Inhibition of monoamine oxidase by (E)-styrylisatin analogues

Article abstract of DOI:10.1016/j.bmcl.2009.03.030

Previous studies have shown that (E)-8-(3-chlorostyryl)caffeine (CSC) is a specific reversible inhibitor of human monoamine oxidase B (MAO-B) and does not bind to human MAO-A. Since the small molecule isatin is a natural reversible inhibitor of both MAO-B

Full text of DOI:10.1016/j.bmcl.2009.03.030

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2509–2513
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of monoamine oxidase by (E)-styrylisatin analogues
Elizna M. Van der Walt a, Erika M. Milczek b, Sarel F. Malan a, Dale E. Edmondson b, Neal Castagnoli Jr. c,
Jacobus J. Bergh a, Jacobus P. Petzer a,
*
a Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Departments of Biochemistry and Chemistry, Emory University, Atlanta, GA 30322, USA
c Department of Chemistry, Virginia Tech and Edward Via College of Osteopathic Medicine, Blacksburg, VA 24061, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous studies have shown that (E)-8-(3-chlorostyryl)caffeine (CSC) is a specific reversible inhibitor of
human monoamine oxidase B (MAO-B) and does not bind to human MAO-A. Since the small molecule
isatin is a natural reversible inhibitor of both MAO-B and MAO-A, (E)-5-styrylisatin and (E)-6-styrylisatin
analogues were synthesized in an attempt to identify inhibitors with enhanced potencies and specificities
for MAO-B. The (E)-styrylisatin analogues were found to exhibit higher binding affinities than isatin with
the MAO preparations tested. The (E)-5-styrylisatin analogues bound more tightly than the (E)-6 ana-
logue although the latter exhibits the highest MAO-B selectivity. Molecular docking studies with MAO-
B indicate that the increased binding affinity exhibited by the (E)-styrylisatin analogues, in comparison
to isatin, is best explained by the ability of the styrylisatins to bridge both the entrance cavity and the
substrate cavity of the enzyme. Experimental support for this model is shown by the weaker binding
of the analogues to the Ile199Ala mutant of human MAO-B. The lower selectivity of the (E)-styrylisatin
analogues between MAO-A and MAO-B, in contrast to CSC, is best explained by the differing relative
geometries of the aromatic rings for these two classes of inhibitors.
Received 26 January 2009
Revised 9 March 2009
Accepted 10 March 2009
Available online 14 March 2009
Keywords:
Monoamine oxidase A
Monoamine oxidase B
Reversible inhibitor
Isatin
(E)-5-Styrylisatin
(E)-6-Styrylisatin
Ó 2009 Elsevier Ltd. All rights reserved.
O
N
O
The oxidative deamination reaction catalyzed by monoamine
oxidase B (MAO-B) is one of the major catabolic pathway of dopa-
mine in the brain. Inhibitors of this enzyme lead to enhanced dopa-
minergic neurotransmission and are currently used in the
symptomatic treatment of Parkinson’s disease (PD).1–4 Further-
more, MAO-B inhibitors also may exert a neuroprotective effect
by reducing the concentrations of potentially hazardous by-prod-
ucts produced by MAO-B-catalyzed dopamine oxidation.5 Consid-
ering that the human brain exhibits an age-related increase in
MAO-B activity, inhibition of this enzyme is especially relevant in
the treatment of PD.6–8 The endogenous small molecule isatin (1)
(Fig. 1) has been reported to be a moderately potent inhibitor of
human MAO-B with an enzyme-inhibitor dissociation constant
3
5
6
N
N
N
O
8
2
N
O
H
Caffeine (2)
Isatin (1)
O
N
N
N
8
Cl
N
O
CSC (3)
Figure 1. The structures of isatin (1), caffeine (2) and CSC (3).
