The nonenzymatic decomposition of guanidines and amidines

Article abstract of DOI:10.1021/ja411927k

To establish the rates and mechanisms of decomposition of guanidine and amidine derivatives in aqueous solution and the rate enhancements produced by the corresponding enzymes, we examined their rates of reaction at elevated temperatures and used the Arrhenius equation to extrapolate the results to room temperature. The similar reactivities of methylguanidine and 1,1,3,3-tetramethylguanidine and their negative entropies of activation imply that their decomposition proceeds by hydrolysis rather than elimination. The influence of changing pH on the rate of decomposition is consistent with attack by hydroxide ion on the methylguanidinium ion (k₂ = 5 × 10 ^-6 M^-1 s^-1 at 25 C) or with the kinetically equivalent attack by water on uncharged methylguanidine. At 25 C and pH 7, N-methylguanidine is several orders of magnitude more stable than acetamidine, urea, or acetamide. Under the same conditions, the enzymes arginase and agmatinase accelerate substrate hydrolysis 4 × 10¹⁴-fold and 6 × 10¹²-fold, respectively, by mechanisms that appear to involve metal-mediated water attack. Arginine deiminase accelerates substrate hydrolysis 6 × 10¹²-fold by a mechanism that (in contrast to the mechanisms employed by arginase and agmatinase) is believed to involve attack by an active-site cysteine residue.

Full text of DOI:10.1021/ja411927k

Article
pubs.acs.org/JACS
The Nonenzymatic Decomposition of Guanidines and Amidines
Charles A. Lewis, Jr. and Richard Wolfenden*
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, United States
*
S Supporting Information
ABSTRACT: To establish the rates and mechanisms of
decomposition of guanidine and amidine derivatives in
aqueous solution and the rate enhancements produced by
the corresponding enzymes, we examined their rates of
reaction at elevated temperatures and used the Arrhenius
equation to extrapolate the results to room temperature. The
similar reactivities of methylguanidine and 1,1,3,3-tetramethylguanidine and their negative entropies of activation imply that their
decomposition proceeds by hydrolysis rather than elimination. The influence of changing pH on the rate of decomposition is
−
6
−1 −1
consistent with attack by hydroxide ion on the methylguanidinium ion (k = 5 × 10
M
s
at 25 °C) or with the kinetically
2
equivalent attack by water on uncharged methylguanidine. At 25 °C and pH 7, N-methylguanidine is several orders of magnitude
more stable than acetamidine, urea, or acetamide. Under the same conditions, the enzymes arginase and agmatinase accelerate
1
4
12
substrate hydrolysis 4 × 10 -fold and 6 × 10 -fold, respectively, by mechanisms that appear to involve metal-mediated water
12
attack. Arginine deiminase accelerates substrate hydrolysis 6 × 10 -fold by a mechanism that (in contrast to the mechanisms
employed by arginase and agmatinase) is believed to involve attack by an active-site cysteine residue.
INTRODUCTION
Scheme 1. Possible Sites (A) and Mechanisms (B) of
Guanidine Cleavage
■
To evaluate the power of an enzyme as a catalyst and its
potential sensitivity to inhibition by transition state analogues,
it is necessary to establish the rate constant of the reaction in
water in the absence of a catalyst. That information is available
for the cleavage of amides, peptides, and urea but not for the
cleavage of guanidines, another group of C−N derivatives that
is widely distributed in nature.
1
2
For guanidines with the general formula RHN−C(NH)−
NH , the products of enzymatic decomposition may include (a)
2
urea and an amine or (b) a substituted urea and ammonia
(
Scheme 1A). Enzymes of the first type, which are classified as
amidinohydrolases, include arginase (EC 3.5.3.1), creatinase
EC 3.5.3.3), and agmatinase (EC 3.5.3.11). Enzymes of the
(
second type are termed iminohydrolases and include arginine
deiminase (EC 3.5.3.6), agmatine deiminase (EC 3.5.3.12),
dimethylarginase (EC 3.5.3.18), and peptidyl/protein-arginine
deiminases (or “PADs”) (EC 3.5.3.15), which participate in the
3
,4
post-translational modification of histones.
In the work described here, we set out to determine the
reactivities of guanidines and amidines in neutral aqueous
solution in the absence of a catalyst. Because the spontaneous
decomposition of these molecules is very slow at ordinary
temperatures, we examined their rates of decomposition at
elevated temperatures and used the Arrhenius relationship to
estimate their rate constants for decomposition at ordinary
temperatures.
