Reversible inactivation of bovine plasma amine oxidase by cysteamine and related analogs

Article abstract of DOI:10.1016/j.bbrc.2010.11.052

Cysteamine (1) was reported many years ago to reversibly inhibit lentil seedling amine oxidase, through the formation of a complex with thioacetaldehyde, the turnover product of 1. Herein, cysteamine (1) and its analogs 2-(methylamino)ethanethiol (3) and 3-aminopropanethiol (6) were found to be reversible inhibitors of bovine plasma amine oxidase (BPAO), but 2-(methylthio)ethylamine (7) was determined to be a weak irreversible inhibitor of BPAO. Based on our results, indicating the necessity of a sulfhydryl-amine for reversible inactivation of BPAO, the failure of inhibited BPAO to recover activity after gel filtration, the first-order kinetics of activity recovery upon dialysis, and 2,4,6-trihydroxyphenylalanine quinine (TPQ) cofactor transformation which indicated from the results of phenylhydrazine titration and substrate protection, we propose a mechanism for the reversible inactivation of BPAO by 1 involving the formation of a cofactor adduct, thiazolidine, between BPAO and 1.

Full text of DOI:10.1016/j.bbrc.2010.11.052

Biochemical and Biophysical Research Communications 403 (2010) 442–446
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Reversible inactivation of bovine plasma amine oxidase by cysteamine
and related analogs
⇑
Heung Bae Jeon , Yujin Jang
Department of Chemistry, Kwangwoon University, Seoul 139-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 October 2010
Available online 19 November 2010
Cysteamine (1) was reported many years ago to reversibly inhibit lentil seedling amine oxidase, through
the formation of a complex with thioacetaldehyde, the turnover product of 1. Herein, cysteamine (1) and
its analogs 2-(methylamino)ethanethiol (3) and 3-aminopropanethiol (6) were found to be reversible
inhibitors of bovine plasma amine oxidase (BPAO), but 2-(methylthio)ethylamine (7) was determined
to be a weak irreversible inhibitor of BPAO. Based on our results, indicating the necessity of a sulfhy-
dryl-amine for reversible inactivation of BPAO, the failure of inhibited BPAO to recover activity after
gel filtration, the first-order kinetics of activity recovery upon dialysis, and 2,4,6-trihydroxyphenylalanine
quinine (TPQ) cofactor transformation which indicated from the results of phenylhydrazine titration and
substrate protection, we propose a mechanism for the reversible inactivation of BPAO by 1 involving the
formation of a cofactor adduct, thiazolidine, between BPAO and 1.
Keywords:
Copper amine oxidase
Reversible inhibitor
Mechanism-based inhibitor
Cysteamine
Thiazolidine
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
iology of atherosclerosis and diabetes [7–9], and brain SSAOs might
play a role in controlling energy balance in adipocytes [10]. In addi-
The quinone-dependent amine oxidases (EC 1.4.3.6), which con-
tain tightly bound Cu(II), catalyze the oxidative deamination of un-
branched primary amines to the corresponding aldehydes. The
recent elucidation of the structures of the quinine cofactors has at-
tracted interest in the mechanistic aspects of oxidative deamina-
tion and in the development of useful inhibitors. Most of these
enzymes utilize a 2,4,6-trihydroxyphenylalanine quinine (TPQ)
cofactor derived from an active site tyrosine to catalyze the oxida-
tive deamination [1,2].
tion, cell surface SSAOs that appear to be involved in regulating
glucose uptake, signaling, and cell–cell adhesion have been identi-
fied [11]. Overall, potent and selective inhibitors could provide
important tools for enzymologic probes of pharmacologic inter-
vention and potential therapeutic agents.