(Ki value) of 3
lM. Isatin also inhibits human MAO-A with a Ki va-
lue of 15
l
M.9 The three-dimensional structure of a complex be-
Another small molecule, caffeine (2), is a weak inhibitor of
MAO-B with a Ki value of 3.6 mM. The inhibition potency of caf-
feine is substantially increased by substitution at C-8 of the caffei-
tween isatin and human recombinant MAO-B shows that isatin
binds within the substrate cavity with the dioxoindolyl NH and
the C-2 carbonyl oxygen hydrogen bonded to ordered water mole-
cules in the active site (Fig. 2).10 This binding mode leaves the en-
trance cavity of MAO-B unoccupied. The structure of isatin bound
to MAO-A has not yet been determined.
nyl ring with
chlorostyryl)caffeine [CSC, (3)] (Ki = 0.086
a
styryl side-chain. For example, (E)-8-(3-
M) is approximately
l
40,000-fold more potent as an inhibitor of baboon liver MAO-B
than is caffeine.11,12 Also of interest is the observation that CSC
does not bind to MAO-A.9 The improved inhibition potency and
selectivity of CSC compared to caffeine has been explained by the
possibility that (E)-styrylcaffeines span both the substrate and en-
trance cavities of MAO-B. This would allow for more productive
* Corresponding author. Tel.: +27 18 2992206; fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.030

2510
E. M. Van der Walt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2509–2513
Figure 2. The binding modes of isatin, 4a and 5 in the active site of MAO-B. The three-dimensional structure of isatin in complex with human recombinant MAO-B (1OJA.pdb)
is displayed in panel A10 while the docking solutions of 4a and 5 obtained with LigandFit are displayed in panel B and C, respectively. The models were generated in Yasara.18
van der Waals contacts with active site residues in both cavities
that would lead to more potent inhibition.11,13 In contrast, caffeine
is expected to bind to either the substrate or entrance cavity, leav-
ing the other cavity unoccupied. Human MAO-A has a single active
site cavity whose geometry differs from that of MAO-B.14 Data in
the literature support the proposal that reversible inhibitors that
span both cavities of MAO-B are more likely to exhibit selectivity
for MAO-B over MAO-A. For example the crystal structures of rel-
atively large reversible inhibitors, such as 1,4-diphenyl-2-butene,10
farnesol9 and safinamide15 in complex with human MAO-B show
that these inhibitors exhibit such a dual binding mode. In the pres-
ent study we investigated the extent to which styryl substitution of
isatin also may enhance isatin’s MAO-B inhibition potency.
Inspection of the complex between isatin and human MAO-B10
reveals that, in the substrate cavity, the dioxoindolyl ring is orien-
tated with the 2-oxo group pointing towards the flavin cofactor
while C-5 is directed in the general direction of the entrance cavity
(Fig. 2). These structural features suggest that styryl substitution at
C-5 would result in structures that traverse both cavities with the
isatin moiety located in the substrate cavity and the C-5 styryl sub-
stituent extending into the entrance cavity. In contrast to the C-5
position, the C-6 position of isatin is directed towards the bottom
of the substrate cavity. Extension of a C-6 styryl side chain into
the entrance cavity would only be permitted if the isatin moiety
adopted a different binding mode from that of isatin as observed
in the human MAO-B crystal structure.10
the side chain of Ile-199, which acts as a ‘gate’ that separates the
entrance cavity from the substrate cavity,10 is rotated out of its
normal conformation to allow for the fusion of the two cavities
and the accommodation of the larger structures. This fused cavity
model of MAO-B allows for the possibility that 4a and 5 may span
both the entrance and substrate cavities. The docking calculations
were performed using the LigandFit application of Discovery Stu-
dio 1.7 according to a previously reported protocol.16,17 The top-
ranked docking solution obtained for 4a indicates that the styryl
side chain extends beyond the boundary defined by the side chain
of Ile-199 into the entrance cavity, while the dioxoindolyl ring is
located in the substrate cavity (Fig. 2). The binding orientation of
the dioxoindolyl ring of 4a is similar to that of isatin with the 2-
oxo and NH functional groups hydrogen bonded to water mole-
cules present in the active site.10 Structure 5 also spans both cavi-
ties with the styryl side chain located in the entrance cavity while
the dioxoindolyl ring binds within the substrate cavity. In contrast
to isatin and 4a, the dioxoindolyl ring of 5 is rotated through ꢀ180°
to allow for access of the C-6 styryl side chain to the entrance cav-
ity (Fig. 2). Based on these considerations, (E)-5-styrylisatin (4a–c)
and (E)-6-styrylisatin (5) analogues (Scheme 1) were synthesized
and evaluated initially as inhibitors of baboon liver mitochondrial
MAO-B and then as inhibitors of purified recombinant human
MAO-B and MAO-A. Their respective inhibition potencies are com-
pared to that of isatin.
