The pH dependence of kinetic isotope effects in
monoamine oxidase A indicates stabilization of the
neutral amine in the enzyme–substrate complex
Rachel V. Dunn
1
, Ker R. Marshall
2
, Andrew W. Munro
1
and Nigel S. Scrutton
1
1 Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, UK
2 Department of Biochemistry, University of Leicester, UK
The mammalian monoamine oxidases (MAO) (EC
1.4.3.4) are flavoproteins localized to the outer mito-
chondrial membrane, and contain a FAD cofactor
covalently linked via the 8a-methyl group to an active
site cysteine residue [1]. They catalyse the oxidative
deamination of neurotransmitters (e.g. dopamine and
serotonin) and exogenous alkylamines, and are there-
fore important pharmaceutical targets for the develop-
ment of antidepressants and neuroprotective agents [2].
The catalytic cycle for monoamine oxidase activity is
shown in Scheme 1.
A number of mechanisms for MAO-catalysed amine
oxidation have been proposed over the years, and sev-
eral reviews are available [3–5]. There are currently
Keywords
kinetic isotope effect; mechanism;
monoamine oxidase; pH dependence
Correspondence

, report-
ing on ionizations in the free enzyme and ⁄ or free substrate) is due to
deprotonation of the free substrate, and the alkaline limb is due to unfa-
vourable deprotonation of an unknown group on the enzyme at high pH.
The pK
a
of the free amine is above 9.3 for all substrates, and is greatly per-
turbed (DpK
a
$ 2) on binding to the enzyme active site. This perturbation
of the substrate amine pK
a
on binding to the enzyme has been observed
with other amine oxidases, and likely identifies a common mechanism for
increasing the effective concentration of the neutral form of the substrate
in the enzyme–substrate complex, thus enabling efficient functioning of
these enzymes at physiologically relevant pH.
Abbreviations
ES, enzyme–substrate; KIE, kinetic isotope effect; MAO, monoamine oxidase; PEA, phenylethylamine; TMADH, trimethylamine
dehydrogenase.
3850 FEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS
three main mechanistic proposals for MAO catalysis.
These comprise: (a) the concerted polar nucleophilic
mechanism; (b) the direct hydride transfer mechanism;
and (c) the single electron transfer mechanism. Recent
support for the concerted polar nucleophilic mecha-
nism has come from kinetic and structural studies on
tyrosine mutants of MAO B [6], and also from compu-
tational studies [7,8]. However, analysis of nitrogen
isotope effects conducted on a related amine oxidase,

are able to function efficiently at physiological pH with
the deprotonated amine substrate, despite the high pK
a
values of common substrates.
Results and Discussion
Catalytically influential macroscopic ionizations
The pH dependence of the catalytic rate was studied
by both stopped-flow and steady-state techniques.
Although the catalytic activity of MAO A has been
shown to be dominated by the reductive half-reaction,
this may change with pH, leading to a different pH
dependence for the reductive half reaction compared
to complete catalytic turnover. Also, a range of sub-
strates were analysed to establish whether the observed
kinetic trends were applicable for all amine substrates.
For example, although benzylamine is a well character-
ized substrate for MAO A, all naturally occurring sub-
strates contain an ethylamine group in the structure.
All steady-state kinetic measurements were per-
formed in air-saturated buffers, which have been
shown to saturate the enzyme with the second sub-
strate, oxygen [16]. The k
cat
values for benzylamine
(see supplementary Fig. S1) and kynuramine exhibit a
sigmoidal dependence upon pH, as shown in Fig. 1A
for kynuramine, indicating the presence of a single
macroscopic ionization with a pK
a
value of 7.9 ± 0.1

