Structural and enzyme activity studies demonstrate that aryl substituted 2,3-butadienamine analogs inactivate Arthrobacter globiformis amine oxidase (AGAO) by chemical derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor

Article abstract of DOI:10.1016/j.bbapap.2010.12.016

Copper amine oxidases (CAOs) are a family of redox active enzymes containing a 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor generated from post translational modification of an active site tyrosine residue. The Arthrobacter globiformis amine oxida

Full text of DOI:10.1016/j.bbapap.2010.12.016

Biochimica et Biophysica Acta 1814 (2011) 638–646
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Structural and enzyme activity studies demonstrate that aryl substituted
2,3-butadienamine analogs inactivate Arthrobacter globiformis amine oxidase
(AGAO) by chemical derivatization of the 2,4,5-trihydroxyphenylalanine quinone
☆
(TPQ) cofactor
Karin Ernberg a, Bo Zhong b,1, Kristin Ko c,1, Larry Miller d,1, Yen Hoang le Nguyen a, Lawrence M. Sayre e,2
,
e,⁎⁎
J. Mitchell Guss a, , Irene Lee
⁎
a
School of Molecular Bioscience, The University of Sydney, NSW 2006, Australia
Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
Department of Chemistry, Westminster College, New Wilmington, PA 16172, USA
b
c
d
e
Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Copper amine oxidases (CAOs) are a family of redox active enzymes containing a 2,4,5-trihydroxypheny-
lalanine quinone (TPQ) cofactor generated from post translational modification of an active site tyrosine
residue. The Arthrobacter globiformis amine oxidase (AGAO) has been widely used as a model to guide the
design and development of selective inhibitors of CAOs. In this study, two aryl 2,3-butadienamine analogs,
racemic 5-phenoxy-2,3-pentadienylamine (POPDA) and racemic 6-phenyl-2,3-hexadienylamine (PHDA),
were synthesized and evaluated as mechanism-based inactivators of AGAO. Crystal structures show that both
compounds form a covalent adduct with the amino group of the substrate-reduced TPQ, and that the chemical
structures of the rac-PHDA and rac-POPDA modified TPQ differ by the allenic carbon that is attached to the
cofactor. A chemical mechanism accounting for the formation of the respective TPQ derivative is proposed.
Under steady-state conditions, no recovery of enzyme activity is detected when AGAO pre-treated with rac-
PHDA or rac-POPDA is diluted with excess amount of the benzylamine substrate (100-fold Km). Comparing
the IC50 values further reveals that the phenoxy substituent in POPDA offers an approximately 4-fold increase
in inhibition potency, which can be attributed to a favourable binding interaction between the oxygen atom in
the phenoxy group and the active site of AGAO as revealed by crystallographic studies. This hypothesis is
corroborated by the observed N3-fold higher partition ratio of PHDA compared to POPDA. Taken together, the
results presented in this study reveal the mechanism by which aryl 2,3-butadienamines act as mechanism-
based inhibitors of AGAO, and the potency of enzyme inactivation could be fine-tuned by optimizing binding
interaction between the aryl substituent and the enzyme active site.
Received 2 November 2010
Received in revised form 23 December 2010
Accepted 30 December 2010
Available online 6 January 2011
Keywords:
Allenic amine
Arthrobacter globiformis amine oxidase
Mechanism-based inactivator
Crystal structure
Inhibition potency
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Copper amine oxidases (CAOs) are a family of redox active enzymes
that catalyze the oxidative deamination of primary amines to aldehydes,
with concomitant reduction of O2 to H2O2 (Scheme 1 [1–3]). CAOs are
nearly ubiquitous in nature, as they are found in plants, most yeasts,
microorganisms, and mammals but somewhat surprisingly not in
archaea. Most CAOs contain a 2,4,5-trihydroxyphenylalanine quinone
(TPQ) cofactor, which is derived from the post-translational modifica-
tion of an active site tyrosine residue [4–5]. During enzyme catalysis, the
primary amine substrate condenses with TPQ to form a substrate Schiff
base adduct (SSB). The SSB then tautomerizes to a product Schiff base
Abbreviations: CAO, copper-containing amine oxidase; AGAO, Arthrobacter globi-
formis amine oxidase; PHDA, racemic 6-phenyl-2,3-hexadienylamine; POPDA, racemic
5-phenoxy-2,3-pentadienylamine; PSB, product Schiff base; SSB, substrate Schiff base;
TPQ, trihydroxyphenylalanine quinone
☆
This work was supported in part by a grant from NIH (GM 48812) and an award
from the American Diabetes Association (1-06-RA-117) to L.M.S. and I.L., and by a
Linkage grant from the Australian Research Council (LP0669658) to J.M.G.
⁎
Corresponding author: Tel.: +61 2 9351 4302; fax: +61 2 9351 5858.
