Inactivation of microbial arginine deiminases by L-canavanine

Article abstract of DOI:10.1021/ja0760877

Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P, aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. coli, B. mallei, and G. intestinalis ADIs.

Full text of DOI:10.1021/ja0760877

Published on Web 01/19/2008
Inactivation of Microbial Arginine Deiminases by
L-Canavanine
Ling Li,† Zhimin Li,† Danqi Chen,† Xuefeng Lu,† Xiaohua Feng,†
Elizabeth C. Wright,† Nathan O. Solberg,† Debra Dunaway-Mariano,*,†
Patrick S. Mariano,*,† Andrey Galkin,‡ Liudmila Kulakova,‡ Osnat Herzberg,‡
Kari B. Green-Church,§ and Liwen Zhang§
Department of Chemistry and Chemical Biology, UniVersity of New Mexico,
Albuquerque, New Mexico 87131, Center for AdVanced Research in Biotechnology, UniVersity of
Maryland Biotechnology Institute, RockVille, Maryland 20850, and Mass Spectrometry and
Proteomics Facility, Campus Chemical Instrument Center, The Ohio State UniVersity,
Columbus, Ohio 43210
Received August 13, 2007; E-mail: mariano@unm.edu
Abstract: Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and
L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for
ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate.
The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine
analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One
such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This
substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and
mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing
pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to
direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads
to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies
to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine
and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the
Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI
partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an
inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli,
Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the
L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible
inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa,
E. coli, B. mallei, and G. intestinalis ADIs.
Scheme 1
Introduction
L-Arginine deiminase (ADI) catalyzes the hydrolysis of
L-arginine to form citrulline and ammonia. This reaction is the
first step in the L-arginine degradation pathway (Scheme 1)1-4
5-9
that is present in a wide range of bacterial species
and in
† University of New Mexico.
‡ University of Maryland.
§ The Ohio State University.
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J. AM. CHEM. SOC. 2008, 130, 1918-1931
10.1021/ja0760877 CCC: $40.75 © 2008 American Chemical Society

Inactivation of Microbial Arginine Deiminases
A R T I C L E S
Figure 1. Stereoscopic representation of the C406A mutant of PaADI active site bound with L-arginine prepared using the graphics program Pymol45 and
the X-ray crystallographic coordinates for the structure of the C406A PaADI (L-arginine) complex (PDB ID 2A9G).19
Scheme 2
For the human parasite G. intestinalis, which lacks mito-
chondria, the L-arginine degradation and the glycolysis pathways
serve as the primary source of energy. Flux studies have shown
that the L-arginine degradation sequence is dominant in the
production of ATP.10 Furthermore, gene silencing experiments
carried out with G. intestinalis indicate that this pathway is
required for survival of the trophozites in culture (Kulakova,
unpublished result). In light of the fact that the pathway is not
present in humans, the enzymes involved represent significant
targets for the development of drugs to treat giardiasis.
The investigation described below focuses on the development
of inhibitors for L-arginine deiminase (ADI), the first enzyme
based strategies for inhibitor design for ADI22 and its close
in the L-arginine degradation pathway. The initial phase of our
relatives, protein arginine deiminase (PAD4)23,24 and dimethy-
studies of the structure and catalytic mechanism of this enzyme
larginine dimethylaminohydrolase (DDAH).25,26 Specifically, the
concentrated on the Pseudomonas areuginosa ADI (PaADI).
The structure determination of PaADI set the stage for work
chemical pathway for catalysis by ADI involves addition of the
active site Cys-thiolate27 to the guanidinium carbon of L-arginine
on inhibitor design. Specifically, structural investigations showed
followed by elimination of ammonia to generate the Cys-
that the active site of PaADI is located in a narrow crevice
alkylthiouronium intermediate (Scheme 2). In the second partial
formed by a network of charged residues (Figure 1).18,19 The
reaction in the catalytic pathway, nucleophilic addition of a His
results of site-directed mutagenesis and substrate analog inves-
activated water molecule to the Cys-alkylthiouronium carbon
tigations demonstrated that ligand binding energy is derived
takes place with ensuing elimination of the Cys-thiolate and
from ion pair and hydrogen bonding interactions between active
formation of the product L-citrulline.19,20
site residues and the guanidinium, carboxylate and ammonium
One strategy we have formulated22 for the design of effective
groups of the substrate.19,20 Thus, it is clear that high affinity
ADI inhibitors is founded on the assumption that differences
inhibitors of PaADI must possess the functional groups of
L-arginine.
in the electronic and steric demands of the two partial reactions
involved in the catalytic process could be used advantageously.
The discovery that PaADI employs an active site Cys in
nucleophilic catalysis21 provided the foundation for mechanism-
Accordingly, the approach would employ L-arginine analogs that
are capable of reacting with the ADI Cys-thiolate anion to form
the thiouronium intermediate but, by appropriate design, these
intermediates would either not, or only slowly, undergo the
hydrolysis partial reaction required to regenerate the active form
of the enzyme. Inhibitors adhering to these design features
should have a high potential for effective in ViVo ADI inhibition
owing to their high water solubility, specific targeting of ADI,
and efficient transport into the pathogen via the L-arginine
transporter.28
(9) Schimke, R. T.; Berlin, C. M.; Sweeney, E. W.; Carroll, W. R. J. Biol.
Chem. 1966, 241, 2228-2236.
(10) Schofield, P. J.; Edwards, M. R.; Matthews, J.; Wilson, J. R. Mol. Biochem.
Parasitol. 1992, 51, 29-36.
(11) Linstead, D.; Cranshaw, M. A. Mol. Biochem. Parasitol. 1983, 8, 241-
252.
(12) Yarlett, N.; Lindmark, D. G.; Goldberg, B.; Moharrami, M. A.; Bacchi, C.
J. J. Eukaryotic Microbiol. 1994, 41, 554-559.
(13) Biagini, G. A.; Yarlett, N.; Ball, G. E.; Billetz, A. C.; Lindmark, D. G.;
Martinez, M. P.; Lloyd, D.; Edwards, M. R. Mol. Biochem. Parasitol. 2003,
128, 11-19.
The L-arginine analog selected initially to test this strategy
is L-canavanine (Scheme 2), a compound that differs from the
(14) Silva. L. M.; Baums, C. G.; Rehm, T.; Wisselink; H. J.; Goethe, R.;
Valentin- Weigand, P. Vet Microbiol 2006 115, 117-27.
(15) Gruening, P.; Fulde, M.; Valentin-Weigand, P.; Goethe, R. J. Bacteriol.
2006 188, 361-9.
(16) Makhlin, J.; Kofman, T.; Borovok, I.; Kohler, C.; Engelmann, S.; Cohen,
G.; Aharonowitz, Y. J. Bacteriol. 2007 189, 5976-86.
(17) Seggewiss, J.; Becker, K.; Kotte, O.; Eisenacher, M.; Yazdi, M. R.; Fischer,
A.; McNamara, P.; Al Laham, N.; Proctor, R.; Peters, G.; Heinemann, M.;
von Eiff, C. J. Bacteriol. 2006, 188, 7765-77.
(22) Lu, X. F.; Li, L.; Feng, X. H.; Dunaway-Mariano, D.; Engen, D.; Mariano,
P. S. J. Am. Chem. Soc. 2005, 127, 16412-16413.
(23) Luo, Y.; Arita, K.; Bhatia, M.; Knuckley, B.; Lee, Y. H.; Stallcup, M. R.;
Sato, M.; Thompson P. R. Biochemistry 2006, 45, 11727-36.
(24) Luo, Y.; Knuckley, B.; Lee, Y. H.; Stallcup, M. R.; Thompson, P. R. J.
