N/A
N/A
1.04 ( 0.04
0.45 ( 0.06
5.96 ( 0.47
3.27 ( 0.11
1.01 ( 0.07
4.46 ( 0.17
0.86 ( 0.21
1.69 ( 0.34
3.59 ( 0.21
1.25 ( 0.20
0.18 ( 0.12
0.32 ( 0.20
0.43 ( 0.14
0.17 ( 0.03
1.75 ( 0.42
1.70 ( 0.21
2.16 ( 0.69
3.27 ( 1.10
1.07 ( 0.23
0.14 ( 0.03
N/A
N/A
N/A
N/A
700 ( 20
1000 ( 40
10 ( 7
300 ( 20
<1
N/A
N/A
N/A
N/A
N/A
N/A
2700 ( 650
400 ( 100
500 ( 150
3400 ( 750
9000 ( 1500
1.83 ( 0.07
0.56 ( 0.06
0.57 ( 0.03
N/A
0.48 ( 0.04
0.48 ( 0.06
0.15 ( 0.03
N/A
N/A
N/A
0.34 ( 0.08
1.08 ( 0.22
N/A
0.86 ( 0.29
0.59 ( 0.21
N/A
400 ( 180
1800 ( 1300
700 ( 200
aKinetic parameters were measured by incubating the enzyme at 37 °C. bN/A = not applicable.
peptide with comparable efficiency to histone H4, thereby
validating the use of histone H4-based tail mimics to probe
PAD substrate specificity. To further probe the substrate speci-
ficity of PADs 1, 3, and 4, arginine substitutions were used to
identify the preferred sites of modification. Additionally, the
effects of peptide length, N-terminal amino acid deletions, and
neighboring residues on substrate recognition were evaluated in
an effort to identify specific substrate recognition elements.
Arginine Substitutions. To identify the preferred sites of
modification in the H4-tail mimics, the steady-state kinetic para-
meters were determined for a series of arginine-substituted pep-
tides; the sequences of these peptides are depicted in Table 1. For
PAD1, the AcH4-21 R3A and R19A peptides retain similar levels
of activity as the AcH4-21 peptide; that is, the kcat/Km values are
3300 and 3700 M-1 s-1, respectively, versus 4000 M-1 s-1 for the
AcH4-21 peptide. In contrast, the activity of the AcH4-21 R17A
mutant peptide is reduced by greater than 5-fold (kcat/Km = 600
M-1 s-1). These results suggest that R17 is the major site
deiminated by PAD1 in the AcH4-21 peptide (Figure 5A). For
PAD3, the kcat/Km values for the deimination of the AcH4-21 and
AcH4-21 R17A peptides are 700 and 300 M-1 s-1, respectively,
whereas the deimination rates for the AcH4-21 R3A and R19A
peptides are both reduced by more than 70-fold, suggesting that R3
and R19 are the major sites of modification by PAD3 (Figure 5A).
Consistent with previous reports for PAD4, the fact that the
kcat/Km for the AcH4-21 R3A peptide is reduced by ∼10-fold
(5300 to 575 M-1 s-1) indicates that this residue is a major site of
in vitro modification. R19 also appears to be preferentially
deiminated, as the kcat/Km for the R19A “mutant” is reduced
by ∼13-fold. As with PAD3, R17 appears to be a minor site of
deimination as the effect of the R17A substitution was relatively
minor (∼2-fold). In summary, PADs 1 and 3 appear to prefer-
entially deiminate these H4-tail mimics at R3 and R17, whereas
PAD1 preferentially modifies R17.
AcH4-21-based C-terminal and N-terminal deletion peptides.