⁎⁎ Corresponding author: Tel.: +1 216 368 6001; fax: +1 216 368 3006.
E-mail addresses: Mitchell.guss@sydney.edu.au (J.M. Guss), IXL13@case.edu (I. Lee).
Present address.
In memory of Professor Lawrence M. Sayre, July 25, 1951 to May 8, 2009.
1
2
(PSB), which upon hydrolysis affords a reduced aminated TPQ (TPQamr
)
1570-9639/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2010.12.016

K. Ernberg et al. / Biochimica et Biophysica Acta 1814 (2011) 638–646
639
Scheme 1. Proposed mechanism for the TPQ-catalyzed oxidation of primary amine in copper amine oxidases.
and an aldehyde. These events are collectively known as the reductive
half-reaction. In the oxidative half-reaction, the TPQamr is oxidized by O2
to form quinonimine, which then undergoes hydrolysis to release
ammonia to regenerate the TPQ cofactor.
study provide a structural framework to guide the design of enzyme
selective mechanism-based allenic amine inhibitors for the respective
enzyme that may benefit pharmaceutical developments.
In designing selective inhibitors against CAOs, a mechanism-based
strategy targeting the reductive half-reaction has been exploited [6].
In particular, the approach of incorporating an electrophile adjacent to
the substrate amino group in primary amines containing an aromatic
group led to the synthesis of 4-aryloxy 2-butynamines as selective
mechanism-based inhibitors [7]. Previous crystallographic studies of
AGAO treated with two 4-aryloxy-2-butynamine analogs revealed a
mechanism of enzyme inactivation involving covalent attachment of
the α,β-unsaturated aldehyde turnover product generated from
amine substrate oxidation to the amino group of TPQamr derived
from the first half-reaction.
To further investigate the kinds of electrophile that can be used
to design mechanism-based inhibitors of CAOs, two aryl 2,3-
butadienamine analogs, racemic 5-phenoxy-2,3-pentadienylamine
(POPDA) and racemic 6-phenyl-2,3-hexadienylamine, (PHDA, Fig. 1)
were synthesized. These two compounds contain the 1-amino-2,3-
butadiene moiety that has been shown by Qiao and coworkers to
inactivate bovine plasma amine oxidase (BPAO) with higher potency
compared to 1-amino-3-butyne amine [8]. As aromatic amines con-
taining the 1-amino-3-butyne amine moiety are mechanism-based
inactivators of AGAO, it is plausible that rac-PHDA and rac-POPDA will
inhibit AGAO. To test this hypothesis, the crystal structures of AGAO
treated with rac-PHDA and rac-POPDA, as well as the enzyme
inhibition profiles were determined. The findings presented in this
2. Materials and methods
2.1. Synthesis of racemic 6-phenyl-2,3-hexadienylamine hydrochloride
(PHDA)
A dioxane solution of the t-boc protected propargylamine was
refluxed with 3-phenylpropanal, diisopropylamine and freshly pre-
pared CuBr under Argon for 12 h using the method reported in by
Casara et al [9]. The reaction was then quenched with 1 N acetic acid
followed by extraction with diethyl ether. The organic layer was dried
with anhydrous Na2SO4, prior to rotary evaporation under reduced
pressure. The resulting crude reaction mixture was purified by silica
gel flash chromatography to produce the t-boc protected 6-phenyl-
hexa-2,3-dienylamine. The t-boc group was deprotected with 3 N HCl
at room temperature. Removal of the solvent in excess HCl under
vacuum afforded 1-amino-2,3-hexadiene hydrochloride with an
overall 20% yield. 1H NMR spectra were obtained using aVarian
400 MHz spectrometers with the chemical shifts being referenced to
TMS or the solvent peak. 1H NMR (CD3OD) δ 7.24 (5H, m), 5.46
(1H, m), 5.29 (1H, m), 3.39 (2H, broad multiplet), 2.75 (2H, m), 2.38
(2H, m); 13C NMR (CDCl3) 206.25, 142.75, 129.74, 129.49, 127.14,
95.22, 86.06, 39.69, 36.20, 31.24; HRFABMS MH+ m/z observed
174.12804, C12H16N calculated 174.12827.
Fig. 1. Chemical structure of 5-phenoxy-2,3-pentadienylamine (POPDA) and 6-phenyl-2,3-hexadienylamine (PHDA). These two compounds were synthesized as described in
Materials and methods as a racemic mixture. Both compounds first act as substrates to reduce the TPQ cofactor in AGAO and then chemically modify the reduced TPQ cofactor to
inactivate enzyme turnover.

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K. Ernberg et al. / Biochimica et Biophysica Acta 1814 (2011) 638–646
Table 1
2.2. Synthesis of racemic 5-phenoxy-2,3-pentadienylamine (POPDA)
X-ray data statistics. Values for outer shell are given in parentheses.