Am. Chem. Soc. 2006, 128, 1092-3.
(18) Galkin, A.; Kulakova, L.; Sarikaya, E.; Lim, K.; Howard, A.; Herzberg,
O. J. Biol. Chem. 2004, 279, 14001-14008.
(25) Stone, E. M.; Schaller, T. H.; Bianchi, H.; Person, M. D.; Fast, W.
Biochemistry 2005, 44, 13744-52.
(19) Galkin, A.; Lu, X. F.; Dunaway-Mariano, D.; Herzberg, O. J. Biol. Chem.
2005, 280, 34080-34087.
(26) Hong, L; Fast, W, J. Biol. Chem. 2007, 282, 34684-34692.
(27) Li, L.; Li, Z.; Wang, C. H.; Xu, D.; Mariano, P. S.; Dunaway-Mariano,
D.; Guo, H. Biochemistry submitted for review.
(20) Lu, X. F.; Li, L.; Wu, R.; Mariano, P. S.; Dunaway-Mariano, D.
Biochemistry 2006, 45, 1162-1172.
(21) Lu, X.; Galkin, A.; Herzberg, O.; Dunaway-Mariano, D. J. Am. Chem.
Soc. 2004, 126, 5374-5375.
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2063-2070.
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A R T I C L E S
Li et al.
Table 1. Kinetic Rate Constants for ADI-Catalyzed Conversion of
L-Canavanine (Can) to O-Ureido-L-homoserine Plus Ammonia and
L-Arginine (Arg) to L-Citrulline Plus Ammonia
Scheme 3
1
1
ADI
pH
substrate
k
cat (s-
)
K
M (mM)
k
cat/KM (M-1s-
)
PaADI
5.6
Can
Arga
Can
Argb
Can
Argc
Can
Argd
Can
Arge
0.62 ( 0.04 0.7 ( 0.1
6.3 ( 0.1 0.14 ( 0.01
0.46 ( 0.04 2.3 ( 0.4
4.4 ( 0.1 0.09 ( 0.01
0.42 ( 0.03 2.1 ( 0.3
3.2 ( 0.1 0.32 ( 0.2
0.09 ( 0.01 1.2 ( 0.1
1.3 ( 0.1 0.09 ( 0.01
0.32 ( 0.04 1.9 ( 0.2
2.6 ( 0.1 0.16 ( 0.01
8.9 × 102
4.5 × 104
2.0 × 102
4.9 × 104
2.0 × 102
1.0 × 104
7.5 × 101
1.4 × 104
1.7 × 102
1.6 × 104
arthritidis,31 Giardia intestinalis4b) as well as a low affinity
competitive inhibitor of other types of L-arginine-utilizing
enzymes.32
BcADI
EcADI
7.0
6.0
The slow turnover of L-canavanine by ADI is suggestive of
an R-effect29 promoted by the N-O moiety. To determine which
step(s) in the chemical pathway (Scheme 2) is most affected
and to determine the generality of the effect, kinetic analyses
were carried out using ADIs obtained from distantly related
microbes. The initial velocities of PaADI, BcADI, BmADI,
EcADI, and GiADI catalyzed transformations of L-canavanine
to O-ureido-L-homoserine and ammonia were determined as a
function of L-canavanine concentration. The initial velocities
(Vo) were found to increase with increasing L-canavanine
concentration until saturation of the ADI occurred. The data
obtained from these experiments were fitted to Vo ) (kcat[S]/
(Km+[S]) (see Experimental section in Supporting Information)
to yield kcat and Km values, defined in the kinetic sequence given
in Scheme 3. Because L-canavanine is a slowly reacting
substrate, the binding steps in these processes can reach
thermodynamic equilibrium, in which case the Km values are
equivalent to dissociation constants of the ADI(L-canavanine)
complexes.
The kcat and kcat/Km values for the ADI-catalyzed L-canavanine
and L-arginine hydrolysis reactions, measured at the optimal
pH for each ADI, are given in Table 1. The data show that for
all ADIs the respective kcat/Km and kcat values for the L-
canavanine reaction are ca. 100-fold and ca. 10-fold smaller
than those for L-arginine hydrolysis. Thus, replacement of the
N-methylene group in the natural substrate by the N-O group
in L-canavanine reduces the binding affinities to all ADIs as
well as the rates of catalytic turnover.
BmADI 5.6
GiADI 7.5
a From ref 20. Reaction solutions initially contained 0.1 µM PaADI, 0.1-
2.0 mM L-arginine, and 20 mM MgCl2 in pH 5.6, 50 mM K+MES buffer
at 25 °C. b Reaction solutions initially contained 0.03 µM BcADI, L-arginine
(0.5-5 Km), 10 mM ketoglutarate, 0.2 mM NADH, 0.9 mg of glutamate
dehydrogenase (Sigma, type II) in pH 7.0, 50 mM K+HEPES buffer at
25 °C. c Reaction solutions initially contained 1.0 µM EcADI, 0.1-5.0 mM
L-arginine, and 20 mM MgCl2 in pH 6.0, 50 mM Bis-Tris-HCl buffer at
25 °C. d Reaction solutions initially contained 1.0 µM BmADI, 0.1-1.0
mM L-arginine, and 20 mM MgCl2 in pH 5.6, 50 mM K+MES buffer at
25 °C. e Reaction solutions initially contained 1.0 µM GiADI and 0.1-2.0
mM L-arginine in pH 7.5, 50 mM K+HEPES buffer at 25 °C.
native substrate by replacement of an N-methylene group by
a N-oxygen moiety. We believed that this modification would
not significantly impair binding to the active site nor would it
prevent reaction with the Cys-thiolate anion. On the other hand,
the N-O for N-CH2 replacement might alter the hydrolysis
step by (1) introducing an R-effect,29 (2) inducing proton loss
from the thiouronium ion intermediate, and/or (3) causing
misalignment of the electrophilic carbon with respect to the His-
bound water nucleophile.
The results of an in depth kinetic and mass spectral study of
the L-canavanine reaction catalyzed by ADIs derived from
Pseudomonas aeruginosa (PaADI), Bacillus cereus (BcADI),
Escherichia coli (EcADI), Burkholderia mallei (BmADI), and
G. intestinalis (GiADI) are described below. In this investiga-
tion, L-canavanine was shown to be a substrate for all of the
ADIs, which promote its conversion to O-ureido-L-homoserine
by using the same pathway employed for transformation of
L-arginine to L-citrulline (Scheme 2). However, in each case
the Cys-alkylthiouronium intermediate derived from L-canava-
nine partitions between product formation and generation (by
proton loss) of the Cys-alkyl-isothiourea adduct, from which
the regeneration of a catalytically active enzyme is slow, yet
finite. Among the ADIs examined, BcADI is unique because
the Cys-alkylthiourea adduct undergoes irreversible formation
of a Cys-alkylthiocarbamate adduct.
Formation of the Cys-S-alkylthiouronium Intermediate
During Single Turnover Reaction between BcADI and
L-Arginine. By carrying out a single turnover reaction of [14C]-
L-arginine with excess BcADI, in which rapid acid quench is
applied at various time points, we have demonstrated that the
BcADI-catalyzed hydrolysis of L-arginine proceeds via a Cys-
S-alkylthiouronium intermediate (Figure 2A). The time course
data for [14C]-L-arginine consumption and [14C]-L-citrulline
formation as well as for the formation and disappearance of
radiolabeled BcADI were fitted to the kinetic mechanism shown
in Scheme 4 using the kinetic simulation program KINSIM33
to define rate constants for formation of the intermediate from
the BcADI(L-arginine) complex (k2) and for hydrolysis of the
intermediate to form the BcADI(citrulline) complex (k3). For
the reaction of 104 µM BcADI with 49 µM [14C]-L-arginine at
pH 7 and 25 °C, k2 is 50 s-1 and k3 is 9 s-1. The amount of the
Cys-S-alkylthiouronium intermediate reaches a maximum value
of 6% of the BcADI(L-arginine) complex at 30 ms, after which
it decreases (Figure 2A).