Analogues 4a–c of (E)-5-styrylisatin and (E)-6-styrylisatin (5)
were synthesized in three steps (Scheme 1). Diethyl 4- or diethyl
3-nitrobenzylphosphonate (6a,b)19 was condensed with the appro-
priate benzaldehyde (7) in the Wittig reaction to yield the 4- or 3-
nitrostilbenes (8a,b), respectively.20 Reduction of the nitrostilbenes
with Sn/HCl led to the corresponding aminostilbenes (9a,b)21 that,
in turn, were treated with diethyl ketomalonate in the presence of
To provide additional insights, molecular docking studies of (E)-
5-styrylisatin (4a) and (E)-6-styrylisatin (5) (Scheme 1) in the ac-
tive site of MAO-B were performed. For this purpose, the crystallo-
graphic structure of the complex of the reversible inhibitor
safinamide and human recombinant MAO-B was selected
(2V5Z.pdb).15 This selection was based on the observation that

E. M. Van der Walt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2509–2513
2511
O2N
H2N
O2N
a
b, c
OHC
+
(OEt)2
P
6a,b
7
R
8a,b
9a,b
O
R
R
O
O
5
R
d, e
or
O
O
R
N
6
N
H
4a: R = H
4b: R = Cl
4c: R= F
H
5: R = H
Scheme 1. Synthetic pathway to analogues 4a–c of (E)-5-styrylisatin and (E)-6-styrylisatin (5). Reagents and conditions: (a) NaOEt (b) SnCl2/HCl, reflux (c) NaOH (d) diethyl
ketomalonate, CH3CO2H, 120 °C (e) air, 120 °C.
acetic acid according to the literature description.22 Following oxi-
dative decarboxylation of the resulting 3-hydroxy-2-oxindolyl
intermediate, the target (E)-styrylisatin analogues (4–5) were
obtained.22,23
MAO-B activity remained unchanged, irrespective of the preincu-
bation period (results not shown), it may be concluded that these
(E)-styrylisatins interact reversibly with the active site of MAO-B.
The inhibition potencies towards baboon liver mitochondrial
MAO-B are presented in Table 1. All of the (E)-styrylisatin ana-
logues were found to be more potent inhibitors of baboon liver
MAO-B than isatin. For example, (E)-5-styrylisatin (4a)
(IC50 = 41.7 nM) and (E)-6-styrylisatin (5) (IC50 = 444 nM) were
The MAO-B inhibitory properties of the (E)-styrylisatin ana-
logues 4–5 initially were investigated using baboon liver mito-
chondrial fractions as a source of MAO-B and 1-methyl-4-(1-
methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine (MMTP) as sub-
strate.12 These procedures have been documented in literature.12
The baboon liver mitochondrial fractions were prepared according
the procedure described in literature for the preparation of beef li-
ver mitochondrial fractions.24 In order to determine if the test
compounds acted as time-dependent inactivators or reversible
inhibitors of the enzyme, 4b (40 nM) was preincubated with the
mitochondrial fractions for periods of 0, 15, 30, and 60 min.17 Since
Table 2
The Ki values for the inhibition of purified recombinant human MAO by isatin (1) and
(E)-styrylisatin analogues (4–5)
MAO-A
MAO-B
MAO-B
selectivityc mutanta
M)
MAO-B I199A
wild-typea
wild-typeb
(l
M)
(lM)
(l
1
Isatin
15d
0.78 0.10
5.6 0.3
3d
5.0
2.5
4.0
12
1.1 0.2
2
4a (E)-5-Styrylisatin
4b (E)-5-(3-
Chlorostyryl)isatin
4c (E)-5-(3-
0.31 0.06
1.4 0.4
Table 1
12.5 0.7
4.5 0.2
2.2 0.4
The IC50 values for the inhibition of baboon liver mitochondrial MAO-B by isatin (1)
and (E)-styrylisatin analogues (4–5)
2.2 0.2
0.60 0.07
0.56 0.02
3.6
Fluorostyryl)isatin
(E)-6-Styrylisatin
IC50 valuea (nM)
5
22
2
39
1
Isatin
8566 530
41.7 1.8
20.7 0.6
30.1 4.6
444 12.2
a
The Ki values were determined spectrophotometrically using p-CF3-benzyl-
4a
4b
4c
5
(E)-5-Styrylisatin
(E)-5-(3-Chlorostyryl)isatin
(E)-5-(3-Fluorostyryl)isatin
(E)-6-Styrylisatin
amine as substrate.
b
The Ki values were determined spectrophotometrically using benzylamine as
substrate.
c
Selectivity for the B isoform is given as the ratio of Ki(MAO-A)–Ki(MAO-B) of the
a
The IC50 values were determined as described previously12 and are expressed as
wild-type enzymes.
d
the means SEM of duplicate determinations.