5-phenoxy-2,3-pentadienylamine was prepared via the custom
synthesis service of Sai Pharmaceutical, Inc. using the same
procedure previously described for the preparation of PHDA, except
2-phenoxyacetaldehyde was used to react with t-boc protected
propargylamine. The identity of the final product was confirmed by
liquid chromatography mass spectrometry (supplementary S1),
infrared spectroscopy (supplementary S2) and 1H NMR (500 MHz;
DMSO-D6; supplementary S3). The melting point of the product is
128.1 °C.
Prior to co-crystallization with purified AGAO, the inhibitors PHDA
and POPDA were dissolved in 20% ethanol to give a final concentration
of 100 mM. During protein work, the ethanol concentration was kept
below 1% to avoid disrupting protein integrity.
AGAO/POPDA
AGAO/PHDA
Radiation wavelength (Å)
Space group
Unit-cell parameters
a, b, c (Å)
1.54178
C2
192.7, 63.0, 158.2
117.4
2
50.00–1.90 (1.97–1.90)
131273 (13143)
98.9 (99.7)
3.8 (3.6)
10.0 (3.5)
0.078 (0.356)
2.96
157.9, 63.3, 92.2
112.1
1
50.00–2.050 (2.12–2.05)
52557 (5229)
99.2 (99.4)
4.3 (3.9)
10.0 (2.9)
0.091 (0.457)
2.97
β (°)
No. of molecules in ASU
Resolution range (Å)
No. of unique reflections
Completeness (%)
Redundancy
I/σ(I)
Rmerge
Matthews coefficient,
VM (Å3 Da−1
)
Solvent content (%)
58
59
2.3. Enzyme purification and crystallization
AGAO was overexpressed as a C-terminal Strep-tagII fusion
protein and purified according to previous protocols [10], with an
added final gel filtration step using a Sepharose 12 column. The
protein was eluted with a solution of 0.1 M Tris–HCl and 0.3 M NaCl at
pH 7.1 and subsequently concentrated to a final concentration of
12.9 mg mL−1 using a 30 kDa MWCO spin column (Millipore). Co-
crystals of protein–inhibitor complexes were grown by vapor-
diffusion in hanging drops at 293 K. Drops contained 2 μL reservoir
solution (150 mM NaCitrate pH 7.0, 800 mM (NH4)2SO4), 2 μL purified
protein (12.9 mg mL-1) and inhibitor (4 mM) and were set up over the
reservoir. Crystals were obtained after two months.
experiments were performed at least in triplicate. Ratios of the initial
rates of inhibited versus uninhibited (vi/vo) AGAO-catalyzed oxida-
tion of 1 mM benzylamine were plotted against the corresponding
rac-PHDA or rac-POPDA concentrations; fitting the plots with Eq. (1)
generated the IC50 values (concentration of inhibitor needed to attain
50% enzyme inhibition), where [I] is the concentration of inhibitor,
and n is the Hill coefficient (N1) introduced to fit the data.
vi
1
=
ð1Þ
ꢀ
ꢁ
n
vo
½Iꢀ
1 +
IC50n
2.4. X-ray data collection and analysis
2.6. Time-dependent inhibition of AGAO by PHDA and POPDA
The crystals were cryoprotected prior to data collection by 5% (v/v)
incremental increases of glycerol to reach a final concentration of 30%
(v/v) and immediately cryocooled in a stream of nitrogen at 100 K. X-
ray diffraction data were collected using an in-house Rigaku RU200
generator (Rigaku, The Woodlands, USA) equipped with a copper
rotating anode target (λ=1.5418 Å) and Osmic mirror optics, and
recorded on a Mar345 image plate detector (Marresearch). The data
were processed using DENZO and SCALEPACK [11].
A previously refined AGAO model at 1.73 Å (PDB code 1SIH [7])
was used as the initial model for both AGAO/inhibitor structures.
Solvent molecules, metal ions, sulfate ions, glycerol molecules and the
TPQ/inhibitor complex were stripped from the model and residue 382
(TPQ) was initially modeled as alanine. Refinement was carried out
using REFMAC5 [12] within the CCP4 program suite [13], with checks
of the electron density maps (Fobs −Fcalc and 2Fobs −Fcalc) and manual
modeling in COOT [14]. TLS refinement was used for AGAO/POPDA
with one TLS group per subunit [15]. Inhibitors were built using the
Dundee PRODRG Server [16].