Results and Discussion
ADI Catalyzed Hydrolysis of L-Canavanine to Form
Ammonia and O-Ureido-L-homoserine. Earlier, Kihara and
Snell30 observed that L-canavanine is a slow substrate for
catalysis by the ADI derived from Streptocococcus faecalis.
Since that time, it has been shown that this L-arginine analog is
a slow substrate for other ADIs (e.g., from Mycoplasma
Because [14C]-L-canavanine is not readily available, a mass
spectrometric based analysis procedure was used to monitor the
(29) (a) Hudson, R. F. Angew. Chem., Int. Ed. 1973, 12, 36-56. (b) Gerstein,
J.; Jencks, W. P. J. Am. Chem. Soc. 1964, 86, 4655-4663. (c) This effect
is proposed to be responsible for cysteine protease inhibition by azapeptide
analogs (ref 29d). (d) Magrath, J.; Abeles, R. H. J. Med. Chem. 1992, 35,
4279-4283.
(31) Smith, D. W.; Ganaway, R. L.; Fahrney, D. E. J. Biol. Chem. 1978, 253,
6016-6020.
(32) Rosenthal, G. A. Q. ReV. Biol. 1977, 52, 155-178.
(33) Barshop, B. A.; Wrenn, R. F.; Frieden, C. Anal. Biochem. 1983, 130, 134-
(30) Kihara, H.; Snell, E. E. J. Biol. Chem. 1957, 226, 485-495.
145.
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1920 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Inactivation of Microbial Arginine Deiminases
A R T I C L E S
Figure 3. (A) Time and concentration dependence of the inactivation
of BcADI by L-canavanine in 50 mM K+HEPES at pH 7.0 and 25 °C
(0.5 mM (]), 2 mM (0), 4 mM (O), 8 mM (b), and 12 mM (4)).
(B) Plot of kobs vs L-canavanine concentration with data fitting to the
equation kobs ) (kinact[L-canavanine]/(KI + [L-canavanine]) to obtain KI and
kinact values.
Scheme 4
Table 2. Kinetic Rate Constants kinact and KI for Inactivation of
ADI by L-Canavanine and the Ratio of kcat for ADI Catalyzed
L-Canavanine Hydrolysis and the kinact for L-Canavanine Induced
ADI Inactivationa
1
ADI
pH
k
inact (s-
)
KI (mM)
kcat/kinact
PaADI
BcADI
EcADI
BmADI
GiADI
5.6
7.0
6.0
5.6
7.5
5.2 ( 0.5 × 10-3
1.7 ( 0.5
5.0 ( 1.0
5.8 ( 0.7
2.2 ( 0.2
2.2 ( 0.2
120
730
370
70
0.63 ( 0.01 × 10-3
1.15 ( 0.03 × 10-3
1.32 ( 0.03 × 10-3
1.63 ( 0.06 × 10-3
200
a Kinetic data are reported in Figure 3 (text) and Figure S3 (Supporting
Information).
time course for formation and consumption of the Cys-S-
alkylthiouronium intermediate during a single turnover reaction
of the BcADI(L-canavanine) complex (see below). Prior to
initiating these experiments, this method was tested on the
BcADI(L-arginine) single turnover reaction. Accordingly,
reaction of 207 µM BcADI with 96 µM L-arginine at pH 7 and
25 °C was carried out in a rapid quench apparatus. The acid-
quenched samples, collected at reaction times of 5, 30, 100,
and 400 ms, were analyzed by using mass spectrometry. As
can be seen in Figure 2B, only BcADI (46 938 Da) and not the
BcADI thiouronium intermediate (47 096 Da) was observed for
mixtures quenched after 5 and 400 ms reaction times. In contrast,
Figure 2. (A) Time courses for the formation and decay of the 14C-labeled
enzyme from the reaction of wild-type BcADI (b) and Cys400Ser mutant
(O). The dotted curve was obtained by fitting the data points to the kinetic
model shown in Scheme 4 by using the program KINSIM33 and k2 ) 50
s-1, k-2 ) 0.1 s-1, k3 ) 9 s-1, and k-3 ) 0.1 s-1. (B) Electrospray mass
spectra of BcADI thiouronium intermediate from the reaction of 207 µM
BcADI and 96 µM L-arginine in 50 mM K+HEPES at pH 7.0 and 25 °C at
5, 30, 100, and 400 ms. The peak at 46 938 Da is from unmodified BcADI
whereas the peak at 47 096 Da is from the thiouronium intermediate formed
from BcADI and L-arginine. (C) Electrospray mass spectra of BcADI
thiouronium intermediate from the reaction of 207 µM BcADI and 9.6 mM
L-arginine in 50 mM K+HEPES at pH 7.0 and 25 °C at 30 ms. The peak
at 46 938 Da is from unmodified BcADI whereas the peak at 47 096 Da is
from the thiouronium intermediate formed from BcADI and L-arginine.
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A R T I C L E S
Li et al.
Figure 4. Determination of the ratio of rate constants for BcADI turnover
and inactivation by L-canavanine. A plot of the ratio of [L-canavanine]0/
[BcADI]0 vs the fraction of BcADI activity remaining, where [BcADI]0 )
6 µM, reactions at pH 7.0 and 25 °C, and 1 h incubation times.
the 47 096 Da BcADI thiouronium intermediate was observed
at a level of 8% of the total BcADI and 17% of the BcADI-
(L-arginine) complex in mass spectra of reaction mixtures
quenched after 30 and 100 ms. The reliability of these
measurements was verified by carrying out duplicate experi-
ments.
Importantly, the mass spectrometry-based time course for
formation of the thiouronium intermediate and that for the
overall BcADI catalyzed L-arginine hydrolysis reaction compare
favorably to those determined by employing the radioisotope
technique (Figure 2A). The mass spectrum of the quenched
sample from reaction of 207 µM BcADI with 9.6 mM L-arginine
(pH 7 and 25 °C) terminated at 30 ms shows that ca. 16% of
the BcADI has been converted to the thiouronium intermediate
(Figure 2C). Unlike the single turnover reaction, in this process
BcADI is saturated with L-arginine and therefore a larger fraction
of the BcADI accumulates as the thiouronium intermediate.
Inactivation of BcADI by L-Canavanine. A striking obser-
vation made during the steady-state kinetic studies with L-
canavanine is that BcADI is irreversibly inactivated during the
course of multiple turnovers to product. This phenomenon was
further investigated by incubating BcADI with L-canavanine and
monitoring the time- and concentration-dependent loss of
enzyme activity (Figure 3A). A plot of the pseudo-first-order
rate constants for inactivation (kobs, calculated from the data
reported in Figure 3A) vs L-canavanine concentration is shown
in Figure 3B. From inspection of this plot it is clear that kobs
increases with increasing L-canavanine concentration until
BcADI becomes saturated. The data were fitted to the kinetic
equation kobs ) (kinact[I]/(KI+[I]) to obtain the KI and kinact values
(Table 2). The ratio kcat/kinact ) 730 defines the relative
efficiencies of the product-forming and the enzyme-inactivating
pathways. This partition ratio was determined independently by
using the titration method.34 Inactivation reactions were carried
out in which the [canavanine]/[enzyme] ratio ([I]0/[E]0) was
varied between 0 and 1000 (Figure 4). The plot of the [I]0/[E]0
vs Vt/V0 yielded a partition ratio of product formation to enzyme
inactivation of 1617 at pH 7.0 and 25 °C.