In the 1970s, Abeles and co-workers first reported that amines
bearing an unsaturated C–C bond at the b-position could inactivate
bovine plasma amine oxidase (BPAO) [12,13]. Recently, inactivation
of BPAO by various propargylamine, homopropargylamine, and
chloroallylamine analogues was studied extensively by Sayre and
co-workers [14–17]. Additionally, cysteamine (1) was reported to
be a good inhibitor of pig kidney amine oxidase [18] and lentil seed-
ling amine oxidase [19]. In these papers, the researchers proposed
that inhibition by cysteamine (1) involved the formation of a stable
enzyme complex with the product of turnover, thioacetaldehyde.
Since little work has been carried out on the interaction of
sulfur-containing amine compounds with BPAO, we investigated
the effects of several aminothiol compounds, including cysteamine
(1), on BPAO and report the results in this paper.
The mechanism of the deamination half-reaction [3] begins
with condensation of the substrate amine with TPQ to give the
‘‘substrate Schiff base’’ (SSB). A conserved active-site catalytic base
(Asp) abstracts the C proton, allowing tautomerization to the
a
‘‘product Schiff base’’ (PSB), which hydrolyzes to yield the aldehyde
product and reductively aminated cofactor (an aminoresocinol).
The latter subsequently is reoxidized at the expense of reduction
of O2 to H2O2, with the hydrolytic release of NH3 or displacement
of NH3 by another substrate amine (Scheme 1).
The human plasma amine oxidases, which are often known as
the soluble semicarbazide-sensitive amine oxidases (SSAOs),
deaminate endogeneous and exogeneous amines and have been
extensively investigated with respect to their role in various path-
ophysiological conditions [4–6]. SSAO-mediated production of
toxic aldehydes has been proposed to contribute to the pathophys-
2. Materials and methods
2.1. General procedure
NMR spectra were obtained at a 1H resonance frequency of
300 MHz (13C NMR at 75 MHz), with chemical shifts referenced
to the solvent peak. UV–Vis spectra were obtained using a jacketed
⇑
Corresponding author. Fax: +82 2 911 8584.
E-mail address: hbj@kw.ac.kr (H.B. Jeon).
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.11.052

H.B. Jeon, Y. Jang / Biochemical and Biophysical Research Communications 403 (2010) 442–446
443
temperature under Ar, 2.8 g (20 mmol) of methyl iodide was added
dropwise, and the reaction mixture was stirred for 24 h. Sodium
iodide that had formed was filteredoff, the filtrate was concentrated,
and the residue was dissolved in aqueous NaOH. The resulting solu-
tion was extracted with chloroform, and the organic layer was sepa-
rated, dried over Na2SO4, and concentrated. To the residue were
added 10 mL of MeOH and 2 mL of concentrated HCl. The mixture
was stirred for 10 min and then concentrated. The residue was
recrystallized from MeOH and Et2O to give 7ꢀHCl at 40% yield: 1H
NMR (D2O) d 2.15 (s, 3H), 2.85 (t, 2H), 3.26 (t, 2H). 13C NMR (D2O) d
13.8, 30.4, 37.7.
O
O
OH
RCH2NH2
H2O
-O
-O
HO
NCHR
H +
O
N=CHR
H2O
.
H
.
B
NH3
NH3
RCH2NH2
RCH=O
H2O
OH
O
H2O2
O2
-O
HO
2.6. Time-dependent inactivation of BPAO by candidate inhibitors
NH2
NH2
+
A 0.9 mL aliquot of a stock solution of candidate inhibitor in
100 mM potassium phosphate buffer, pH 7.2, was mixed with
BPAO (0.1 mL) suspension (Sigma, 100 U/g of protein, final concen-
Scheme 1.
tration ꢁ2
lM) and aerobically incubated at 30 °C. Aliquots
(temperature-controlled) cell compartment, from a Perkin-Elmer
Lambda 3 spectrophotometer. All column chromatography was
carried out with flash-grade silica gel. BPAO (100 U/g of protein)
was purchased from Sigma.