AGAO (2.5 μM) was incubated with 0-, approximately 5-, 10-, and
15-times the IC50 of PHDA or POPDA at 30 °C in 50 mM sodium
phosphate, pH 7.2 for 0, 2, 5, 15 and 30 min prior to diluting 10-fold
into 5 mM benzylamine in the phosphate reaction buffer. Upon
dilution, the rates of benzylamine oxidation were monitored at
250 nm. The percentage enzyme activity remaining after inhibition
was defined by dividing the rate of reaction containing inhibitor with
the rate of uninhibited reaction at each time point. The partition ratios
(r) for PHDA and POPDA were obtained by plotting the fractional
Table 2
Crystallographic refinement statistics. Values for outer shell are given in parentheses.
AGAO/POPDA
AGAO/PHDA
No. of reflections used in refinement for Rcryst
No. of reflections for Rfree
Average B factors
Protein atoms (Å2)
Solvent molecules (Å2)
Metal ions (Å2)
124675 (8967)
6538 (438)
49890 (3529)
2660 (169)
24.4
32.5
25.5
38.6
34.3
40.5
31.6
44.8
Validation of the final structures was carried out using several
complementary programs: the MOLPROBITY server [17] for geometric
analysis of the model; SFCHECK [18] in the CCP4 suite for model vs.
data analysis; and, POLYGON [19] for comparison with structures of
similar resolution.
TPQ side chain (Å2)
Final model
Total no. of atoms
Protein residues
Residues with alternate confomers
Metal ions
10715
1235
11
2 Cu, 2 Na
4
5882
618
8
1 Cu, 1 Na
1
Sulfate ions
2.5. Inhibition kinetics of AGAO by PHDA and POPDA
Glycerol molecules
Rcryst
Rfree
10
11
0.176 (0.230)
0.218 (0.280)
0.178 (0.305)
0.221 (0.343)
Using the generation of benzylaldehyde as an activity reporter of
AGAO, the inhibition parameters of PHDA and POPDA were deter-
mined by monitoring the time courses of benzylamine oxidation in
the presence of various concentrations of PHDA or POPDA [6]. The
enzyme used in the kinetic studies was provided by the Dooley lab at
the Montana State University. The steady-state rates were determined
from tangents to the linear steady-state portions of the reaction time
courses using the computer program KaleidaGraph (Synergy, Inc.). All
Ramachandran data
Allowed (%)
Favored (%)
99.8
97.1
0.2
99.8
96.7
0.2
Outliers (%)
R.m.s.d. from ideal geometry
Bond lengths (Å2)
Bond angles (°)
0.01
1.5
0.01
1.5
PDB ID
3KII
3KN4

K. Ernberg et al. / Biochimica et Biophysica Acta 1814 (2011) 638–646
641
3. Results and discussion
The structures of AGAO in complex with PHDA and POPDA were
refined to 2.05 and 1.90 Å resolution, respectively. X-ray data
collection and refinement statistics can be found in Tables 1 and 2.
Overall the structure of AGAO is not significantly different from that
found in crystals of the native enzyme. The r.m.s.d. for Cα atoms
between either of the complexes reported in this paper and the native
structures previously reported (PDB codes 1W6G and 1W6C [20])
range from 0.2 to 0.4 Å. Four other structures of AGAO with a
covalently bound inhibitor have been published (PDB codes 1SIH and
1SII [7], 1W4N and 1W5Z [21]). When compared with these models,
the complexes reported here align with a slightly lower r.m.s.d for Cα
atoms than when compared to native structures. This is expected
since the active site residues move slightly upon substrate binding.
The electron density maps for the AGAO/PHDA and the AGAO/POPDA
complexes revealed both inhibitors to be covalently bound to the
enzyme through the C5 carbon of the TPQ cofactor as reported
previously for 4-aryloxy-2-butynamine compounds [7] as well as
benzylhydrazine and tranylcypromine [21]. Overall, the active site
cavities are very similar to each other in the two structures as well as
to other AGAO inhibitor complexes [7,21]. The O5 atom on C5 of TPQ is
modeled as a nitrogen atom, linking to the Cγ and Cβ of PHDA and
POPDA respectively (Fig. 2), and the chemical mechanism accounting
for the formation of each adduct is presented in Scheme 2. As
discussed below, the differences in the binding interaction between
AGAO with rac-PHDA and rac-POPDA could cause the observed
difference in adduct formed between the respective inhibitor and
TPQamr. The B-factors of the inhibitors are slightly higher on average
(~60Å2) than the surrounding residues, suggesting that the sites may
not be fully occupied in either inhibitor complex. In the absence of an
inhibitor, the TPQ in AGAO is normally disordered suggesting that any
uncomplexed TPQ in the crystals would simply not be observed.
Fig. 2. Chemical structures of the complex formed between the reduced TPQ and
inhibitor as revealed by the respective crystal structure of enzyme-inhibitor complex.