Figure 5. (A) Electrospray mass spectra of BcADI present in aliquots taken
from an incubation mixture initially containing 10 µM BcADI and 12 mM
L-canavanine in 50 mM K+HEPES at pH 7.0 and 25 °C. Aliquots were
withdrawn at time 0 s and 23 h, quenched with HCl, and then passed through
a 10-KD filter to remove unreacted L-canavanine. The peak at 46 938 Da
is from unmodified BcADI whereas the peak at 47 098 Da is from the
covalent-adduct formed from BcADI and L-canavanine. (B) A plot of the
percent remaining catalytic activity and product O-ureido-homoserine
formation in BcADI incubated with L-canavanine vs the time period of the
incubation. The incubation mixture initially contained 10 µM BcADI and
12 mM L-canavanine in 50 mM K+HEPES at pH 7.0 and 25 °C. The fraction
of native BcADI remaining is calculated from kinetic (O) and mass
spectrometric (b) data, respectively. The formed product O-ureido-
homoserine (]) was measured using the fixed-time, colorimetric assay
described in the Experimental section (Supporting Information). (C) Time
course for formation of the BcADI S-alkylthiouronium intermediate (E-I),
calculated from mass spectrometric data (b) by assuming [E-I] ) [Etotal]-
Elabeled/(Elabeled + Eunlabeled) (Scheme 4). The curve was simulated with
KinTek. (D) Data obtained by analysis of aliquots of reaction mixtures
initially containing 10 µM BcADI and 12 mM L-canavanine in 50 mM
K+HEPES at pH 7.0 and 25 °C. Time course for formation of BcADI
S-alkylthiocarbamate adduct (E-I*) calculated from mass spectrometric data
(O) and kinetics (O) by assuming [E-I*] ) [Etotal]Elabeled/(Elabeled + Eunlabeled
)
and [E-I*] ) [Etotal] - [Etotal](Vt/V0) (Scheme 7). The curve was simulated
with KinTek.
analysis of acid-quenched reaction mixtures of BcADI (10 µM)
and L-canavanine (12 mM) (pH 7, 25 °C). With increasing
reaction time, the intensity of the mass peak corresponding to
native BcADI (46 938 Da) decreases and that associated with
the covalent adduct (47 098 Da) increases (see Supporting
Information Figure S1). The difference between the masses of
native and inactivated BcADI was found to be 160 Da (Figure
5A and Supporting Information Figure S1).
In Figure 5B is shown an overlay of progress curves for (1)
the formation of the O-ureido-L-homoserine, (2) the fraction of
native BcADI remaining (peak height at 46 938 Da/(peak height
at 47 098 Da + peak height at 46 938 Da)), and (3) the fraction
Comparison of the Reaction Pathways Leading to O-
Ureido-L-homoserine and Ammonia and to Inactived BcADI.
The structure of the covalent adduct present in the L-canavanine
inactivated BcADI was investigated by using mass spectrometric
(34) Silverman, R. Mechanism-based Enzymes InactiVators: Chemistry and
Enzymology; Academic Press: Boca Raton, FL, 1988.
9
1922 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Inactivation of Microbial Arginine Deiminases
A R T I C L E S
Table 3. MALDI-MS Peaks of Proteolytic Cleavage of BcADI with
of BcADI activity remaining. The parallel nature of the latter
two profiles demonstrates that BcADI inactivation correlates
with the formation of the 47 098 Da enzyme adduct. The
progress curve for product formation shows that the L-canava-
nine is consumed within the first 6 h of the reaction. The mass
spectrum measured at 23 h (Figure 5A) reveals that 95% of the
BcADI is present as the 47 098 Da adduct. The reversion of
inactivated BcADI to the native enzyme is estimated to occur
Trypsin Endoproteinasea
fragment
observed mass (Da)
calculated mass (Da)
394-399
246-250
175-180
175-181
400-407
127-134
193-199
400-407b
82-89
599.29
636.30
759.34
915.45
936.45
559.3378
636.3469
759.3936
915.4941
936.4464
948.5002
976.4999
936.4464
948.52
976.47
at a rate <6 × 10-5 min-1
.
1096.47
1063.51
1064.48
1071.53
1141.65
1149.58
1272.58
1279.55
1292.66
1319.75
1335.62
1417.76
1417.76
1452.66
1584.82
1796.93
1805.04
1825.95
2138.18
2230.22
2230.22
2273.33
2542.43
2561.40
2711.64
3121.46
3138.48
3978.02
Taken together, the data demonstrate that L-canavanine
inactivation of BcADI is associated with irreversible formation
of an enzyme adduct with a mass of 47 098 Da that takes place
at a rate that is ca. 1000-fold slower than the conversion of
L-canavanine to O-ureido-L-homoserine. The inactivated
BcADI has the same molecular mass as that of the BcADI
Cys-S-alkylthiouronium species (Scheme 2), the reactive inter-
mediate in the catalytic pathway. However, the inactive adduct
must differ from the thiouronium intermediate because its rate
of formation is ca. 1000-fold slower than that of product
formation.
1063.5531
1064.5413
1071.5952
1141.6767
1149.6164
1272.6544
1279.6071
1292.6529
1319.7323
1319.7323
1417.8167
1417.8274
1292.6529
1584.8572
1796.9394
1805.0397
1825.9311
2138.1333
2230.2447
2230.1371
2273.2691
2542.3393
2561.3034
2711.5395
3122.5484
3122.5484
3980.0558
400-408
175-182
257-266
200-208
183-192
51-60
400-410
61-71
61-71c
82-92
239-250
400-410b
181-192
378-393
235-250
183-196
181-196
61-80
By using the mass spectrometry-based methodology em-
ployed to monitor formation of the Cys-S-alkylthiouronium
intermediate in the L-arginine catalyzed reaction, the short time
period (0-20 s) time course of the appearance of the Cys-S-
alkylthiouronium intermediate in reaction of BcADI with
L-canavanine was determined (Figure 5C) along with the longer
period (0-800 min) time course associated with the BcADI
inactivation reaction (Figure 5D). The observed burst, associated
with formation of the Cys-S-alkylthiouronium intermediate of
mass 47 098 Da, rises to a level of 16% (Figure 5C) at a rate
of 37 min-1. This burst is followed by the accumulation
of the 47 098 Da inactivated enzyme and a concurrent
depletion of the native enzyme (Figure 5D) at a rate of 4.7 ×
183-199
1-20
181-199
294-315
1-24
267-293
267-293c
200-234
a See Experimental section in Supporting Information for details.
b Peptides generated from L-canavanine inactivated BcADI. c Difference
caused by oxidation of the methionine residue, a common occurrence
with this technique. During the MALDI analysis, partially oxidized
methionines undergo facile loss of the methyl sulfoxide to generate a triplet
peaks in which the adjacent pairs are separated by 16 and 48 Da. Three
peptides, M175-R180, N294-R315 and A267-K293, formed by cleavage
of inactivated BcADI undergo Met oxidation. The peaks at 759.3936,
2561.3034, and 3122.5484 Da correspond to the three unmodified
peptides whereas the peaks at 1319.7323, 2577.2983 and 3138.5433 Da
correspond to the same peptides transformed to the corresponding methion-
ine sulfoxides.
10-2 min-1
.