In principle, these reactions might proceed by nucleophilic
attack at the central carbon atom or by elimination to yield a
carbodiimide as the immediate product (Scheme 1B). We
sought to obtain an indication of the actual mechanism from
the thermodynamics of activation by comparing the behavior of
methylguanidine with that of 1,1,3,3-tetramethylguanidine, in
which methyl substitution precludes elimination. For com-
parative purposes, we conducted similar experiments on
pivalamidine (2,2,2-trimethylacetamidine) and pivalamide
(
2,2,2-trimethylacetamide). The results permit quantitative
comparison of the rates and mechanisms of decomposition of
guanidines, amidines, amides, and urea in neutral solution and
the extent to which these reactions are enhanced by hydrolytic
5
enzymes.
Received: May 14, 2013
Published: December 12, 2013
©
2013 American Chemical Society
130
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Article
Table 1. Kinetic and Thermodynamic Constants for the Decomposition of Guanidines, Amidines, Ureas, and Amides
(Observed Values Are Not Italicized, While Calculated Values Are Italicized)
pKa
k25 (s−1)
ΔH⧧ (kcal/mol)
TΔS⧧ (kcal/mol)
Methylguanidines
13
MG (pH 7)
5.3 × 10−
34.7
20.1
33.1
14.7
0.6
−5.3
0.7
−
6
MG (uncharged)
M4G (pH 7)
13.4
12.8
1.1 × 10
9.2 × 10−
1.3 × 10−
12
6
M4G (uncharged)
−9.8
Acetamidines
9
acetamidine (pH 7)
4.3 × 10−
28.6
13.2
28.6
13.2
−0.2
−8.0
−1.0
−8.9
−
3
acetamidine (uncharged)
pivalamidine (pH 7)
pivalamidine (uncharged)
12.5
12.5
1.4 × 10
1.1 × 10−
9
3.7 × 10−
4
Ureas
urea
2.3 × 10−
13
29.7
22.0
−4.9
−10.6
6
−12
M4U
4.2 × 10
Acetamides
7.0 × 10−
5.4 × 10−
11
acetamide
pivalamide
23.0
25.7
−8.2
−7.0
12
MATERIALS AND METHODS
over the temperature range from 100 to 200 °C, traces of N-
methylurea (<2%) made a transitory appearance and then
disappeared, consistent with the somewhat more rapid
decomposition of ureas by elimination under these conditions
(Table 1 and Figure S4 in the Supporting Information). An
Arrhenius plot of the rate constants observed for methyl-
guanidine decomposition was linear (Figure 1), and extrap-
■
1
,1-Dimethylurea, N-methylguanidine hydrochloride, tetramethylurea,
and urea were purchased from ACROS Organics. Pivalamide (2,2,2-
trimethylacetamide), pivalamidine (2,2,2-trimethylacetamidine) hydro-
chloride, and 1,1,3,3-tetramethylguanidine were purchased from TCI
America. Guanidine hydrochloride and acetamidine hydrochloride
were purchased from Spectrum Chemical Manufacturing Corp.
Kinetic experiments were conducted using solutions of guanidines,
amidines, and amides (0.02 M) in potassium acetate, phosphate,
borate, and carbonate buffers (0.1 M), as indicated in the Results.
Solutions were flushed with argon, sealed under vacuum in quartz
tubes, heated for various time intervals in Thermolyne 47900 and
48000 furnaces, and cooled with ice. Samples of the product mixtures
(
0.025 mL) were transferred to 5 mm NMR tubes and diluted with
2
D O (0.5 mL) containing added pyrazine to serve as an internal
reference for calibrating chemical shifts (pyrazine = 8.60 ppm) and
1
integrated signal intensities. H NMR spectra were acquired using a
Bruker AVANCE 500 MHz spectrometer equipped with a cryoprobe,
usually with four transients. At each temperature, triplicate samples
were heated long enough to allow the reaction to proceed to between
10% and 90% completion. The integrated intensities of the reactants
and products were used to calculate their percentages in each product
mixture. The chemical shifts observed for the reactants and products
are shown in Table S1 in the Supporting Information.