(0.1 mL) were periodically withdrawn using disposable calibrated
Drummond micropipets and diluted with 1.0 mL of benzylamine
(10 mM in 50 mM sodium phosphate buffer, pH 7.2) in a 1 cm cuv-
ette (1.5 mL). The rate of oxidation of benzylamine to benzalde-
hyde was measured by recording the increase in absorbance at
250 nm for 1 min, and compared to the rate of benzylamine oxida-
tion for a companion control solution of enzyme without the inhib-
itor. The concentration of active BPAO was estimated from the rate
2.2. Thiazolidine hydrochloride (2ꢀHCl)
To a solution of 4.0 g (35 mmol) of 2-aminoethanethiol hydro-
chloride in 25 mL of water was added 3.0 mL of 37% formaldehyde
solution. The mixture was stirred overnight at room temperature.
The reaction mixture was then concentrated in vacuo to give a
white solid, which was recrystallized from EtOH to give 2ꢀHCl at
80% yield: 1H NMR (DMSO-d6) d 3.09 (t, 2H), 3. 39 (t, 2H), 4.21
(s, 2H). 13C NMR (DMSO-d6) d 29.2, 47.3, 47.9.
of benzylamine oxidation (1 U oxidizes 1.0 lmol of benzylamine to
benzaldehyde per minute at 25 °C), using an activity of 0.48 U/mg
protein for the pure enzyme of molecular weight 85,000 Da and
De
250 = 12,800 Mꢂ1 cmꢂ1 for benzaldehyde (corresponding to a
A250 of 12.8/min/U of activity for 1 mL volume).
2.7. Irreversibility of BPAO inhibition
2.3. 2-(Methylamino)ethanethiol hydrochlodide (3ꢀHCl)
(a) By gel filtration. The irreversibility of the inhibition for inac-
tivators was checked by applying enzyme preparations (0.5 mL)
In a portion-wise manner, 0.85 g of LiAlH4 was added to a solu-
tion of 2.0 g (16 mmol) of thiazolidine (2) in anhydrous THF
(80 mL) under Ar. The mixture was heated at reflux for 2 h under
Ar. To decompose unreacted hydride, 10 mL of water was carefully
added, and the mixture was filtered using a Celite pad. The filtrate
was dried over Na2SO4 and concentrated in vacuo. The residue was
recrystallized from EtOH and Et2O to give 3ꢀHCl at 63% yield: 1H
NMR (D2O) d 2.75 (s, 3H), 3.01 (t, 2H), 3.31 (t, 2H).
that were 50–90% inhibited to
a PDX G. F. 25 column
(1 ꢃ 7.8 cm) equilibrated with 100 mM sodium phosphate buffer
(pH 7.2) to separate non-covalently bound small molecules. Per-
cent activity following gel filtration was determined by comparing
the activity between two gel-filtered incubations of enzyme in the
absence and presence of inhibitor. The same initial protocol had to
be worked out each time the column was changed.
(b) By dialysis. The irreversibility of the inhibition for inactiva-
tors was also checked by adding 0.5 mL enzyme preparations that
were 50–90% inhibited to dialysis tubing (6.4 mm Spectrum Spec-
tro/Por membrane, MW cutoff 12,000–14,000), followed by dialy-
sis against 100 mM sodium phosphate buffer, pH 7.2, at room
temperature for up to 24 h. Percent activity was determined by
comparing enzyme activity between two dialysis experiments fol-
lowing incubation of enzyme in the absence and presence of
inhibitor.
2.4. 3-Aminopropanethiol hydrochlodide (6ꢀHCl)
To a solution of 40 mL of CS2, 2.0 g (11.5 mmol) of 3-bromopro-
pylamine hydrochloride was added. The mixture was stirred for
2 h, and the solid that formed was filtered to give perhydro-1,3-thi-
azine-2-thione (5). Compound 5 (1.2 g, 9.0 mmol) was heated at
reflux for 1 h in a 2N aqueous NaOH solution (20 mL). The reaction
mixture was concentrated in vacuo, and the residue was parti-
tioned between CH2Cl2 and brine. The organic layer was separated,
dried over Na2SO4, and concentrated. To the residue, 10 mL of
MeOH and 2 mL of concentrated HCl were added. The mixture
was stirred for 10 min and then concentrated. The residue was
recrystallized from MeOH and Et2O to give 6ꢀHCl at 75% yield: 1H
NMR (D2O) d 2.06 (m, 2H), 2.78 (t, 2H), 3,08 (t, 2H). 13C NMR
(D2O) d 28.6, 36.4, 40.7.