Top: TPQ/PHDA. Bottom: TPQ/POPDA.
enzyme velocity, defined by dividing the percentage enzyme activity
remaining with and without inhibitor treatment, against the
corresponding [I]/[AGAO] ratio. The r value was determined by
subtracting 1 from the intercept on the ‘fractional enzyme velocity’
axis obtained by extrapolating a straight line fit of the data.
TPQamr
H
AGAO
AGAO
OH
AGAO
O
N+
B
BH
O
B
H
H
TPQamr
O
O
NH
O
OH
Ph
C
H2N
Ph
CH
O
Ph
CH
OH
interact with
AGAO to
TPQamr
oritent the
aldehyde
AGAO
AGAO
AGAO
B
H
BH
O
B
H
O
O
Ph
C
OH
Ph
C
Ph
C
N+
H2N
TPQamr
NH
TPQamr
H
OH
H
TPQamr
Top: POPDA attack mode
Bottom: PHDA attack mode
Scheme 2. Proposed mechanism for the inactivation of AGAP by POPDA (top) and PHDA (bottom).

642
K. Ernberg et al. / Biochimica et Biophysica Acta 1814 (2011) 638–646
position [22], see Fig. 4. This ~90° rotation accommodates the TPQ/
inhibitor adduct and has been observed for other inhibitor structures
of AGAO [7,21,23]. This movement is accompanied by movements of
Leu 137 and of the sidechain of Pro 136. In active “off-Cu” AGAO, the
tyrosine is in its open conformation allowing access to the TPQ, whereas
in inactive “on-Cu” AGAO, it is in a closed conformation blocking
substrate access to the active site, hence its designation as a ‘gate’.
Interestingly, in AGAO/PHDA there is only poor electron density for Tyr
296 and the side chain has high B-factors, indicating that it is
disordered. This can be compared to AGAO/POPDA where Tyr 296 has
side chain B-factors of about 35–40 Å2 in both subunits and is well
ordered. The disorder of Tyr 296 in AGAO/PHDA is surprising and has
only been observed in one other structure where the TPQ is ordered
and in an “off-Cu” conformation (PDB code 1W6C, [20]). The movement
of Pro 136, which results from inhibitor binding and the movement of
Tyr 296, allows its carbonyl oxygen atom to form a hydrogen bond with
Tyr 302 OH. The Tyr 296 O to Tyr 302 OH distance of ~2.9–3.0 Å
compares with ~4 Å in the native structure.
3.2. Mode of inhibitor binding
The residues that line the cavity surrounding the inhibitors (b 3.5 Å),
include Ile 379 and Gly 380 on the top side of the non-aromatic part of
thesubstrate, Leu 137 and Tyr 296 below and Tyr 302 on theside(Fig. 5).
Phe 407 lies on the opposite side of the wall facing Tyr 302. Other
residues interact with the phenyl ring of the inhibitor molecules,
including Phe 105 at the end of the inhibitor channel, Ala 135 and Pro
136 below the phenyl ring and Trp 168 on the side opposite Phe 105. Leu
358 and Trp 359 from the other subunit in the dimer lie at the very end
of the channel and also interact with the inhibitor phenyl ring. The
phenyl groups of the inhibitors extend out into the active site cavity
between residues Pro 136 and Gly 380 (Fig. 5). The position of the
inhibitor causes Phe 105 to move significantly (~45° rotation) in order
to accommodate the large molecule in the active site, something that
has been reported previously for similar large inhibitor molecules [7,21].
However, in AGAO/POPDA chain A as well as AGAO/PHDA, Phe 105 is
disordered and not visible in the electron density maps.
For both inhibitors, the terminal amine group is modeled as an
aldehyde, resulting from prior oxidation by the enzyme, consistent
with the proposed reaction mechanism and previous reports
[7,21,23,24]. However, in the structure of the PHDA complex, the
electron density associated with the aldehyde group and adjacent
atoms is very weak, which together with the very high B-factors,
indicates that this “arm” is highly disordered and does not make any
significant interactions with nearby protein residues. While it is clear
that an adduct has formed with PHDA, disorder of Tyr 296, Phe 105
and the aldehyde end of the inhibitor, prevent a detailed discussion of
Fig. 3. The 2Fo–Fc map at 1.0 (light blue) and 1.5 (dark blue) sigma for TPQ/PHDA
(yellow) and TPQ/POPDA B (blue). The active site copper is shown, coordinated by His
431, 433 and 592 and the water molecule linking the copper to the TPQ molecule.
3.1. TPQ and the active site
In both structures reported here, the TPQ cofactor is in its active
“off-Cu” conformation (Fig. 3) which is required for adduct formation
with the inhibitors. The copper atom is coordinated by three histidine
residues, His 431, His 433 and His 592, and one water molecule. This
water molecule also forms a hydrogen bond to the O2 atom of TPQ.