In each of these reactions, the disappearance of starting material
followed first-order kinetics to at least 90% completion, and the results
were used to calculate the rate constant. The logarithms of the
observed rate constants were then plotted as a function of the
reciprocal of absolute temperature. Linear regression was used to
obtain the slope and intercept of the resulting Arrhenius plot and the
associated correlation coefficient. To determine the pH dependence of
the decomposition of methylguanidine, initial rates were determined at
Figure 1. Rate constants for decomposition of methylguanidine (0.02
M) in potassium phosphate buffer (0.1 M, pH 7) plotted
logarithmically as a function of the reciprocal of absolute temperature
over the range from 100 to 200 °C. Linear regression yielded (5.3 ±
−
13 −1
⧧
2
0
.5) × 10
s
and ΔH = 34.7 ± 1.0 kcal/mol (R = 0.997).
1
40 °C in potassium acetate-d , potassium phosphate, sodium borate,
3
and sodium carbonate buffers (0.1 M) after the pH of each reaction
mixture was determined at 25 °C with a glass electrode. The
corresponding pH values at 140 °C were calculated from the values
determined at 25 °C on the basis of the known heats of ionization of
these buffers (see the Results and the Supporting Information).
olation to 25 °C yielded an estimated rate constant of k = (5.3
2
5
−13 −1
⧧
2
±
0.5) × 10
s
at pH 7 with ΔH = 34.7 ± 1.0 kcal/mol (R
0.997). In these experiments, as in the others described
=
below, the coefficients of variation of the logarithms of the rate
constants at each temperature were less than 5%.
Under the same conditions, 1,1,3,3-tetramethylguanidine
(M4G) decomposed to give mainly dimethylamine along with a
small amount (<10%) of 1,1,3,3-tetramethylurea. The resulting
rate constants yielded a linear Arrhenius plot over the
temperature range from 100 to 180 °C with k = (9.2 ±
RESULTS
■
Rate Constants and Thermodynamics of Activation
for Decomposition of Methylguanidine, Acetamidine,
Acetamide, and Their Methyl-Substituted Derivatives at
pH 7. At early stages in the decomposition of methylguanidine
2
5
1.0) × 10−
12
s
−1
and ΔH = 33.1 ± 1.0 kcal/mol (R = 0.994)
⧧
2
(
MG) (0.02 M) in potassium phosphate buffer (0.1 M, pH 7.0)
(Figure S1 in the Supporting Information).
1
31
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Acetamidine under the same conditions decomposed several
orders of magnitude more rapidly than methylguanidine,
yielding acetamide, which then decomposed more slowly to
acetate (Figure 2). Rate constants for the disappearance of
Figure 2. Disappearance of acetamidine (red) and formation of
acetamide (blue) and finally acetate (green) after heating for 70 h from
Figure 3. Observed logarithm of the rate constant for methylguanidine
decomposition at 140 °C plotted as a function of pH. The 0.1 M
buffers used in this experiment were 0.1 M potassium acetate-d3,
potassium phosphate, sodium borate, and sodium carbonate.
50 to 120 °C in potassium phosphate buffer (0.1 M, pH 7).
acetamidine, monitored from 50 to 100 °C, yielded a linear
−
9
Arrhenius plot and an extrapolated rate constant of 4.3 × 10
−1
⧧
2
Scheme 2. Alternative Pathways of Decomposition of
Methylguanidine
s
at 25 °C with ΔH = 28.6 kcal/mol (R = 0.991). Similar
experiments yielded a linear Arrhenius plot for acetamide
decomposition over the temperature range from 80 to 180 °C
−
11 −1
with an extrapolated rate constant of 7.0 × 10
s
at 25 °C
⧧
2
and ΔH = 28.6 kcal/mol (R = 0.986) (Figure S2 in the
Supporting Information)
Under the same conditions, pivalamidine (2,2,2-trimethyla-
cetamidine) decomposed to pivalamide over the temperature
range from 70 to 130 °C. The Arrhenius plot, which was linear,
−9
−1
2
yielded k25 = 1.1 × 10
s
at 25 °C (R = 0.994). The
decomposition of pivalamide to pivalate, observed over the
temperature range from 120 to 200 °C, yielded a linear
Arrhenius plot and an extrapolated rate constant at 25 °C of 5.4
−
12
−1
×
10
s
at 25 °C (Figure S3 in the Supporting
Information).
effort to distinguish between these alternatives, we examined
the thermodynamics of activation for this reaction and the
effects of replacing the exchangeable hydrogen atoms with
methyl groups.
Influence of pH on the Decomposition of Methyl-
guanidine at 140 °C. The logarithm of the rate constant
observed for the decomposition of methylguanidine was found
to increase linearly over the range from pH 3.6 to pH 10 at 140
The negative entropy of activation observed for the
⧧
°C based on the pH values measured at 25 °C (blue data in
decomposition of methylguanidine (TΔS = −5.3 kcal/mol)
Figure 3). After those pH values had been corrected to 140 °C
using the heats of ionization of the conjugate acids of the
corresponding buffers (see the Supporting Information), these
results yielded a line with a steeper slope (red data in Figure 3).