Structure of the L-Canavanine Inactivated BcADI. The
C400S BcADI mutant was used to show that the active site
Cys400 is required for formation of the L-canavanine inactivated
enzyme. As anticipated, kinetic assays for formation of L-
citrulline or O-ureido-L-homoserine from L-arginine or L-
canavanine, respectively, reveal that the BcADI C400S mutant
does not catalyze either deimination reaction. Specifically, 50
µM BcADI C400S, incubated with 10 mM L-arginine or 10
mM L-canavanine at pH 7 and 25 °C for 23 h does not produce
detectable levels (kcat < 1 × 10-6 s-1) of either product.
Likewise, mass spectrometric monitoring of the reaction of
50 µM BcADI C400S with 12 mM L-canavanine at pH 7 and
25 °C for 6 h shows that an enzyme adduct of 160 Da higher
mass is not produced.
As shown in Figure S2 (Supporting Information) and Table 3,
protonated molecular ions (M+H+) for 33 peptides were
detected by using MALDI-TOF analysis of the tryptic digest,
achieving an overall protein sequence coverage of 65%. Based
initially on the X-ray crystal structure of the published PaADI
structure18,19 and now on the recently determined structure of
the BcADI C265S mutant (Galkin and Herzberg, unpublished
data), we know that the BcADI residues Ser402, His270,
Asp272, Glu215, Asp218, and Glu219 are potential nucleophilic
residues located in close proximity to Cys400. The peptides
containing these six residues are included in the 33 tryptic digest
peptides, and the mass spectrometric data show that none of
them are modified.
One possible explanation of the formation of inactivated
BcADI in the reaction with L-canavanine is that the Cys400-
S-alkylthiouronium intermediate in the catalytic pathway un-
dergoes rearrangement by migration of the thiouronium group
to a neighboring nucleophilic residue. This possible scenario
was ruled out by using MALDI-TOF and ESI-MS/MS mass
spectrometry to unambiguously assign the site at which modi-
fication takes place in the L-canavanine inactivation reaction.
L-Canavanine inactivated BcADI was subjected to tryptic
digestion and the resulting peptides were analyzed and se-
quenced by using ESI MS/MS. Individual peptides were
identified by comparing their observed and theoretical masses.
Major peptides containing the native and modified forms of
the active site Cys400 BcADI are observed by mass spectro-
metric analysis of the tryptic digest of L-canavanine inactivated
BcADI (Table 3). Included in this group are C400
-
MSMPIVRKDI410 (mass of unmodified 1292 Da and modified
1452 Da), C400MSMPIVR407 (mass of unmodified 936 Da and
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 1923

A R T I C L E S
Li et al.
Figure 6. ESI-MS/MS spectrum of modified peptide C400MSMPIVR407obtained after tryptic digestion of L-canavanine inactivated BcADI containing several
b-ions, which gain 160 Da mass units due to the covalent adduct formation at Cys400.
Figure 7. MALDI-TOF mass spectra of peptides C400MSMPIVRKDI410 (mass of unmodified 1293 Da and modified 1453 or 1455 Da) obtained from
tryptic digestion of inactivated enzyme generated by treatment of BcADI with L-canavanine in (A) 1:1 H218O and H216O, (B) H218O, and (C) H216O. The
respective 300 µL solutions of 100 µM BcADI in 50 mM K+HEPES (pH 7.0, 1 mM DTT) and 120 mM L-canavanine in 50 mM K+HEPES (pH 7.0) were
first lyophilized to remove all water. Then 300 µL of H218O were added to each solution prior to mixing and incubating for 7 h. Parallel reactions were run
in an identical fashion in H216O and a 1:1 mixture of H218O and H216O. The enzymes obtained in each case were subjected to tryptic digestion followed by
MALDI-TOF analysis of the Cys400 containing peptide C400MSMPIVRKDI410
.
modified 1096 Da). The observation of Cys400 containing
peptide fragments with a 160 Da higher mass is consistent with
Cys400 being the site of L-canavanine promoted modification.
Quantitative analysis of the peaks associated with the unmodi-
fied and modified peptides indicates that ca. 16% of the protein
is modified by L-canavanine. This observation suggests that the
modification is partially reversed during the tryptic digestion
process.
9
1924 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Inactivation of Microbial Arginine Deiminases
A R T I C L E S
Scheme 5
Collision induced dissociation (CID) MS/MS analysis35,36 was
carried out on the C400MSMPIVR407 peptide in order to
demonstrate unambiguously that Cys400 is the site of adduct
formation in L-canavanine inactivated BcADI (Figure 6). The
daughter ions most commonly seen by using this technique are
produced by cleavage of peptide amide bonds with charge
retention at the N-terminal (b ions) and C-terminal (y ions)
positions.37 The mass difference between b or y ions in a given
series corresponds to the mass of the amino acid residue. The
set of all LC/MS/MS data collected on L-canavanine inactivated
BcADI was processed by using the Mascot Distiller software
with standard data processing parameters and searched by
MASCOT (Matrix Science, Boston, MA) and PEAKS (Bioin-
formatics Solutions Inc., Waterloo, ON, Canada). Results from
the best match for the peptide C400MSMPIVR407, which were
manually verified, suggest that canavanine modification occurs
at Cys400. Several y-type ions are observed, including m/z
274.05, 387.33, 484.41, 615.36, 702.48, and 833.50 (y2-y7,
respectively). Five b-type ions, including m/z 235.03, 395.18,
613.35, 823.43, and 922.37 (b2, b2*, b4, b6, b7, respectively)
are also observed with a mass difference of 160 Da, which
corresponds to the attachment of the thiouronium group of
canavanine. The remaining b-type ions are also shifted to 160
Da higher mass. These observations demonstrate that Cys400
is the only site in BcADI at which covalent adduct formation
takes place upon treatment with L-canavanine.
can be seen by viewing these spectra, L-canavanine modification
conducted in H218O results in the production of inactivated
The results of the ESI-MS/MS studies described above give
solid support for the conclusion that the covalent modification
present in inactivated BcADI is at the same Cys residue that
functions as the nucleophile in the catalytic conversion of
L-canavanine to O-ureido-L-homoserine. In order to gain ad-
ditional information for characterization of the inactivated
enzyme, mass spectrometric analysis was carried out on the
peptide C400MSMPIVRKDI410 (mass of unmodified peptide is
1292 Da; mass of modified peptide is 1452 Da) obtained from
tryptic digestion of inactivated enzyme generated by treatment
of BcADI with L-canavanine in H218O, in H216O or in a 1:1
mixture of H218O and H216O. The resulting protein mixtures
were subjected to tryptic digestion followed by MALDI-TOF
analysis of the Cys400 containing peptide C400MSMPIVRKDI.410
In Figure 7 are displayed mass regions for C400MSMPIVRKDI410
generated from native and L-canavanine inactivated BcADI. As
Figure 8. (A) Electrospray mass spectra of PaADI present in aliquots taken
from an incubation mixture initially containing 10 µM PaADI and 1 mM
L-canavanine in 50 mM K+MES at pH 5.6 and 25 °C. Aliquots were
withdrawn at time 0 s, 31.5 min, and 22.5 h and quenched with HCl then
passed through a 10-KD filter to remove unreacted L-canavanine. The peak
at 46 305 Da is due to native PaADI and the peak at 46 465 Da is associated
with the covalent adduct formed between PaADI and L-canavanine (Scheme
7). (B) Percent remaining PaADI activity in reaction of 10 µM PaADI and
1 mM L-canavanine at pH 5.6 and 25 °C. The fraction of native PaADI
remaining is calculated from kinetic (O) and mass spectrometric (b) data.