Values obtained from Ref. 9.
1.50
1.25
1.00
0.75
0.50
0.25
0.00
2.5
2.0
1.5
1.0
0.5
0.0
0.00
0.01
0.02
0.00
0.01
0.02
1/[Benzylamine] (µM-1)
1/[p
-CF3-Benzylamine] (µM-1)
Figure 3. Lineweaver–Burk plots of the recombinant human MAO-A (left) and MAO-B (right). The activity of MAO-A (left) was assayed at varied concentrations of p-CF3-
benzylamine with constant inhibitor concentrations of: (j) 0.0 M of 4a, (s) 1.0 M of 4a, (N) 2.0 M of 4a, (.) 3.0 M of 4a.The activity of MAO-B (right) was assayed at
varied concentrations of benzylamine with constant inhibitor concentrations of: (j) 0.0 M of 4a, (s) 0.3 M of 4a, (N) 0.7 M of 4a, (.) 1.0 M of 4a.
l
l
l
l
l
l
l
l

2512
E. M. Van der Walt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2509–2513
Figure 4. The docking solutions obtained with LigandFit for the binding of 4a (A) and 5 (B) in the active site of MAO-A (2Z5X.pdb).14 The models were generated in Yasara.18
approximately 200- and 19-fold more potent than isatin
(IC50 = 8566 nM), respectively.
pared to that of 4a. Comparison of the structures of CSC with those
of 4a and 5 after energy minimization shows that CSC exhibits a
planar structure where both rings occupy the same planar orienta-
tion (Fig. 5). In contrast, the styrylisatin analogues show the aro-
matic styryl ring is not coplanar with the isatin ring. For MAO-A
inhibition by styrylisatins and styrylcaffeines a relatively larger de-
gree of conformational freedom may be a requirement since the
aromatic rings of 4a and 5 (Fig. 4) adopt non-coplanar conforma-
tions in the MAO-A active site. Rigid coplanar styrylcaffeines such
as CSC are therefore not expected to be MAO-A inhibitors.
In conclusion, (E)-5-styrylisatin and (E)-6-styrylisatin analogues
have been identified as promising new probes of the binding sites
of MAO-B and of MAO-A. The findings of this study support the
hypothesis that the inhibition potencies of small molecule inhibi-
tors of these flavoenzymes may be improved by substitution with
side chains that promote binding to both the entrance and sub-
strate cavities of MAO-B. The side chain of the Ile-199 ‘gate’ in
MAO-B appears to play an important role in the recognition of
The MAO-B inhibitory properties of the (E)-styrylisatin ana-
logues were also investigated using purified recombinant human
MAO-B.25 For all analogues tested, the modes of inhibition were
found to be competitive (Fig. 3). As shown in Table 2 the (E)-sty-
rylisatin analogues are 2–10-fold more potent than isatin as inhib-
itors of recombinant human MAO-B. These data provide evidence
that potent MAO-B inhibitors result from structures that bind to
both the entrance and substrate cavities. It was previously re-
ported that the Ile-199 ‘gate’ residue has significant functional
importance in inhibitor binding and may be a determinant for
the specificity of reversible MAO-B inhibitors.9 The role of Ile-199
in the binding of 4–5 and of isatin to MAO-B was investigated using
the human MAO-B I199A mutant protein. The results (Table 2) doc-
ument that isatin and all of the (E)-styrylisatin analogues are
slightly weaker inhibitors of the human MAO-B I199A mutant en-
zyme when compared to wild-type MAO-B. The Ile-199 residue
therefore plays an important role in the binding interactions of
these analogues. These data are consistent with the binding of
(E)-styrylisatins in close proximity to Ile-199, a binding mode pos-
sible if the inhibitors traverse both active site cavities. Since the
binding affinity of isatin to the I199A mutant protein is also re-
duced, hydration and/or structural alterations to the binding site
cannot be ruled out and must await structural studies on this mu-
tant form of MAO-B. According to the molecular docking studies,
the functional groups of the inhibitors that may interact with Ile-
199 are the styryl moieties of compounds 4–5.