The values shown in blue are based on buffer pH values
falls toward the lower end of a range of values that have been
reported for conventional hydrolytic processes (−4 to −11
6
kcal/mol). However, its value is considerably more negative
than the near-zero or positive values that have been reported
⧧
for most elimination reactions (e.g., TΔS = +2.0 kcal/mol for
−
1
2
measured at 25 °C and are related by the line log (k/s ) =
the elimination of ammonia from urea).
10
2
0
.69(pH) − 9.85 with R = 0.99. The values shown in red
represent the same results after the pH values measured at 25
C were corrected by taking into account the heats of
ionization of the buffer acids as described in the Supporting
A further indication of the probable mechanism arises from
the observed effects of methyl substitution. When the
exchangeable protons of methylguanidine were replaced by
methyl groups in 1,1,3,3-tetramethylguanidine, the enthalpies
and entropies of activation for decomposition were very similar
to those observed for methylguanidine (Scheme 3 and Table
1). Since elimination is precluded in the case of 1,1,3,3-
tetramethylguanidine, that similarity suggests once again that
both methylguanidine and 1,1,3,3-tetramethylguanidine decom-
pose by hydrolysis rather than elimination.
°
−1
Information. The equation for the red line is log (k/s ) =
10
2
0
.81(pH) −10.39 with R = 0.99. Figure S5 in the Supporting
Information shows the equations and R values for rate
constants observed for each buffer system.
2
DISCUSSION
■
Methylguanidine Decomposition: Elimination or
Hydrolysis? In earlier work, urea was shown to decompose
mainly by elimination rather than hydrolysis. In principle,
Depending on which C−N bond is cleaved, hydrolysis of
1,1,3,3-tetramethylguanidine would be expected to yield one of
two alternative sets of reaction products: tetramethylurea +
2
methylguanidine might decompose by either elimination (A or
B in Scheme 2) or hydrolysis (C or D in Scheme 2). In an
NH (route 1 in Scheme 4) or 1,1-dimethylurea + dimethyl-
amine (route 2 in Scheme 4). Because the two dimethylamine
3
1
32
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Scheme 3. Effect of Methyl Substitution on Guanidine
Decomposition
the fraction that is present in uncharged form at 25 °C (4.0 ×
−
7
10 ).
When the decomposition of methylguanidine was examined
at 140 °C at various pH values, the logarithm of the rate
constant was found to increase linearly with increasing pH over
the pH range from 3.6 to 10 with a slope of 0.81 after
correction for the effect of temperature on the buffer pK values
a
(
Figure 3). In view of the approximations involved in
estimating pH values at 140 °C from values measured at 25
C based on heats of buffer ionization, it seems reasonable to
°
8
suppose that this slope is actually unity. That behavior would
−
be consistent with OH attack on methylguanidinium ion, or
with the kinetically indistinguishable decomposition of un-
charged methylguanidine, via the same tetrahedral intermediate
as shown in Scheme 5. The second-order rate constant for the
Scheme 5. Decomposition of Methylguanidine
Scheme 4. Alternative Pathways of Hydrolysis of 1,1,3,3-
Tetramethylguanidine
reaction of OH with methylguanidinium ion is 5.3 × 10 M−1
−
−6
−
1
s
at 25 °C, whereas the first-order rate constant for
−6
−1
decomposition of the uncharged species is 1.1 × 10
see the Supporting Information for calculations).
Mechanisms of Decomposition of Acetamidine and
s
(
moieties are equally likely be removed in route 2, that route
would be expected to be preferred over route 1 by a factor of 2
for statistical reasons alone. In fact, dimethylamine was
generated as the major product (∼80%) along with a minor
fraction (10%) of tetramethylurea and ammonia. 1,1-
Dimethylurea decomposes relatively rapidly to dimethylamine
and carbamate under the conditions of these experiments
Acetamide. In neutral solution at temperatures between 50
and 120°, acetamidine was found to decompose rapidly to
acetamide, which then underwent relatively slow hydrolysis
(Figure 2). Table 1 shows that pivalamidine, in which
elimination is precluded by methylation, decomposes with a
heat and entropy of activation very similar to those of
acetamidine. That behavior suggests that acetamidineslike
guanidines but unlike ureasdecompose mainly by hydrolysis
rather than elimination. Scheme 6 illustrates the blocking of
elimination reactions in amidines and amides. Likewise, the rate
and temperature dependence of the decomposition of
pivalamide are similar to those of acetamide (Table 1). That
accords with the generally accepted view that amides
decompose in water by hydrolysis rather than elimination, as
implied by the results of the 18O9 exchange experiments on
benzamide by Bender and Ginger.