The results of these analyses indicate that, for PADs 1and 4, there
was little variability in the rates at which the C-terminally
truncated peptides were modified (Table 4). For example, the
differences in kcat/Km between the AcH4-21 peptide and shorter
peptides (AcH4-16, AcH4-15, AcH4-13, and AcH4-5) are at
most 2-fold, suggesting that long-range interactions play only a
minimal role in substrate recognition. A less than 4-fold decrease
in kcat/Km was also observed with the N-terminal deletion
peptides (AcH4-21 N(1) and AcH4-21 N(2)). In total, peptide
length seems to only slightly influence substrate recognition by
PADs 1 and 4 (Figure 5B). In contrast, for PAD3, the kcat/Km for
the AcH4-21 peptide is 5.4-fold higher than the best small
molecule PAD3 substrate (i.e., BAA), again suggesting that
longer range interactions are important for substrate recognition
by this isozyme. Given that similarly high rates of deimination
were observed with the AcH4-5 peptide, these results suggest
that the residues immediately surrounding the major sites of
deimination contribute the most to this effect. Consistent with
this hypothesis is the fact that the increase in kcat/Km is mostly
driven by a decrease in Km.
Site-Directed “Mutagenesis”. Alanine scanning “mutagen-
esis” was used to evaluate the specific contributions of neighbor-
ing residues to substrate recognition. First, a series of single lysine
to alanine mutant peptides (i.e., AcH4-21 K16A and AcH4-21
K20A) were tested as substrates for PADs 1, 3, and 4. The results
show a modest ∼3-fold increase in the deiminating activity of
PAD4, as compared to the AcH4-21 peptide, suggesting that
neighboring positively charged residues negatively influence the
deiminating activity of PAD4. In contrast, no such trend was
observed for PADs 1 or 3; that is, less than a 3-fold decrease in
activity was measured for these peptides (Table 4, Figure 5C). In
addition to these mutant peptides, we measured the rates of
citrullination using a histone AcH4-21 peptide variant in which
all of the Lys residues are acetylated; these experiments were
performed because these residues are known to be acetylated in
vivo (55). For PAD1, the results of these analyses indicate that the
Effect of Peptide Length on Substrate Recognition. We
next examined the effect of peptide length on substrate recogni-
tion by measuring the kinetic parameters for a series of histone

4860 Biochemistry, Vol. 49, No. 23, 2010
Knuckley et al.
F
IGURE 5: Plots of the kcat/Km data shown in Table 4 were generated to provide a visual depiction of the results of the substrate specificity studies
on histone H4 tail mimics. Comparisons of the data to show (A) the major site of deimination as determined by Arg substitution, (B) the effects of
peptide length on substrate recognition, and (C, D) the effects of site-directed “mutagenesis” experiments.
kcat/Km values for the AcH4-21 peptide and AcH4-21 all K-Ac
peptide are very similar to one another, suggesting that a
reduction in charge on residues that are near the sites of
modification does not affect substrate binding. This result is
consistent with the relatively minor effects observed when the
neighboring Lys residues were converted to Ala (see above). For
PAD3, the kcat/Km value for the AcH4-21 all K-Ac peptide
increased by ∼2.5-fold, again suggesting that acetylation does not
substantially affect substrate recognition. In contrast to the
results for PADs 1 and 3, the kcat/Km for PAD4 with this peptide
is reduced by ∼2-fold. Although this decrease would appear to
contradict the results of the Lys f Ala substitutions, i.e., that
positive charge diminishes substrate recognition, the increased
steric bulk of the acetyl group may give rise to steric clashes that
hinder substrate binding. Nevertheless, it should be noted that
most of the effects on catalysis are relatively minor, suggesting
that the presence or absence of positively charged residues near
the site of modification has minimal effects on catalysis.