Interestingly, the (E)-styrylisatin analogues are also competitive
inhibitors of recombinant human MAO-A,26–28 with 4a exhibiting
the most potent inhibition (Ki = 0.78
lM). (E)-6-Styrylisatin (5)
was the least potent inhibitor of MAO-A (Ki = 22
lM), and dis-
played the highest selectivity for MAO-B (39-fold) of all the isatin
analogues tested. These results are surprising since relatively large
reversible inhibitors, such as 1,4-diphenyl-2-butene and CSC that
occupy both the entrance and substrate cavities of MAO-B, exhibit
selective inhibition of the MAO-B isoform and do not to bind to
MAO-A.9 To provide additional insights into this problem, molecu-
lar docking of 4a and 5 in the active site of MAO-A were performed
using LigandFit16 as described before.17 For this purpose, the crys-
tallographic structure of the complex of human recombinant MAO-
A with the reversible inhibitor harmine was selected (2Z5X.pdb).14
As shown in Figure 4, in the top-ranked docking solutions the
dioxoindolyl rings of 4a and 5 are positioned close to the FAD with
the styryl side chains extending towards the entrance of the active
site cavity. These binding modes are similar to those observed with
MAO-B. Also analogous to the docking results obtained with MAO-
B is the dioxoindolyl ring of 5 which is rotated through ꢀ180° com-
Figure 5. Energy-minimized structures of 4a (top), 5 (middle) and CSC (bottom).
These structures and their energy-minimized conformations were constructed
using Chem-3D (CambridgeSoft). The relative conformations of all structures are
pictured with the isatin or caffeine rings being perpendicular to the plane of the
page. Oxygen atoms are in red, nitrogen atoms in blue and chlorine atoms in green.

E. M. Van der Walt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2509–2513
2513
17. Ogunrombi, M. O.; Malan, S. F.; Terre’Blanche, G.; Castagnoli, N., Jr.; Bergh, J. J.;
Petzer, J. P. Bioorg. Med. Chem. 2008, 16, 2463.
18. Yasara Structure, Yasara Biosciences, Graz, Austria. 2008, http://
www.yasara.org.
19. Lee, Y.-B.; Woo, H. Y.; Yoon, C.-B.; Shim, H.-K. J. Mater. Chem. 1999, 9, 2345.
20. Kuo, E. A.; Hambleton, P. T.; Kay, D. P.; Evans, P. L.; Matharu, S. S.; Little, E.;
McDowall, N.; Jones, C. B.; Hedgecock, C. J.; Yea, C. M.; Chan, A. W.; Hairsine, P.
W.; Ager, I. R.; Tully, W. R.; Williamson, R. A.; Westwood, R. J. Med. Chem. 1996,
39, 4608.
21. Hanna, P. E.; Gammans, R. E.; Sehon, R. D.; Lee, M. K. J. Med. Chem. 1980, 23,
1038.
22. Langenbeck, V. W.; Rühlmann, K.; Reif, H. H.; Stolze, F. J. Prakt. Chem. 1957, 4,
136.
reversible inhibitors possibly by interacting with the styryl side
chains of compounds 4–5 as well as other factors that will require
additional structural information to identify. The surprising finding
that (E)-styrylisatins are also competitive inhibitors of MAO-A, in
contrast to results with CSC, shows that consideration of the rela-
tive geometries are factors important in the design of MAO
inhibitors.
Acknowledgments
23. Compound 4a: yield 77%; mp 254–255 °C, lit. 264–266 °C;22 1H NMR (Varian
Gemini 300, DMSO-d6) d 6.92 (d, 1H, J = 8.1 Hz), 7.21 (s, 1H), 7.25 (m, 2H), 7.35
(t, 2H, J = 7.2 HZ), 7.56 (d, 2H, J = 7.2 Hz), 7.75–7.81 (m, 2H), 11.12 (s, 1H); 13C
NMR (DMSO-d6) d 112.38, 118.22, 122.03, 126.34, 126.96, 127.51, 127.75,
128.61, 132.12, 136.16, 136.90, 149.69, 159.49, 184.35; EIMS (AutoSpec ETOF,
Micromass) m/z 249 (M+); HR-EIMS m/z calcd 249.078979, found 249.080332.