(
Figure S4 in the Supporting Information) and was therefore
not expected to accumulate.
Influence of pH on Guanidine Hydrolysis. Of the
ionizable groups that are present in biological molecules,
guanidine derivatives are distinguished by the high pK values
a
of their conjugate acids (e.g., 13.6 for unsubstituted
guanidinium ion). However, because the heats of ionization
of guanidinium ions are much larger than those of other
functional groups in biological molecules (e.g., 18.2 kcal/mol
7
for guanidinium ion compared with 13.2 kcal/mol for
methylammonium ion and 8.8 kcal/mol for imidazolium ion),
Effects of Neutral versus Charged Species. Figure 4
compares the rates of decomposition of amides, amidines,
guanidines, and urea in dilute aqueous solution at 25 °C. In the
case of amidines and guanidines, rate constants are shown for
the uncharged species of the substrate and also for the substrate
in all forms that are present at pH 7 (for details, see the
their pK values are extremely sensitive to temperature (e.g.,
a
pK for unsubstituted guanidinium ion decreases from 13.6 at
a
2
5 °C to 9.7 at 140 °C). Accordingly, the fraction of
methylguanidine that is present in the reactive uncharged
form at pH 7 is 1.9 × 10 at 140 °C, which is much larger than
−3
1
33
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Journal of the American Chemical Society
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Scheme 6. Effects of Methyl Substitution on the
Decomposition of Acetamidine (A) and Acetamide (B)
Figure 4. Scale of rate constants for C−N cleavage.
Supporting Information). For both methylguanidine and
acetamidine, protonation is seen to reduce the rate of
hydrolysis by more than 5 orders of magnitude.
Scheme 7. Amidinohydrolases: Water Attack
Comparisons with Enzymatic Reactions. Schemes 7 and
summarize the variety of reactions that are known to be
8
catalyzed by amidinohydrolases and iminohydrolases. Notably,
a single substrate, arginine, decomposes in the presence of
different enzymes to yield different products (ornithine and
citrulline, respectively).
In principle, these reactions might by proceed by direct water
attack or a double displacement mechanism. Amidinohydro-
lases (Scheme 7) are believed to catalyze direct water attack on
their substrates. Thus, arginase and agmatinase use two Mn2+
ions to activate a water molecule for direct attack on the
1
0,11
guanidino group,
whereas creatinase uses an active-site
12
histidine to activate the attacking water molecule. In that
respect, they resemble the mechanisms employed by cytidine
deaminase and the carboxypeptidases, which are also
metalloenzymes. In such cases, the enzyme’s formal affinity
for the altered substrate in the transition state is given by the
1
3
14
6
reciprocal of (k /K )/k . For human arginase, the strongest
cat
m
non
known reversible inhibitor, 2-amino-6-boronohexanoic acid
ABH), has been shown to displace a water molecule from its
(
2
+
10,11
position between the two Mn atoms.
value of ABH for human arginase (5 × 10 M) is much
larger than the value expected for an ideal transition state
analogue [5 × 10
proficiency (k /K )/k in Table 2], suggesting that there
The reported K
i
15
−9
16
17
nase, agmatine deiminase, dimethylarginine dimethylami-
18
nohydrolase (DDAH), and protein-arginine deiminase
(PAD) are believed to act by double displacement
mechanisms. The crystal structures of arginine deiminase
−
18
19
M, the reciprocal of the catalytic
20
cat
m
non
is considerable room for improvement.
In contrast with the enzymes mentioned above, the
iminohydrolases (Scheme 8), which include arginine deimi-
and agmatine deiminase are consistent with the intervention of
a reaction intermediate in which ammonia or dimethylamine is
displaced by the thiol group of an active-site cysteine residue to
1
34
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Journal of the American Chemical Society
Article
Scheme 8. Iminohydrolases: Double Displacement
other C−N-cleaving enzymes that include urease, cytidine
deaminase, and also carboxypeptidase B, the most proficient
proteolytic enzyme for which information is available.