To evaluate the effects of residues directly adjacent to R3, we
generated a series of peptides based on the sequence of the
AcH4-5 peptide, in which each of the individual residues was
mutated to Ala. Although the data did not reveal any major
trends for either PADs 1 or 4, it is noteworthy that the AcH4-5
K5A peptide showed a greater than 3-fold increase in deiminating
activity, as compared to the AcH4-5 peptide, again suggesting
that a positive charge C-terminal to the site of modification is not
preferred. In contrast, the data for PAD3 revealed several
interesting findings. First, the kcat/Km for the AcH4-5 K5A
peptide is reduced by ∼20-fold relative to the AcH4-5 peptide,
suggesting that a positively charged residue at the R þ 2 position
is important for substrate recognition. Second, the fact that
the replacement of Ser1 with an Ala residue reduces kcat/Km by
∼4.5-fold suggests that a hydrogen bond to the Ser hydroxyl
contributes to substrate recognition. Third, greater conforma-
tional flexibility appears to be important for the binding of PAD3
to its substrates as the replacement of Gly4 with an Ala residue
reduces kcat/Km by 12.5-fold; the greater flexibility of the AcH4-5
peptide may allow other residues, e.g., the substrate Arg or,
alternatively, Lys5, to adopt a confirmation that is preferentially
recognized by this enzyme (Table 4, Figure 5D).
Inhibition Experiments with F- and Cl-amidine. As de-
scribed in the introduction, we have previously reported the
design and synthesis of F- and Cl-amidine, which are the most
potent PAD4 inhibitors described to date (19, 25, 33). To assess
the selectivity of these compounds, and possibly gain insights that
could guide the design of inhibitors with greater selectivity, we
evaluated their ability to inhibit PADs 1 and 3. Initially, IC50
values were determined (Figure 6A, top panel). The values
obtained for Cl-amidine were 0.8 ( 0.3 and 6.2 ( 1.0 μM for
PADs 1 and 3, respectively. For PAD1, this represents an ∼5-fold
increase in potency, whereas for PAD3, the value is roughly
comparable to that obtained for PAD4. For F-amidine, the IC50
obtained with PAD1 (i.e., 29.5 ( 1.31 μM) is also quite similar to
that obtained for PAD4 (i.e., 21.6 ( 2.10 μM(25)). Interestingly, the
IC50 value obtained for F-amidine with PAD3 was significantly
higher, i.e., ∼350 μM, than that obtained for either PAD1 or PAD4.
These data suggest that Cl-amidine is a pan PAD inhibitor, whereas
F-amidine is a PAD1/4 selective inhibitor. The possible reasons for
the lack of F-amidine potency toward PAD3 are discussed below.
Inhibition by F-ethyl- and Cl-ethylamidine. The fact that
the PAD3 kcat/Km value for BAEE is approximately 5-fold lower
than that obtained for BAME (see above) suggested that it might
be possible to generate an inhibitor that selectively inhibits PADs
1 and 4 over PAD3 by adding an ethyl group to the backbone

Article
Biochemistry, Vol. 49, No. 23, 2010 4861
FIGURE 6: (A) Inhibition of PADs 1, 3, and 4 by (A) F- and Cl-amidine (top), F- and Cl-ethylamidine (middle), and F4- and Cl4-amidine
(bottom). (B) Plot of kobs versus the concentration of F-amidine (top) and Cl-amidine (bottom) for the inactivation of PAD3.
amide of F- and Cl-amidine. As such, F- and Cl-ethylamidine
were synthesized (Figure 1B), and their ability to inhibit PADs 1,
3, and 4 was evaluated (Figure 6A, middle panel). The IC50 values
of Cl-ethylamidine for PADs 1, 3, and 4 are 2.8 ( 0.3, 26 ( 4.6,
and 4.1 ( 1.7 μM, respectively. The IC50 values of F-ethylamidine
for PADs 1, 3, and 4 are 40 ( 19, 157 ( 58.0, and 24.3 ( 22.3, μM,
respectively. Gratifyingly, the IC50 values for PADs 1 and 4 for
both F-ethyl- and Cl-ethylamidine were quite similar to those
obtained for F- and Cl-amidine. Somewhat surprisingly, how-
ever, the addition of the ethyl group did not lead to a significant
improvement in inhibitor selectivity. The reasons for the lack of
enhanced selectivity are unclear and require further investigation.