There is no significant residual electron density for a second water
molecule that is frequently observed to coordinate the copper atom in
many other CAO structures. The position of the TPQ in its active
conformation allows it to hydrogen bond via its O4 atom with the OH
group of Tyr 284. Asn 381 tilts down towards the TPQ ring moiety,
making contacts of ~3.5 Å between its Oδ and Nδ atoms and the C3 and
C5 atoms of TPQ. This interaction combined with the hydrogen bonds
effectively restricts the movement of the TPQ.
The shift of the TPQ from its inactive to its active conformation
upon binding of the inhibitors is accompanied by the movement of the
‘gate’ residue, Tyr 296, which lies in the active site channel, to its “open”
Fig. 4. The active site residues that have moved significantly upon inhibition are displayed for AGAO/PHDA (yellow), AGAO/POPDA (blue) and native protein (white, PDB ID 1W6G).
When the protein is inactive, the TPQ is on-copper; whereas in active AGAO, the TPQ is off-copper. The change from on-copper TPQ to its off-position switches the active site gate
reside Tyr 296 from a closed conformation to an open. This movement pushes Leu 137 and Pro 136 around, and allows Tyr 302 to interact with the backbone of Pro 136. Phe 105
moves in order to interact with the large phenyl ring of the inhibitor. Dashed lines indicate the interactions between the aldehyde arms of PHDA and POPDA and the catalytic base
Asp 298.

K. Ernberg et al. / Biochimica et Biophysica Acta 1814 (2011) 638–646
643
Fig. 5. TPQ/PHDA (yellow) and TPQ/POPDA (blue) interacts with mainly hydrophobic residues where it sits in the active site. A loop from the second subunit in the dimer reaches in
and interacts with the inhibitor phenyl ring through residues 358 and 359 (magenta).
its interactions. In the complex with POPDA, however, the aldehyde
moiety is clearly visible and interacts with Asp 298, the conserved
catalytic base in the enzymatic reaction (Fig. 4). The interaction with
the aldehyde “arm” in AGAO/POPDA causes a ~90° rotation of the side
chain of Asp 298 in both subunits of AGAO/POPDA when compared to
native structures and other inhibitor complexes. This movement
allows Asp 298 to hydrogen bond to the aldehyde oxygen of POPDA
with the consequence that the aspartate must be protonated. The
second carboxyl oxygen atom of Asp 298 forms a hydrogen bond to
Tyr 384 OH. The specific interaction between Asp 298 and the
aldehyde arm of POPDA may help to retain the oxidized aldehyde
product in the active site of AGAO, thereby facilitating the inactivation
of TPQamr by the mechanism proposed in Scheme 2. Overall, the two
AGAO inhibitor structure active sites are apparently very similar but
the disorder seen in the PHDA complex indicates that there are fewer
stabilizing interactions than in the POPDA complex. This observation
would suggest that the ratio of aldehyde release to aldehyde retention
would be higher in the PHDA complex than in the POPDA complex as
discussed below in relation to the partition ratios.
to favor nucleophilic attack of the TPQamr at the γ-carbon of the
oxidized PHDA aldehyde product as proposed in the bottom panel of
Scheme 2.
3.4. Potency of POPDA and PHDA as inhibitors of AGAO
Using benzylamine as substrate, the inhibitory effects of POPDA and
PHDA on AGAO were compared by steady-state kinetic techniques. The
fractional activity of AGAO activity determined in the presence of
various concentrations of inhibitor was plotted against the
corresponding inhibitor concentration to yield the dose–response
curves shown in Fig. 7. Fitting the average data of each plot with
Eq. (1) (see Materials and methods) provides an IC50 of 1.2 0.1 μM for
racemic POPDA. PHDA, which contains a methylene group rather than
an ether oxygen juxtaposed to the phenyl ring, has an IC50 of 5.6
0.3 μM, which is ~4-fold less potent than the aryloxy analog POPDA.
When fitting the data to extract the IC50 values, the data shown in Fig. 7
fit best to Eq. (1), with an apparent Hill coefficient of 2.3 0.3 for rac-
PHDA, and 2.1 0.3 for rac-POPDA. The data fits poorly if the Hill
coefficient is set to one. As AGAO is a dimer, the Hill coefficient detected
3.3. Mode of enzyme inhibition
As indicated above PHDA and POPDA link to the TPQ by different
carbon atoms; Cγ and Cβ of PHDA and POPDA, respectively (Fig. 2).