2.8. Phenylhydrazine titration of active site after inactivation of BPAO
with inactivators
Incubation of the enzyme with the test inactivator in 100 mM
phosphate buffer, pH 7.2, was allowed to proceed at 30 °C until
there was more than 80% loss of activity after gel filtration or dial-
ysis. A control reaction was run for the same time. The gel filtered
2.5. 2-(Methylthio)ethylamine hydrochlodide (7ꢀHCl)
or dialyzed enzyme fractions were titrated with 4 lL of phen-
ylhydrazine hydrochloride (2 mM) in H2O, and the absorbance at
440 nm was measured. The percent decrease in the absorbance
at 440 nm was determined compared to the control experiment.
Under Ar, 1.0 g of sodium was added to a solution of 1.2 g
(15.6 mmol) of 2-aminoethanethiol (1) in absolute EtOH (25 mL).
After complete dissolution of sodium by stirring for 30 min at room

444
H.B. Jeon, Y. Jang / Biochemical and Biophysical Research Communications 403 (2010) 442–446
Table 1
H
N
BPAO inhibition data for the various compounds.
CH2O
CS2
LiAlH4
NHCH3
NH2
HS
HS
Br
a
Compound IC50
After PDX
G.F.b
After
S
2
dialysisc
1
3
1
12.5
lM
Partition ratio = 21
KI = 0.39 mM
No recovery
No recovery
No recovery
Recovery
Recovery
Recovery
S
kinact = 0.72 minꢂ1
NaOH
S
NH
3
4
6
5 mM
HS
NH2
NH2
d
–
0.3 mMe KI = 1.81 mM
6
kinact = 0.035 minꢂ1
5
7
8
1.5 mM
5 mM
No recovery
n.d.f
No recovery
n.d.
NaOCH3
CH3I
NH2
NH2
HS
S
a
The number listed represents the concentration needed to achieve 50% inhibi-
7
1
tion at the point of plateau (30–180 min).
b
Enzyme activity was measured following gel filtration over PDX G.F. 25.
Enzyme activity was measured following dialysis for 10 and 22 h against pH 7.2
c
NH2
N(CH3)2
sodium phosphate buffer at room temperature. The event of recovery is shown in
Fig. 3.
HO
HS
d
8
4
No inhibition seen at 5 mM.
The concentration needed to achieve 50% inhibition in 110 min (no apparent
e
Scheme 2.
plateau behavior).
f
No detection.
3. Results
3.1. Synthesis
100
Our initial studies revealed that cysteamine (1) is a time-depen-
dentinactivatorofBPAO. Toinvestigatethestructuraldependenceof
this effect, we wanted to compare the secondary and tertiary amine
analogs 3 and 4, the homolog 6, the thioether analog 7, and the oxy-
gen-containing analog 8. We prepared 2-(methylamino)ethanethiol
(3), 3-aminopropanethiol (6), and 2-(methylthio)ethylamine (7), as
shown in Scheme 2. 2-(N,N-Dimethylamino)ethanethiol (4) and
2-aminoethanol (8) were used in their commercially available form.
To prepare 2-(methylamino)ethanethiol (3), 2-aminoethane-
thiol (1) was reacted with formaldehyde to give thiazolidine (2),
followed by reduction with LiAlH4 [20]. 3-Aminopropanethiol (6)
[21] was prepared by the basic hydrolysis of the intermediate per-
hydro-1,3-thiazine-2-thione (5) [22], which was obtained from the
reaction of 3-bromopropylamine with carbon disulfide. For 2-
(methylthio)ethylamine (7), 2-aminoethanethiol (1) was reacted
with sodium methoxide and methyl iodide [23].