(C) Product O-ureido-homoserine formation in PaADI incubated with
L-canavanine vs the time period of the incubation. The incubation mixture
initially contained 10 µM PaADI and 1 mM L-canavanine in 50 mM K+-
MES at H 5.6 and 25 °C. O-Ureido-homoserine was measured using the
fixed-time, colorimetric assay described in the Experimental section
(Supporting Information). (D) Time course for formation and consumption
of the inactive enzyme calculated from inactivation kinetic (O) and mass
spectrometric (b) data by assuming [E-I*] ) [Etotal] - [Etotal](Vt/V0) and
[E-I*] ) [Etotal]Elabeled/(Elabeled + Eunlabeled). The curve was simulated with
KinTek.
(35) Raymackers, J.; Daniels, A.; De Brabandere, V.; Missiaen, C.; Dauwe, M.;
Verhaert, P.; Vanmechelen, E.; Meheus, L. Electrophoresis 2000, 21, 2266-
2283.
(36) Gustafsson, E.; Thoren, K.; Larsson, T.; Davidsson, P.; Karlsson, K. A.;
Nilsson, C. L. Rapid Commun. Mass Spectrom. 2001, 15, 428-432.
(37) Biemann, K. Appendix 5. Nomenclature for peptide fragment ions (positive
ions). Methods Enzymol. 1990, 193, 886-887.
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 1925

A R T I C L E S
Li et al.
Figure 9. MALDI-TOF mass spectra of peptides GGGHC406MTCPIVRDPIDY418 obtained by tryptic digestion of PaADI, partially inactivated by reaction
with L-canavanine in (A) 1:1 H218O and H216O, (B) H218O, and (C) H216O, followed by alkylation of unmodified cysteines with iodoacetamide. Respective
450 µL solutions of 100 µM PaADI in 50 mM MES (pH 5.6, 20 mM MgCl2) and 20 mM L-canavanine in 50 mM MES (pH 5.6, 20 mM MgCl2) were first
lyophilized to remove all water. Then 450 µL of H218O were added to each solution prior to mixing and incubating for 7 min. Parallel reactions were run
in an identical fashion in H216O and a 1:1 mixture of H218O and H216O. The protein mixtures obtained were subjected to tryptic digestion followed by
MALDI-TOF analysis of the Cys406 containing peptide GGGHC406MTC409PIVRDPIDY418 in which Cys409 contains an S-acetamido group (mass 57).
BcADI that yields a modified peptide, which is 2 mass units
higher than that generated by tryptic digestion of inactivated
BcADI formed in H216O.
PaADI, EcADI, BmADI, and GiADI Reactions with
L-Canavanine. To test the generality of L-canavanine inactiva-
tion of ADI, inactivation reactions of PaADI, EcADI, BmADI,
and GiADI were monitored at the pH optimum for catalysis of
L-arginine hydrolysis to determine the time and concentration
dependent loss of enzyme activity (Supporting Information
Figure S3). The plots of the pseudo-first-order rate constants
of inactivation (kobs) as a function of L-canavanine concentration
are hyperbolic, showing that the rate of inactivation is slower
than the rate of formation of the ADI(L-canavanine) complex
(Figure S3 Supporting Information). The data were used to
obtain the KI and kinact values provided in Table 2. Although
some variation can be seen in the KI and kinact values for the
five ADIs, no correlation with either structure divergence or
pH optima appears to exist. The kinact value for each ADI is
considerably smaller than the value of kcat for product formation
(Tables 1 and 2).
The most remarkable finding arising from these studies is
that, in contrast with BcADI where inactivation by L-canavanine
is essentially irreversible, inactivation of PaADI, EcADI,
BmADI, and GiADI is reversible. A thorough study of the
PaADI catalyzed reaction of L-canavanine was carried out to
define the kinetics of the inactivation and reactivation processes.
The results, summarized in Figure 8, were obtained for the
reaction of PaADI (10 µM) with a sub-saturating concentration
(1 mM) of L-canavanine at pH 5.6 and 25 °C. In Figure 8A are
shown selected mass spectrometric data measured for reaction
mixtures quenched at various times (the complete data set is
shown in Supporting Information Figure S4). The changes,
Taking into account the molecular mass of inactivated BcADI,
the requirement for participation of Cys400 in its formation,
and the fact that a water derived oxygen is incorporated into
the adduct, we assign the structure of the inactivated enzyme
the BcADICys400-alkylthiocarbamate (2, Scheme 5). We envi-
sion the formation of the thiocarbamate to occur from the
tetrahedral adduct (1, Scheme 5) upon elimination of ammonia.
The tetrahedral adduct 1 might be formed through a pathway
that branches from the chemical pathway leading from the
substrate L-canavanine to the product O-ureido-L-homoserine
or alternatively, it might be formed through a pathway that
originates with the product O-ureido-L-homoserine. Either route
would lead to formation of a Cys400-S-alkyl thiocarbamate
derivative 2 of BcADI, which has a mass of 47 098 Da which
is 160 (from reaction in H216O) or 162 (from reaction in H218O)
mass units higher than that of the native enzyme. To distinguish
between these two possibilities, conditions for the occurrence
of the back reactions were simulated by incubating BcADI with
O-ureido-L-homoserine and ammonia for 1 h under the same
conditions used for inactivation of BcADI by L-canavanine
(Figure 3A). The observation that the activity of BcADI remains
unchanged (data not shown) under these conditions leads to the
conclusion that inactivated BcADI is not formed by the reaction
of the BcADI with the O-ureido-L-homoserine, and therefore
that it is formed via a minor pathway which branches from the
pathway leading from L-canavanine to O-ureido-L-homoserine.
9
1926 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Inactivation of Microbial Arginine Deiminases
A R T I C L E S
Figure 10. ESI-MS/MS spectrum of the peptide G402GGHC406MTC409PIVRDPIDY418 after L-canavanine inactivation of PaADI, Cys409 alkylation with
iodoacetamide, and tryptic digestion.
taking place over the 0-23 h time course of the reaction, reveal
the formation of an enzyme adduct of mass 46 465 Da, which
corresponds to the calculated mass of a PaADI Cys-alkylthiou-
rionium intermediate (Scheme 2). This adduct accumulates to
ca. 70% maximally and then slowly reverts to native PaADI.
Shown in Figure 8B is a plot of the fraction of the remaining
activity of PaADI vs reaction time. Enzyme activity is first lost
and then regained. Also shown in Figure 8B is the time profile
of the fraction of native PaADI (46 305 Da) remaining as
determined by mass spectrometric analysis of acid quenched
aliquots. The activity loss and build-up of the 46 465 Da mass
species are closely correlated as are the regeneration of activity
and the conversion of the 46 465 Da enzyme adduct back to
the native PaADI.
The progress curve for formation of the O-ureido-L-ho-
moserine product reaches saturation at 30 min (Figure 8C),
suggesting that the level of inactive enzyme is determined by
(1) the prevailing concentration of canavanine, (2) the rates at
which the thiouronium intermediate partitions to product and
to inactive enzyme, and (3) the rate at which the inactive enzyme
returns to the thiouronium ion intermediate and/or undergoes
hydrolysis to regenerate active enzyme. The time course for
the formation and consumption of the inactive enzyme species,
calculated from the mass spectrometric and the kinetic data
(Figure 8B), are shown in Figure 8D. The time course data
(Figures 8C and 8D) were fitted using the simulation program
KinTek (KinTek Corp; Austin, Texas) and the calculated the
kcat, kinact, Km, and KI values (Tables 1 and 2) to set limits on
the individual rate constants. This treatment gave rate constants
for the conversion of the PaADI(L-canavanine) complex to the
ADI-thiouronium intermediate (k2 ≈ 40 min-1) and for conver-
sion of the ADI-thiouronium intermediate to product (k3 ≈ 40
min-1). Because we do not have a direct measurement of the
formation of the ADI-thiouronium intermediate, the values of
these rate constants are not well constrained.