Inactivation of PAD3 by F- and Cl-amidine. We were
intrigued by the large disparity in the inhibition constants
obtained for PAD3 with F- and Cl-amidine and how these values
compared to those obtained for PAD4. To gain insight into this
disparity, we characterized the kinetics of PAD3 inactivation by
both F- and Cl-amidine. Briefly, the pseudo-first-order rate
constants of inactivation, i.e., kobs, were obtained for various
concentrations of inactivator at the pH optimum and subse-
quently plotted versus inactivator concentration. Because the
plots are hyperbolic, an indication of a two-step mechanism of
decrease in kinact/KI is primarily due to a decrease in kinact (∼20-fold
as compared to the previously reported value for PAD4 (25)). For
Cl-amidine, the kinact is 0.056 ( 0.005 min-1 and the KI is 28 (
7.3 μM, resulting in a kinact/KI of 2000 M-1 min-1 (Figure 6B).
Relative to PAD4, these data indicate that kinact/KI is decreased by
>6-fold. Similarly to the results obtained for F-amidine, this
decrease in kinact/KI is driven by a decrease in kinact (∼40-fold as
compared to the previously reported value for PAD4 (25)).
The decrease in kinact observed for both compounds with PAD3
is most easily explained by an inability to properly position the
haloacetamidine warhead for nucleophilic attack by the active site
Cys. To examine this possibility, we determined the IC50 values
for derivatives of F- and Cl-amidine that incorporate a longer side
chain; we reasoned that the longer side chain would allow for the
proper positioning of the warhead for nucleophilic attack and
would thus exhibit greater selectivity for PAD3. The structures of
these compounds, denoted F4-amidine and Cl4-amidine, are
depicted in Figure 1, and we have previously reported that they
are relatively weak PAD4 inhibitors (IC50 g 640 μM) (19). The
IC50 values are at least partially consistent with this hypothesis
(Figure 6A, bottom panel). For example, the IC50 of F4-amidine
for PAD3 was reduced by 5.7-fold, from 367 ( 189 to 64.4 ( 24.4
μM, whereas the IC50 values obtained for PADs 1 and 4 with this
compound are increased by 270- and 30-fold, respectively.
Although a less significant effect was observed with Cl4-amidine,
the same trends emerge; Cl4-amidine shows >20-fold and
50-fold selectivity for PAD3 over PAD1 and PAD4, respectively.
inactivation, the data were fit to eq 5 to obtain values for kinact
,
KI, and kinact/KI (Figure 6B). For F-amidine, these values are
0.05 ( 0.01 min-1, 293 ( 193 μM, and 170 M-1 min-1, respec-
tively. Interestingly, the kinact/KI value is decreased >15-fold
relative to the previously reported value for PAD4 (25). This

4862 Biochemistry, Vol. 49, No. 23, 2010
Knuckley et al.
Thus, F4- and Cl4-amidine represent the first PAD3-selective
inhibitors to be described in the literature, and they will undoubtedly
serve as useful chemical probes to discern the physiological func-
tions of this enzyme. It should be noted that dialysis experiments
were used to demonstrate that these compounds irreversibly
inactivate PAD3 (Supporting Information Figure S3).
2. Moscarello, M. A., Mastronardi, F. G., and Wood, D. D. (2007) The
role of citrullinated proteins suggests a novel mechanism in the
pathogenesis of multiple sclerosis. Neurochem. Res. 32, 251–256.
3. Ishigami, A., Ohsawa, T., Hiratsuka, M., Taguchi, H., Kobayashi,
S., Saito, Y., Murayama, S., Asaga, H., Toda, T., Kimura, N., and
Maruyama, N. (2005) Abnormal accumulation of citrullinated pro-
teins catalyzed by peptidylarginine deiminase in hippocampal extracts
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128.