80
5 µM
60
10 µM
15 µM
40
25 µM
20
50 µM
100 µM
0
0
20
40
60
80
100
120
140
Time, Min
3.2. Inactivation of BPAO by cysteamine (1) and its analogs
Fig. 1. Effect of incubation time on inactivation of BPAO, at various concentrations
of 1.
All compounds were evaluated for their ability to inactivate the
benzylamine oxidase activity of BPAO (1.2
and the data are listed in Table 1.
lM) at 30 °C, pH 7.2,
100
Cysyeamine (1) produced a time- and concentration-dependent
inactivation of BPAO (Fig. 1). As shown, the loss of activity occurred
in the first 30 min at all concentrations measured, but was incom-
plete, suggesting a significant amount of turnover. The plateau IC50
is estimated from Fig. 1 to be 12.5 lM, and a re-plot of the plateau
0.3 mM
0.5 mM
values allowed us to estimate a partition ratio of 21. At concentra-
tions achieving >50% inactivation, the loss of enzyme activity fol-
lowed pseudo first-order kinetics. KI and kinact values were
determined by the method of Kitz and Wilson to be 0.39 mM and
0.72 minꢂ1, respectively [24].
The secondary amine 2-(methylamino)ethanethiol (3) was a
much weaker inhibitor, effecting 50% inactivation at 5 mM in
120 min. In contrast, the tertiary amine 2-(N,N-dimethyl-
amino)ethanethiol (4) exhibited no inhibition of BPAO even at
5 mM. These findings suggest that the primary amino group in 1
is obligatory for achieving potent inactivation.
0.75 mM
1 mM
10
2 mM
0
50
100
150
Time, Min
200
250
300
350
As shown in Fig. 2, the homologous primary amine 3-amino-
propanethiol (6) yielded 50% inactivation of BPAO at 0.3 mM in
110 min. The inactivation was slow, but higher concentrations
Fig. 2. Effect of incubation time on inactivation of BPAO, at various concentrations
of 6.

H.B. Jeon, Y. Jang / Biochemical and Biophysical Research Communications 403 (2010) 442–446
445
100
80
60
40
20
0
100
50
0
5
10
15
20
25
0
5
10
15
20
25
Time, Hr
Time, Hr
Fig. 4. Re-plot of the recovery of activity following incubation of BPAO with 1, 3,
and 6. See the legend to Fig. 3 for details.
Fig. 3. Loss and recovery of activity upon incubation of BPAO with 1, 3, and 6.
Following a 2 h incubation of BPAO with the inactivators, the solutions were
dialyzed against 100 mM sodium phosphate buffer (pH 7.2). Enzyme activity was
measured after 10 and 22 h. Ç, 0.1 mM compound 1, j, 10 mM compound 4, d,
2 mM compound 6.
of enzyme activity in these cases may arise from hydrolytic rever-
sal of a covalent modification to the quinine cofactor or a residue at
the active site.
In contrast to compounds 1, 3, and 6, inactivation by 2-(methyl-
thio)ethylamine (7) was not reversed by dialysis. This finding is
consistent with 7 following a different pathway for inactivation
on account of its lacking a free sulfhydryl group. Therefore, it is
clear that the sulfhydryl moiety of sulfhydryl-amine inactivators
plays an important role in the mechanism of inactivation of the en-
zyme. Even though a putative covalent adduct of the enzyme and
the inactivators is stable during gel filtration, it is not stable during
dialysis.
followed pseudo first-order kinetics. By the method of Kitz and
Wilson, KI and kinact values were determined to be 1.81 mM and
0.035 minꢂ1, respectively. Using kinact/KI as a measure of inactivat-
ing efficiency, 3-aminopropanethiol (6) is a weaker inhibitor of
BPAO than is cysteamine (1) by a factor of about 90.