These different “attack modes” cause the inhibitor in AGAO/POPDA
to stretch ~0.8 Å further out towards the opening of the active site
cavity (Fig. 4). To account for the formation of the POPDA and PHDA
derivatized TPQ, the mechanisms depicting the reaction between
TPQamr with the aldehydes generated from oxidation of the respective
compounds are proposed in Scheme 2. The two mechanisms differ by
the position at which the C5 amino group of TPQamr attacks the
electrophilic carbon in the allenic aldehydes generated from initial
oxidation of the primary amine in the two compounds. The electro-
static potential maps of the allenic aldehydes generated from amine
oxidation are shown in Fig. 6. The two structures differ principally by
the orientation of the allenic aldehyde arms but not by electrophilicity
at the β- and γ-carbons. Therefore, the observed differences in the
mode of TPQ derivation could be attributed to the way by which the
respective allenic group is positioned with respect to the C5 amino
group of TPQamr. In POPDA, the phenoxy oxygen is surrounded by a
hydrophilic patch in AGAO. The oxygen is replaced by a hydrophobic
carbon in PHDA. Such interactions may position the aldehyde product
of POPDA oxidation to react favorably at the β-carbon position as
shown in the top panel of Scheme 2. The lack of such interactions in
PHDA may allow a slightly different orientation of the diene in PHDA
Fig. 6. Spartan models of the allenic aldehydes generated by the AGAO catalyzed
oxidation of POPDA (top) and PHDA (bottom).

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K. Ernberg et al. / Biochimica et Biophysica Acta 1814 (2011) 638–646
1.2
1
further clarify this issue. Alternatively, the apparent cooperativity can be
attributed to the buildup of a certain amount of aldehyde product
needed to inactivate AGAO. At low levels of allenic amine, the aldehyde
product readily diffuses out of the enzyme active site whereas at high
levels of the compound, enough of the aldehyde product is generated to
stay bound and then inactivate the enzyme's TPQamr. At this time, the
mechanistic feature giving rise to the observed Hill coefficient cannot be
conclusively assigned. Therefore, additional work will be needed to
define this issue.
0.8
0.6
0.4
0.2
0
According to the structural data described above, AGAO/POPDA is
better-ordered in the electron density and displays interactions with
active site residues (e.g. Asp 298) not observed in AGAO/PHDA. The
disorder of Tyr 296 in AGAO/PHDA could further indicate less effective
inhibitor binding (Fig. 5). The interactions observed between AGAO and
POPDA are likely to provide a more productive binding mode in
facilitating the modification of TPQ, which accounts for the lower IC50 of
POPDA (Table 3). Fig. 2 shows the structures of TPQ modified by PHDA
and POPDA. The mechanisms proposed for their formation are
reminiscent of the one proposed for aryloxy-2-butyn-1-amines, which
irreversibly inactivate AGAO by covalently modifying reduced TPQ
through formation of an α,β-unsaturated aldehyde product. Therefore,
experiments were conducted to evaluate whether AGAO is irreversibly
inactivated by PHDA and POPDA. Fig. 8 shows the time courses of
enzyme inactivation by different concentrations of PHDA or POPDA. In
both cases, irreversible inactivation is indicated by the lack of recovery
of activity upon dilution of the inhibitor-treated AGAO into 100-fold
the Km of benzylamine substrate [6], and by the completion of
inactivation within 2 min of mixing with the inhibitors. These results
are consistent with the mode of covalent enzyme inactivation revealed
by the structures of the complexes. Furthermore, the inactivation
appears to be completed within 2 min of reaction mixing, corroborating
the observation that linear time courses were observed in the rac-PHDA
and rac-POPDA treated reaction time courses (S4). It is likely that the
chemical inactivation step was completed within the mixing timing of
the reaction; the linear time courses detected the activity of the
unreacted enzyme.
0
2
4
6
8
10
12
14
16
[Inhibitor] µM
Fig. 7. Dose-dependent plot of the inhibition of AGAO by PHDA (□) and POPDA (●). All
reactions were performed at least in triplicate. The fractional activities of AGAO-
catalyzed the oxidation of benzylamine were determined by monitoring the formation
of benzylaldehyde at 250 nm by UV spectroscopy as described in Materials and
methods. The solid black line is the fit of the data with Eq. (1). All experimental values
were within 25% or less deviation from the averaged values reported here.
Table 3
Racemic
PHDA
Racemic
POPDA
4-phenyoxy-2-butyn-1-amine
(PBA)
IC50 (μM)
Partition ratio
5.6 0.3
1.2 0.1
15.9 0.9
50*
12*
52
1
*Value obtained from O'Connell et al., Biochemistry 43, 10965–10978 (2004).