The rate constant, derived from the data of Figure 8, for the
conversion of the PaADI(L-canavanine) complex to the inactive
enzyme is defined as 4.0 × 10-1 min-1, whereas the rate
constant for conversion of the inactive enzyme to active enzyme
is the product complex is 8.5 × 10-3 min-1. The difference
between these rate constants and between the corresponding rate
constants measured for the BcADI reaction (4.7 × 10-2 min-1
and <6 × 10-5 min-1, respectively) nicely accounts for the
observed L-canavanine time-controlled inhibition of PaADI and
the irreversible inactivation of BcADI.
In order to characterize the structure of the covalent adduct,
mass spectrometric analysis was carried out on the peptides
GGGHC406MTCPIVRDPIDY418, obtained by tryptic digestion
of PaADIs that are partially inactivated by reaction with
L-canavanine in H218O, in H216O or in a 1:1 mixture of H218O
and H216O. The protein mixtures were treated with iodoaceta-
mide to alkylate the unmodified cysteine residues, before
subjecting them to tryptic digestion and MALDI-TOF analysis
of the Cys406 containing peptide GGGHC406MTC409
-
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 1927

A R T I C L E S
Li et al.
Figure 11. (A) Percent remaining GiADI activity in reaction of 5 µM GiADI and 2 mM L-canavanine at pH 7.5 and 25 °C. (B) Product O-ureido-
homoserine formation catalyzed by GiADI incubated with L-canavanine vs the time period of the incubation. The incubation mixture initially contained 5
µM GiADI and 2 mM L-canavanine at pH 7.5 and 25 °C. O-Ureido-homoserine (b) was measured using the fixed-time, colorimetric assay described in the
Experimental section (Supporting Information). (C) Time course for formation and consumption of the inactive enzyme calculated from inactivation kinetic
data by assuming [E-I*] ) [Etotal] - [Etotal](Vt/V0). The curve was simulated with KinTek.
Table 4. Simulated Rate Constants for the Formation of the
Inactive ADI Adduct (*kinact) and for Its Conversion to Product
a
(kreact
)
1
1
ADI
*kinact (min-
)
k )
react (min-
PaADI
BcADI
EcADI
BmADI
GiADI
0.4
0.0085
0.047
0.073
0.08
0.1
<5 × 10-5
0.05
0.02
0.0032
a Fitted kinetic data are shown in Figures 5, 8, and 11 of the text and
Figure S3 of the Supporting Information.
Scheme 6
PIVRDPIDY418. This peptide contains the acetamido group
derived from iodoacetamide (mass 57) and the urea or carbamate
group derived from L-canavanine. As can be seen by viewing
the spectra of the peptides displayed in Figure 9, the peptides
derived from PaADI reacted with L-canavanine in H218O vs
H216O have the same mass. Therefore, the covalent adduct does
not contain a solvent derived oxygen atom.
Figure 12. pH profiles for kinact (A) and KI (B) of L-canavanine induced
BcADI inactivation and for kcat (C) and Km (D) of BcADI catalyzed
L-canavanine hydrolysis to O-ureido-homoserine and ammonia. For details
see the Experimental section (Supporting Information).
For the purpose of demonstrating that the inactivation reaction
of L-canavanine with PaADI is not due to translocation of the
thiouronium group from its initial Cys 406 position to Cys409
or another nucleophilic site, collision induced dissociation (CID)
canavanine thiouronium model described in the following
section, suggests that the modified PaADI is the Cys406-S-
alkylthiourea adduct formed by proton loss from the Cys406-
S-alkylthiouronium ion intermediate.
MS/MS analysis35,36 was carried out on the G402GGHC406
-
MTC409PIVRDPIDY418 peptide. Results from the best match
for the peptide (Figure 10), which were manually verified in a
manner described above, show that the L-canavanine modifica-
tion occurs at the catalytic Cys406. The low pKa of a L-
The kinetics of the L-canavanine inactivation process opera-
tive in the other ADIs were briefly examined. GiADI is of
particular interest because of its sequence divergence from the
bacterial ADIs, and its optimal activity pH range from 5 to 7.5,
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1928 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Inactivation of Microbial Arginine Deiminases
A R T I C L E S
Figure 13. Superpositions of BcADI and PaADI structures. (A) Stereoscopic view of a ribbon representation of the superposed PaADI and BcADI monomers.
PaADI is depicted in pink color and BcADI is depicted in gray color. The regions exhibiting the largest difference associated with the active site correspond
to two loops. For PaADI, these loops are highlighted in red. For BcADI, the loops are largely disordered and the ordered terminal residues are labeled and
highlighted in blue. Residue numbering corresponds to BcADI. The catalytic cysteine residue is shown in stick model. (B) Stereoscopic view of the superposed
PaADI and BcADI active site region using the same color scheme as in (A). Atomic colors are as follows: oxygen, red; nitrogen, blue; sulfur, yellow;
carbon, light blue for PaADI and pink for BcADI. Residue numbers correspond to BcADI, except that Arg401 of PaADI is labeled R401-Pa (the corresponding
Arg397 in BcADI is disordered). The figures were generated usin the X-ray crystallographic coordinates for the structure of the C406A PaADI (L-arginine)
complex (PDB ID 2A9G)19 and the apo BcADI C265S mutant (Galkin and Herzberg, unpublished results).
Scheme 7
which distinguishes both GiADI and BcADI from PaADI,
BmADI, and EcADI (optimal activity pH range <6).20,27 The
fact that BcADI and PaADI have pH optima of 7.0 and 5.6,
respectively require that measurements of their inactivation
kinetics be carried out under different pH conditions. Conse-
quently, studies with GiADI provide information about whether
differences observed between L-canavanine inactivation of
BcADI and PaADI is a result of differences in reaction pH. In
Figure 11A is shown the time course for the loss and regain of
GiADI (5 µM) activity in reaction with 2 mM L-canavanine in
50 mM K+HEPES buffer (pH 7.5) at 25 °C. The time course
for the L-canavanine to O-ureido-L-homoserine conversion
(Figure 11B) shows that the activity of GiADI decreases by ca.
45% with incubation time and following the depletion of
L-canavanine (ca. 40 min) 100% of the original enzyme activity
is slowly regained. The time course for formation and consump-
tion of the inactive enzyme (shown in Figure 11C), was fitted
using KinTek to define the rates of GiADI thiouronium ion
intermediate formation and conversion to O-ureido-L-homoserine
equivalent at ∼61 min-1, the rate of GiADI inactivation at 1 ×
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J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 1929

A R T I C L E S
Li et al.
10-1 min-1, and the rate of reactivation at 3.2 × 10-3 min-1
.
Scheme 8
Thus, like PaADI, GiADI undergoes L-canavanine promoted
time controlled inhibition, not irreversible inactivation, sup-
porting the conclusion that the solution pH is not the factor
determining the unique behavior of BcADI. The same conclu-
sion is evident from the pH profile for L-canvanine inactivation
which shows efficient irreversible inactivation at pH 5.6 (see
below).
Scheme 9
Similar kinetic characteristics were observed for BmADI
(reaction mixtures of 20 µM BmADI and 5 mM L-canavanine
at pH 5.6) and EcADI (20 µM EcADI with 10 mM L-canavanine
at pH 6.0) (Figure S5, Supporting Materials). Like with PaADI
and GiADI, in both cases L-canavanine inactivation is reversible.
The individual rate constants for inactivation and reactivation
for BmADI and EcADI are listed in Table 4 along with those
for the other ADIs. The ratios of these rate constants define the
efficiency of L-canavanine inhibition of each ADI.