Notably, when the sulfhydryl group of cysteamine (1) was chan-
ged to a hydroxyl group, the inhibitory potency was markedly
reduced. 2-Aminoethanol (8) showed 50% inhibition in 175 min at
5 mM. When the sulfhydryl group in 1 was changed to the methyl
thioether in order to investigate the necessity of sulfhydryl group
forinactivation,2-(methylthio)ethylamine(7)showed50%inactiva-
tion at 1.5 mM in 60 min. Although both 7 and 8 exhibited time-
dependent inhibition, the inhibitory potencies were very weak com-
pared to 1. Considering all the data, it is clear that both amino and
sulfhydryl groups of 1 are required for highly potent inactivation
and that having two rather than three methylenes between them
is optimal.
3.4. Substrate protection from inactivation by cysteamine (1)
The loss of the characteristic spectral behavior of the active site
carbonyl in BPAO inactivated by cysteamine (1) raises the possibil-
ity of cofactor transformation. Nonetheless, we wanted to deter-
mine whether enzyme inactivation by 1 satisfied the full set of
criteria, which is expected of a mechanism-based inactivator. If
cysteamine (1) is a mechanism-based inactivator, it must be com-
petitive with the normal substrate for the active site. Conse-
quently, the rate of inactivation should be slowed in the presence
of substrate.
3.3. Reversible inactivation of BPAO by cysteamine (1) and its analogs
3 and 6
In an attempt to show substrate protection against inactivation
by cysteamine (1), BPAO was incubated for 1 h with 0.25 mM cys-
teamine at 30 °C, in the absence or presence of 20 mM benzyl-
amine and catalase. When the enzyme activity was measured
after separation of inhibitor and reaction products (benzaldehyde
and inhibitor metabolites) by gel filtration of the incubation solu-
tion, the aliquot of enzyme incubated in the absence of benzyl-
amine exhibited 5% of the control activity. In contrast, the aliquot
of enzyme incubated in the presence of 20 mM benzylamine exhib-
ited 42% of the control enzyme activity. Thus, substrate does offer
protection against inactivation by 1.
Inhibition by the cysteamine (1) was found to be irreversible on
the basis of re-assay of enzyme activity following gel filtration and
was accompanied by loss of the 480 nm chromophore ascribed to
the TPQ cofactor. The latter finding, along with our inability to
see the normal development at A440 following reaction with phen-
ylhydrazine, suggests that the TPQ cofactor was transformed to an
adduct by a chemical reaction with cysteamine (1). Analogs 3 and 6
were also found to be irreversible inhibitors, based on gel filtration.
Following inactivation, however, when the pre-incubation mix-
tures for these three compounds were dialyzed for 10 or 22 h
against 100 mM sodium phosphate buffer (pH 7.2) at room tem-
perature, the enzyme activity slowly recovered as shown in
Fig. 3. In these experiments, the concentrations of 1, 3, and 6 were
chosen to achieve 70–95% inactivation of BPAO in 2 h at 30 °C prior
to dialysis. As shown in Fig. 3, enzyme activity recovered in all
three cases, but was very slow in the case of 6.
4. Discussion
Although it has been reported that cysteamine (1) inhibits lentil
seedling amine oxidase through the formation of a complex with
thioacetaldehyde, the turnover product of 1 [19], our structure–
activity results with BPAO suggest other possibilities. We propose
that BPAO may be inactivated by forming a cofactor adduct with 1
before the turnover process is complete, in competition with the
The recovery of enzyme activity in the case of inactivation by
cysteamine (1) and its analogs 3 and 6 showed first order kinetics,
as displayed in Fig. 4, where the enzyme activity on the y-axis of
Fig. 3 was re-plotted as percent enzyme inhibition. The recovery

446
H.B. Jeon, Y. Jang / Biochemical and Biophysical Research Communications 403 (2010) 442–446
O
O
SH
+
H2N
HO
HO
NH
HO
O
SH
H2O
H2O2
+H2O
-
O2
OH
N
O
N
O
OH
_
HS
CHO
HO
HO
slow
HO
HN
HO
S
NH2
SH
SH
10
9
Scheme 3.
independent marker of carotid atherosclerosis, Clin. Chim. Acta 323 (2002)
139–146.