here reflects the extent of subunit communication during enzyme
inhibition. However, the oxidation of benzylamine by AGAO is not
cooperative [6]. Since benzylaminebears significantstructural similarity
with the inhibitors, it is not clear if the N1 Hill coefficient indeed reflects
the level of cooperativity in AGAO. Additional studies will be needed to
According to the mechanism proposed in Schemes 1 and 2, PHDA
and POPDA will be converted to an α,β-unsaturated aldehyde
product, which either diffuses out of the active site to enable
subsequent enzyme turnover, or reacts with the reduced TPQ to
1.2
1
1.2
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
minutes pre-incubated with inhibitor
minutes pre-incubated with inhibitor
Fig. 8. Time courses of irreversible inactivation of AGAO by PHDA (left) and POPDA (right). AGAO (2.5 μM) was incubated with 0- (●), 22.5- (▲), 45- (□) and 67.5- (X) μM of PHDA or
0- (●), 7.5- (▲), 15- (□) and 22.5- (X) μM of POPDA for 0, 2, 5, 15 and 30 min prior to diluting 10-fold into 5 mM benzylamine in the phosphate reaction buffer as described in
Materials and methods. Upon dilution, the rates of benzylamine oxidation were monitored at 250 nm. The percentage enzyme activity remaining is standardized against the rate of
benzylaminie oxidation in the absence of inactivator. To reduce clattering of the plots, the error bars for the 22.5 μM of PHDA and 7.5 μM of POPDA, which are within 25% are not
shown.

K. Ernberg et al. / Biochimica et Biophysica Acta 1814 (2011) 638–646
645
1
0.8
0.6
0.4
0.2
0
nism-based inhibitors of AGAO. These two compounds first act as
substrates, which upon oxidation, become α,β-unsaturated aldehyde
products that are highly reactive electrophiles. Consequently, the
unsaturated aldehydes form a covalent adduct with the reduced TPQ
cofactor (Scheme 2) to sequester the catalytic functions of AGAO,
thereby eliminating enzyme turnover. Comparison of the structures of
AGAO/PHDA and AGAO/POPDA complexes reveal binding interactions
are present only in the latter enzyme complex. As POPDA is a more
potent inhibitor than PHDA, with at least a 4-fold lower in IC50, we
attribute the observed potency to the binding interactions of POPDA
by AGAO as revealed by the structural data. Furthermore, the phenoxy
oxygen in POPDA contributes to a lower partition ratio in enzyme
inhibition by interacting with a cluster of hydrophilic residues in the
active site of AGAO. Taken together, this work demonstrates the utility
of installing a 1-amino-2,3-butadiene unit as an electrophile in the
design of potent mechanism-based inhibitors of AGAO. Given that the
two inhibitors were generated and tested as a racemic mixture, it will
be of interest to further determine if enzyme inhibition is also
enantioselective. If so, the stereochemistry of allenic amines could be
exploited as an additional parameter for future design of CAO
inhibitors. Since unsubstituted 1-amino-2,3-butadiene inhibits other
CAOs and monoamine oxidases, the results presented in this study
should provide structural guidance in the design of selective and
potent inhibitors for other oxidases.
fraction PHDA
POPDA
0
10
20
30
40
50
[I]/[AGAO]
Fig. 9. Partition ration plot for the inactivation of AGAO by PHDA (●) and POPDA (▲).
The fractional AGAO activity at the corresponding [I]/[AGAO] was determined by
monitoring the oxidation of benzylamine to benzylaldehyde at 250 nm as described in
Materials and method. In this plot, I represents PHDA or POPDA. All assays were
performed at least in triplicates and experimental deviations were within 20% or less
from the average fractional enzyme activity reported here. The partition ratio of the
respective inactivator was extrapolated by subtracting 1 from the X-intercept of the
corresponding compound.
Acknowledgement
We thank Edward Motea for assistance in the preparation of Fig. 6.
render enzyme inactivation. The partition between the two processes
can be quantified by the partition ratio, a value obtained by plotting
residual enzyme activity with [I]/[E] as shown in Fig. 9. Fitting the data
shown in Fig. 9 to a linear function generates the partition ratios for
rac-PHDA and rac-POPDA (Table 3). For comparison, the partition
ratio of 4-phenoxy-2-butyn-1-amine (PBA), which contains the same
aryloxy substituent as POPDA, is included. It can be seen from Table 3
that the two inhibitors containing the phenoxy substituent have
comparable partition coefficients, and that each is lower than the
value found for rac-PHDA. The structural data shown in Figs. 4 and 5
reveal that the oxygen of the phenoxy group in POPDA could interact
with Tyr 302 OH, Gly 380 N and Pro 136 O in AGAO, which are all
within a distance of 4–5 Å. Since such interactions are not found in
PHDA, it is possible that POPDA is more productively aligned with the
amino group of reduced TPQ to facilitate the crosslinking reaction.
Furthermore, the aldehyde product generated from POPDA oxidation
may be better retained in the active site of AGAO due to the additional
interactions between the phenoxy oxygen and the active site residues.
This may account for the relatively lower partition ratio of POPDA
(15.9) than PHDA (52).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.bbapap.2010.12.016.
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