Compound 4b: yield 28%; mp 252 °C; 1H NMR (DMSO-d6) d 6.94 (d, 1H,
J = 8.1 Hz), 7.22 (1H, d, J = 16.5 Hz), 7.28–7.34 (m, 3H), 7.39 (t, 1H, J = 7.9 HZ),
7.52 (d, 1H, J = 7.8 Hz), 7.65 (s, 1H), 7.77–7.78 (m, 1H), 7.80 (dd, 1H, J = 8.2,
1.7 Hz); 13C NMR (DMSO-d6) d 113.63, 119.41, 123.46, 126.35, 127.05, 127.46,
128.69, 130.03, 131.77, 132.87, 134.88, 137.78, 140.91, 151.28, 161.18, 185.92;
EIMS m/z 283 (M+); HR-EIMS m/z calcd 283.04001, found 283.03829.
Compound 4c: yield 5%; mp 230–235 °C; 1H NMR (DMSO-d6) d 6.94 (d, 1H,
J = 8.1 Hz), 7.06–7.09 (m, 2H), 7.24 (d, 1H, J = 16.5 HZ), 7.31 (d, 1H, J = 16.5 Hz),
7.39–7.43 (m, 3H), 7.77 (m, 1H), 7.80 (dd, 1H, J = 8.2, 1.7 Hz); 13C NMR (DMSO-
d6) d 113.55 (d), 115.35 (d), 119.55, 123.44, 124.06, 127.79, 129.91, 131.85 (d),
133.05, 137.75, 141.07 (d), 151.39, 161.04, 163.28, 164.90, 185.99; EIMS m/z
267 (M+); HR-EIMS m/z calcd 267.069557, found 267.069991. Compound 5:
yield 66%; mp 257–258 °C, lit. 261–265 °C.22 1H NMR (DMSO-d6) d 7.09 (s, 1H),
7.29–7.43 (m, 6H), 7.50 (d, 1H, J = 7.8 Hz), 7.67 (dd, 2H, J = 7.1, 1.5 Hz), 11.10 (s,
1H); 13C NMR (DMSO-d6) d 109.34, 116.77, 121.03, 125.14, 127.15, 127.61,
128.66, 128.74, 133.05, 136.22, 147.05, 151.19, 159.95, 183.20; EIMS m/z 249
(M+); HR-EIMS m/z calcd 249.078979, found 249.079214.
24. Salach, J. I.; Weyler, W. Meth. Enzymol. 1987, 142, 627.
25. Newton-Vinson, P.; Hubálek, F.; Edmondson, D. E. Protein Exp. Purif. 2000, 20,
334.
26. Li, M.; Hubálek, F.; Newton-Vinson, P.; Edmondson, D. E. Protein Exp. Purif.
2002, 24, 152.
27. Recombinant human liver MAO-A and MAO-B were expressed in Pichia pastoris
and purified as described previously.25,26 The human MAO-B I199A mutant
protein was constructed via site-directed mutagenesis. A manuscript is in
preparation detailing the construction, expression, and purification protocol
for the human MAO-B I199A mutant protein.
28. MAO activity measurements were performed spectrally using, as substrates, p-
CF3-benzylamine (MAO-A and MAO-B I199A mutant) and benzylamine (MAO-
B) and following the formation of their respective aldehyde products at 243
and 250 nm, respectively.29 Incubations were carried out at 25 °C in a 50 mM
phosphate buffer (pH 7.5) containing 0.5% (w/v) reduced Triton X-100. Ki
values were determined by measuring the initial rates of substrate oxidation at
Financial support from the National Research Foundation and
Medical Research Council, South Africa is acknowledged. This work
was supported by National Institutes of Health Grant GM-29433
(D.E.E.) and National Institute of Neurological Disorders and Stroke
award number F31NS063648 (to E.M.M.). The content of this pub-
lication is solely the responsibility of the authors and does not nec-
essarily represent the official views of the National Institute of
Neurological Disorders and Stroke or the National Institutes of
Health.
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