[8] K.C. Mathys, S.N. Ponnampalam, S. Padival, R.H. Nagaraj, Semicarbazide-
sensitive amine oxidase in aortic smooth muscle cells mediates synthesis of a
methylglyoxal-AGE: implications for vascular complications in diabetes,
Biochem. Biophys. Res. Commun. 297 (2002) 863–869.
[9] J. Ekblom, Potential therapeutic value of drugs inhibiting semicarbazide-
sensitive amine oxidase: vascular cytoprotection in diabetes mellitus,
Pharmacol. Res. 37 (1998) 87–92.
[10] T. Obata, Semicarbazide-sensitive amine oxidase (SSAO) in the brain,
Neurochem. Res. 27 (2002) 263–268.
[11] S. Jalkanen, M. Salmi, Cell surface monoamine oxidases: enzymes in search of a
function, EMBO J. 20 (2001) 3893–3901.
[12] R.C. Hevey, J. Babson, A.L. Maycock, R.H. Abeles, Highly specific enzyme
inhibitors. Inhibition of plasma amine oxidase, J. Am. Chem. Soc. 95 (1973)
6125–6127.
[13] R.H. Abeles, A.L. Maycock, Suicide enzyme inactivators, Acc. Chem. Res. 9
(1976) 313–319.
[14] C. Qiao, H.B. Jeon, L.M. Sayre, Selective inhibition of bovine plasma amine
oxidase by homopropargylamine, a new inactivator motif, J. Am. Chem. Soc.
126 (2004) 8038–8045.
metabolism of 1 that eventually consumes it, thus explaining why
the loss of activity reaches a plateau and is indicative of a partition-
ing between inactivation and turnover. A possible mechanism is
shown in Scheme 3, where substrate Schiff base 9 can either un-
dergo transamination, leading to turnover, or cyclize into thiazoli-
dine 10. We propose that the adduct 10 is stable during gel
filtration but eventually reverts to active enzyme during dialysis,
either through turnover or hydrolysis. Also, it is considered that
the same mechanism my hold for sulfhydryl-amines 3 and 6.
Our mechanistic proposal in Scheme 3 is supported by the
reversible formation of thiazolidine previously reported [25,26].
The formation and eventual hydrolysis of a thiazolidine (Eq. 1)
may explain the inactivation of the enzyme and the recovery of
the inhibited enzyme activity during dialysis.
O
-
H2O
SH
HN
R
S
R'
+
ð1Þ
H2N
+H2O
[15] H.B. Jeon, Y. Lee, C. Qiao, H. Huang, L.M. Sayre, Inhibition of bovine plasma
amine oxidase by 1,4-diamino-2-butenes and -2-butynes, Bioorg. Med. Chem.
11 (2003) 4631–4641.
R
R'
[16] H.B. Jeon, G. Sun, L.M. Sayre, Inactivation of bovine plasma amine oxidase by 4-
aryloxy-2-butynamines and related analogs, Biochim. Biophys. Acta 1647
(2003) 343–354.
Acknowledgment
[17] H.B. Jeon, L.M. Sayre, Highly potent propargylamine and allylamine inhibitors
of bovine plasma amine oxidase, Biochem. Biophys. Res. Comm. 304 (2003)
788–794.
This work was supported by a 2010 research grant from Kwang-
woon University.
[18] C. DeMarco, B. Mondovi, D. Cavallini, Temporary suppression of diamine
oxidase (histaminase) activity by cysteamine, Biochem. Pharmacol. 11 (1962)
509–514.
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