SUMMARY
4. Chen, C. C., Isomoto, H., Narumi, Y., Sato, K., Oishi, Y., Kobayashi,
T., Yanagihara, K., Mizuta, Y., Kohno, S., and Tsukamoto, K. (2008)
Haplotypes of PADI4 susceptible to rheumatoid arthritis are also
associated with ulcerative colitis in the Japanese population. Clin.
Immunol. 126, 165–171.
5. Chang, X., Han, J., Pang, L., Zhao, Y., Yang, Y., and Shen, Z. (2009)
Increased PADI4 expression in blood and tissues of patients with
malignant tumors. BMC Cancer 9, 40.
6. Chang, X., and Han, J. (2006) Expression of peptidylarginine deimi-
nase type 4 (PAD4) in various tumors. Mol. Carcinog. 45, 183–196.
7. Jones, J. E., Causey, C. P., Knuckley, B., Slack-Noyes, J. L., and
Thompson, P. R. (2009) Protein arginine deiminase 4 (PAD4):
Current understanding and future therapeutic potential. Curr. Opin.
Drug Discovery Dev. 12, 616–627.
8. Kinloch, A., Lundberg, K., Wait, R., Wegner, N., Lim, N. H.,
Zendman, A. J., Saxne, T., Malmstrom, V., and Venables, P. J.
(2008) Synovial fluid is a site of citrullination of autoantigens in
inflammatory arthritis. Arthritis Rheum. 58, 2287–2295.
Dysregulated protein deimination is associated with the onset
and progression of various diseases, most notably rheumatoid
arthritis (RA) (7, 56). Several studies have identified PAD4 as the
disease-implicated enzyme, and it has recently become a major
target for the development of an RA therapeutic (7). In addition
to PAD4, dysregulated PAD2 activity may also play a role in
several diseases, including RA and multiple sclerosis (MS).
Furthermore, the expression of PADs 2 and 4, as well as PADs
1 and 3, is known to overlap (10). Therefore, in order to discern
the specific contributions of individual PAD isozymes to human
disease, PAD-specific inhibitors are needed. In addition, such
compounds will be useful for addressing the question of whether
the inhibition of a single isozyme, or, alternatively, multiple
isozymes, is required to treat these diseases. Finally, the devel-
opment of PAD-selective inhibitors will undoubtedly provide
powerful chemical tools that can be used to characterize the
normal physiological roles of these enzymes.
9. Chavanas, S., Mechin, M. C., Takahara, H., Kawada, A., Nachat, R.,
Serre, G., and Simon, M. (2004) Comparative analysis of the mouse
and human peptidylarginine deiminase gene clusters reveals highly
conserved non-coding segments and a new human gene, PADI6. Gene
330, 19–27.
10. Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J., and Pruijn, G.
J. (2003) PAD, a growing family of citrullinating enzymes: Genes,
features and involvement in disease. BioEssays 25, 1106–1118.
11. Tarcsa, E., Marekov, L. N., Mei, G., Melino, G., Lee, S. C., and
Steinert, P. M. (1996) Protein unfolding by peptidylarginine deiminase.
Substrate specificity and structural relationships of the natural sub-
strates trichohyalin and filaggrin. J. Biol. Chem. 271, 30709–30716.
12. Lee, Y. H., Coonrod, S. A., Kraus, W. L., Jelinek, M. A., and
Stallcup, M. R. (2005) Regulation of coactivator complex assembly
and function by protein arginine methylation and demethylimination.
Proc. Natl. Acad. Sci. U.S.A. 102, 3611–3616.
13. Suzuki, A., Yamada, R., Chang, X., Tokuhiro, S., Sawada, T., Suzuki,
M., Nagasaki, M., Nakayama-Hamada, M., Kawaida, R., Ono, M.,
Ohtsuki, M., Furukawa, H., Yoshino, S., Yukioka, M., Tohma, S.,
Matsubara, T., Wakitani, S., Teshima, R., Nishioka, Y., Sekine, A., Iida,
A., Takahashi, A., Tsunoda, T., Nakamura, Y., and Yamamoto, K.