Scheme 10
group, and the active site His in the BcADI thiouronium ion
intermediate. Consequently, the pH profiles cannot be used to
define the role played by proton loss from the L-canavanine
derived thiouronium intermediate in the inactivation process.
In any event, assuming that the thiouronium ion formed in
the ADI active site by reaction with L-canavanine has a pKa
that is similar to that of the model 5, it is likely that
deprotonation of the intermediate to form a corresponding
isothiourea would be facile.
Chemical Models of the L-Arginine and L-Canavanine
Derived Thiouronium Intermediates. The ADI catalyzed
deimination reaction pathway shown in Scheme 2 contains a
hydrolysis step that involves attack of a bound water molecule
on a thiouronium ion intermediate.19,20,38 Because the pKa of
the model conjugate acid of S-methylisothiourea (3, Scheme
6) is 9.8,39 it is reasonable to expect that the L-arginine derived
ADI Cys-alkylthiouronium ion intermediate will be protonated
at the pH optimum for ADI catalysis (pH 5-7.5).20,27 Cor-
roboration for this hypothesis was sought by examining the
model compounds S-benzyl-N-benzylthiouronium bromide (4)
and its N-benzyloxy analog 5 (Scheme 6). The syntheses of
these substances are described in the Supporting Information.
The thiouronium pKa values of 4 and 5 were determined by
measuring 13C NMR chemical shifts for the corresponding
thiouronium carbon atoms as a function of solution pH (pH
profiles shown in Figure S6A and B). The L-arginine thiouro-
nium model 4 was shown to have a pKa value of 8.5 ( 0.2
which is close to the value determined for the conjugate acid
of S-methylisothiourea. In contrast, 5, a model of the L-
canavanine thiouronium salt, has a remarkably lower pKa value
of 3.0 ( 0.1. Thus, the replacement of the N-CH2 by an N-O
moiety has a pronounced effect on the pKa of the thiouronium
group. Although inexplicably large, this change is consistent
with differences between the pKa of L-arginine (12.5) and
L-canavanine (6.6).40 By using the 13C NMR monitored pH
titration method (Figure S6C), the L-canavanine pKa was
determined to be 7.8 ( 0.1, a value not too different from the
one measured earlier using a potentiometric based method.37
pH Profiles for L-Canavanine Inactivation of BcADI. The
pH profiles for kinact, KI, kcat, and Km of the L-canavanine reaction
with BcADI are not easily interpreted (Figure 12). To the extent
that KI and Km measure L-canavanine-BcADI binding affinities,
the pH profiles reflect contributions made by ionization of the
uncomplexed L-canavanine guanidinium group and ionizable
groups in the active site of the enzyme. Also, the kinact and kcat
pH profiles are composites of ionization associated with the
L-canavanine guanidinium group, the active site ionizable groups
in the BcADI(L-canavanine) complex, the Cys-alkylthiouronium
Conclusion
A unified model which accounts for the reversible L-
canavanine covalent modification of PaADI, EcADI, BmADI,
and GiADI (which leads to time controlled inhibition) and the
irreversible L-canavinine covalent modification of BcADI,
(which leads to permanent inhibition) is presented in Scheme
7. Based on an analysis of the acid dissociation constants of
the chemical models (Scheme 6), it is likely that the Cys-alkyl-
thiouronium ion formed in the reaction of L-canavanine will
undergo ready deprotonation in competition with its reaction
with His-activated water to form the Cys-alkyl-isothiourea
adduct. We expect that this less electrophilic species will react
more slowly with water than the Cys-alkylthiouronium inter-
mediate to from O-ureido-L-homoserine and the active form of
the enzyme.
In the reaction of BcADI with L-canavanine, an alternative
pathway is available to the isothiourea generated by deproto-
nation of the initially formed Cys-alkyl-thiouronium intermedi-
ate. This process leads to generation of a thiocarbamate adduct
(Scheme 7). Although unique among the L-canavanine + ADI
reactions explored in this investigation, hydrolytic reactions of
this type have precedent in the chemistry of thiouronium salts
and closely related thioimidate esters. For example, in contrast
to the typical reactions of thiouronium salts in basic solution,
which form thiol and urea products, Klayman and his co-
workers41 observed that treatment of the N-electron withdrawing
group substituted S-methyl analogs (6 in Scheme 8) with water
results in formation of the corresponding thiocarbamates 7. In
a related fashion, studies42 of the pH dependent hydrolysis of
thioimidates (8, Scheme 9) have shown that thioesters and
amines are produced predominantly at low pH and that amides
(38) Das, K.; Butler, G. H.; Kwiatkowski, V.; Clark, A. D., Jr.; Yadav, P.;
Arnold, E. Structure 2004, 12, 657.
(39) Albert, A.; Goldacre, R.; Phillips, J. J. Chem. Soc. 1948, 2240.
(40) Tomiyama, T. I. J. Biol. Chem. 1935, 111, 45; Boyar, A.; Marsh, R. E. J.
Am. Chem. Soc. 1982, 104, 1995-1923.
(41) Klayman, D. L.; Shine, R. J.; Bower, J. D. J. Org. Chem. 1972, 37, 1532-
1537.
(42) Chaturvedi, R. K.; MacMahon, A. E.; Schmir, G. L. J. Am. Chem. Soc.
1967, 89, 6984-6993.
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1930 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Inactivation of Microbial Arginine Deiminases
A R T I C L E S
and mercaptans are the major products generated at high pH.
These findings suggest that tetrahedral intermediates (9, Scheme
10) produced by water addition to thiouronium salts/isothioureas
can undergo loss of either thiols and amines, depending on the
nature of the environment or substituents.
binding residues present in the active site of PaADI are
conserved in the active site of BcADI. However, the active site
of BcADI exhibits two disordered loop regions that span
residues 394-395 and residues 33-40. In contrast, these regions
are ordered in the structure of PaADI. Assuming that the crystal
structures reflect the mobility of a loop that is in solution, the
observations indicate that the active site of BcADI is more
accessible to bulk solvent than that of PaADI. This difference
might underlie the irreversible partitioning of the L-canavanine
+ BcADI derived isothiourea intermediate to the inert thiocar-
bamate adduct. Clearly, more work is required to clarify this
issue.
Perhaps more relevant to the observations made in our studies
with BcADI are the results of investigations of active site Cys
thiolate mediated nitrile hydrolysis catalyzed by papain and by
asparagine synthetase B. These studies have shown that the
tetrahedral intermediate formed by attack of water on protonated
thioimidate intermediates can eliminate either ammonia or the
Cys thiolate, depending on the particular involvement of active
site residues in wild type and mutant enzymes.43,44 These
findings prompted us to carry out a comparison of the active
site of PaADI18,19 with that of BcADI, the latter of which has
only recently become available (Galkin and Herzberg, unpub-
lished data) (Figure 13). As expected, the catalytic and substrate
Acknowledgment. Support for this study by NIH (DDM, OH,
PSM: AI-059733; KBG-C: CA-16058) is gratefully acknowl-
edged. We also thank Ken Johnson for helpful suggestions and
for use of the pilot KinTek simulation program.
Supporting Information Available: Experimental section,
Figures S1-6, and NMR spectra of 4 and 5. This material is
available free of charge via the Internet at http://pubs.acs.org.
(43) Dufour, E.; Storer, A. C.; Menard, R. Biochemistry 1995, 34, 16382-16388.
(44) Boehlein, S. K.; Rosa-Rodriguez, J. G.; Schuster, S. M.; Richards, N. G.
J. Am. Chem. Soc. 1997, 119, 5785-5791.
(45) http://pymol.sourceforge.net, DeLano Scientific LLC, San Carlos, California.
JA0760877
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