CONCLUSION
■
The decomposition of guanidines, amidines, and amides in
water proceeds by hydrolysis rather than elimination. The
influence of changing pH on the rate of methylguanidine
hydrolysis is consistent with hydroxide attack on the
protonated species or the kinetically indistinguishable hydrol-
ysis of uncharged methylguanidine. Under physiological
conditions, guanidines are hydrolyzed 2 orders of magnitude
more slowly than amides and peptides and 4 orders of
magnitude more slowly than amidines. The rate enhancements
produced by enzymes that catalyze these reactions range from 3
10
14
×
10 -fold to 4 × 10 -fold.
ASSOCIATED CONTENT
■
*
S
Supporting Information
Proton chemical shifts of reactants and products; Arrhenius
plots for 1,1,3,3-tetramethylguanidine, acetamidine, acetamide,
pivalamidine, pivalamide, and 1,1-dimethylurea decomposi-
tions; calculations of the rate constants and thermodynamics
of activation for uncharged guanidines and acetamdines; and
the detailed pH dependence plot for methylguanidine
decomposition. This material is available free of charge via
the Internet at http://pubs.acs.org.
form a covalently bound intermediate, which then undergoes
2
1
hydrolysis. In the hands of Dunaway-Mariano and her
2
2
associates, a detailed mechanism for the action of arginine
deiminase has emerged from experiments involving the
mutation of residues in the enzyme’s catalytic core, which
showed that an aspartate residue and a histidine residue
participate as general acid/base catalysts in the generation and
breakdown of an S-alkyluronium ion intermediate. Moreover,
agmatine deiminase and PAD share a common backbone fold
and catalytic scaffold with arginine deiminase, consistent with a
common mechanism for all three enzymes.
AUTHOR INFORMATION
■
Corresponding Author
richard_wolfenden@med.unc.edu
In double displacement reactions involving the formation of
a covalently bound intermediate, it is not possible to estimate
transition state affinity in the usual sense. However, as pointed
Author Contributions
C.A.L. and R.W. contributed equally to the preparation of the
paper.
2
3
out by Lienhard, it is possible to estimate an equilibrium
constant for “transition state transfer” by comparing the value
of k /K for the enzyme reaction with the second-order rate
Notes
The authors declare no competing financial interest.
cat
m
constant for reaction with a model nucleophile resembling the
nucleophilic residue at the enzyme’s active site.
ACKNOWLEDGMENTS
■
(
This work was supported by the National Institutes of Health
Grant GM-18325).
Table 2 compares the rates of the present guanidine-cleaving
reactions at pH 7 (knon) with the kcat and k /K values that
cat
m
have been reported for their enzyme-catalyzed reaction
counterparts. Arginase, agmatinase, and arginine deiminase
are comparable in catalytic proficiency [(k /K )/knon] with
REFERENCES
(1) Wolfenden, R. Annu. Rev. Biochem. 2011, 80, 645.
■
cat
m
Table 2. Rate Enhancements by C−N-Cleaving Enzymes in Neutral Solution
enzyme
kcat (s−1) k /K (M−1 s−1
)
knon (s−1
)
rate enhancement k /k
catalytic proficiency (k /K )/k (M−1
)
cat M non
cat
M
cat non
Noncovalent Mechanisms
a
1.03 × 105
−13
14
12
13
12
13
17
arginase
100
3
5.3 × 10
1.9 × 10
5.7 × 10
4.7 × 10
1.1 × 10
1.3 × 10
2 × 10
4 × 10
4 × 10
1 × 10
3 × 10
b
3
−13
15
13
16
17
agmatinase
2.1 × 10
5.3 × 10
c
1.9 × 101
−13
creatinase
25
5.3 × 10
d
6
−10
cytidine deaminase
carboxypeptidase B
300
240
2.7 × 10
2.7 × 10
e
6.0 × 106
−11
1.8 × 10
Covalent Mechanisms
f
1.3 × 102
1.0 × 104
2.5 × 10
4.1 × 103
−13
10
12
11
11
14
16
15
15
dimethylargininase
0.014
5.3 × 10
2.6 × 10
6.0 × 10
1.5 × 10
8.5 × 10
3 × 10
2 × 10
5 × 10
8 × 10
g
−13
arginine deiminase
3.2
5.3 × 10
5.3 × 10
h
3
−13
agmatine deiminase
0.082
0.45
i
−13
protein-arginine deiminase (PAD 1)
5.3 × 10
a
b
c
d
e
f
g
h
Data from ref 24. Data from ref 25. Data from ref 26. Data from ref 27. Data from ref 28. Data from ref 29. Data from ref 30. Data from ref
i
3
1. Data from ref 32.
1
35
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