(2003) Functional haplotypes of PADI4, encoding citrullinating enzyme
peptidylarginine deiminase 4, are associated with rheumatoid arthritis.
Nat. Genet. 34, 395–402.
14. Cuthbert, G. L., Daujat, S., Snowden, A. W., Erdjument-Bromage,
H., Hagiwara, T., Yamada, M., Schneider, R., Gregory, P. D.,
Tempst, P., Bannister, A. J., and Kouzarides, T. (2004) Histone
deimination antagonizes arginine methylation. Cell 118, 545–553.
15. Wang, Y., Wysocka, J., Sayegh, J., Lee, Y. H., Perlin, J. R., Leonelli,
L., Sonbuchner, L. S., McDonald, C. H., Cook, R. G., Dou, Y.,
Roeder, R. G., Clarke, S., Stallcup, M. R., Allis, C. D., and Coonrod,
S. A. (2004) Human PAD4 regulates histone arginine methylation
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To further aid in the development of isozyme-specific PAD
inhibitors, we initiated studies on PADs 1 and 3, with the goal of
identifying specific differences between isozymes that could be
exploited for the development of such compounds. These studies
revealed that, like PAD4, PADs 1 and 3 (i) are bona fide PADs, (ii)
require supraphysiological concentrations of calcium for activity,
similarly to previous reports (32), and (iii) utilize a reverse
protonation mechanism. The results of substrate specificity
studies also indicate that there are few PAD-specific recognition
elements. For example, peptide length, acetylation status, and the
presence or absence of charged residues proved, in most cases,
to have only minor effects on the deiminating activity of PADs 1
and 4. The fact that peptide length has little influence on the
deiminating activity of these isozymes is in stark contrast to other
histone-modifying enzymes, e.g., protein arginine methyltransfer-
ase 1 (PRMT1), lysine-specificdemethylase 1 (LSD1), and histone
deacetylase 8 (HDAC8), where long-range interactions are critical
for substrate recognition (57-59). The one exception is that
longer range interactions do appear to contribute to PAD3
substrate recognition, and one key interaction appears to be the
presence of a positively charged residue at the R þ 2 position. This
information will undoubtedly aid the future development of a
PAD3-specific inhibitor. These studies have also shown that Cl-
amidine and, to a lesser extent, F-amidine are pan PAD inhibitors
and, more importantly, have led to the identification of F4- and
Cl4-amidine as PAD3-selective inhibitors.
16. Thompson, P. R., and Fast, W. (2006) Histone citrullination by
protein arginine deiminase: Is arginine methylation a green light or
a roadblock? ACS Chem. Biol. 1, 433–441.
17. Hagiwara, T., Hidaka, Y., and Yamada, M. (2005) Deimination of
histone H2A and H4 at arginine 3 in HL-60 granulocytes. Biochem-
istry 44, 5827–5834.
18. Nakashima, K., Hagiwara, T., and Yamada, M. (2002) Nuclear
localization of peptidylarginine deiminase V and histone deimination
in granulocytes. J. Biol. Chem. 277, 49562–49568.
19. Luo, Y., Arita, K., Bhatia, M., Knuckley, B., Lee, Y. H., Stallcup, M. R.,
Sato, M., and Thompson, P. R. (2006) Inhibitors and inactivators of
protein arginine deiminase 4: Functional and structural characterization.
Biochemistry 45, 11727–11736.
SUPPORTING INFORMATION AVAILABLE
Supplementary methods, Tables S1 and S2, and Figures
S1-S3. This material is available free of charge via the Internet
at http://pubs.acs.org.
20. Wang, J. S., and Chiu, Y. T. (2009) Systemic hypoxia enhances
exercise-mediated bactericidal and subsequent apoptotic responses
in human neutrophils, J. Appl. Physiol. (in press).
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