tude change (20–30% at most) and did not vary with
substrate concentration, only the substrate dependence
of the fast phase was analysed further. As expected, the
pH dependence of the kinetic parameters for the reduc-
tive half-reaction of benzylamine oxidation exhibited
Scheme 1. Catalytic cycle of monoamine oxidase.
R. V. Dunn et al. Isotope effects and their pH dependence in MAO A
FEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS 3851
similar pH profiles to those obtained for the equivalent
steady-state parameters (see supplementary Fig. S2). At
each pH, the value of k
red
was found to be less than
that of k
cat
, which has been observed previously in
kinetic studies with MAO A [12]. This was attributed to
aggregation of the detergent solubilized enzyme at the
high concentrations required for stopped-flow assays.
To minimize this potential effect, the same concentra-
tion of MAO A was used in all stopped-flow experi-
ments. The k
red
exhibited a single ionization with a
corresponding pK
a
of 7.4 ± 0.1, and the k
red
⁄ K
s

ionizable residues that influence the correct orientation
for catalysis and affect the resulting pH profile. The
k
red
⁄ K
s
data also exhibit a bell-shaped pH profile, but
meaningful pK
a
values cannot be determined due to the
large errors associated with these data (Fig. 1D). A
summary of all pK
a
values is given in Table 1.
pH dependence of KIEs identifies substrate
ionization in the ES complex
As the amine substrates are able to ionize over the pH
range investigated, some of the observed macroscopic
0.0
0.5
1.0
1.5
2.0
2.5
3.0
AB
CD
k
cat
(s

(s
–1
)
pH
6.0
6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
-20
0
20
40
60
80
100
120
140
k
red
/K
s
(s
–1
mM
–1
)
pH
Fig. 1. (A, B) pH dependence of the steady-
state kinetic parameters of MAO A-cataly-
sed oxidation of kynuramine at 20 °C. (C, D)
pH dependence of the reductive half-reac-

a
; in part due to: (a) the
shorter C-D bond length leading to a greater charge
density on the carbon and hence greater nitrogen lone
pair availability and (b) the greater reduced mass of
the deuterated analogue for the N-H stretching fre-
quency, causing it to lie lower in the asymmetric
potential energy well (lower zero point energy) relative
to the protiated substrate [18–20]. The pH dependence
of the reductive half-reaction of benzylamine oxidation
was determined at a saturating substrate concentration
of 5 mm, for both the protiated and deuterated forms.
The pH profile of k
red
for both substrates is shown in
Fig. 2, and a small alkaline shift is observed for deu-
terated benzylamine relative to protiated benzylamine,
which results in a decrease of the calculated KIE from
13 to 8 with increasing pH (Fig. 2, inset). A similar
effect has been seen in studies with trimethylamine
dehydrogenase (TMADH), where substrate perdeutera-
tion caused a shift in the observed macroscopic ioniza-
tion in the ES complex, resulting in a strong
dependence of the KIE upon pH [21]. This result, com-
bined with mutagenesis work on TMADH, led to the
assignment of the ionization as that of bound sub-
strate. It is likely that a similar effect is observed with
MAO A, where the observed macroscopic ionization is
due to deprotonation of the bound amine substrate.
The effect of substrate deuteration was more signifi-

ESH
+
form relative to ES at a given pH. Therefore,
the observed KIE will appear inflated at low pH, and
be greater than that due purely to bond breakage
effects.
Perturbation of amine pK
a
mechanism of
monoamine oxidase
The accuracy of the derived pK
a
values from the bell-
shaped pH profiles for k
red
⁄ K
s
or k
cat
⁄ K
m
is quite low.
This is partly due to the error associated with fitting
the particular functions to narrow plots because the
width of the curve is relatively insensitive to the differ-
ence in pK
a
values when pK
a1
)pK

mately 2 relative to the free substrate. Such an effect
has been seen with other amine oxidases. For example,
6 7 8 9 10
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
p
H
k
red
(s
–1
)
0.000
0.002
0.004
0.006
0.008
0.010
0.012
6.5 7.0 7.5 8.0 8.5 9.0 9.5
6
8

A
K
A
S
ES
k
red
+
ESH
+
E + S ES E + P
Scheme 2. Control of flavin reduction by substrate ionization.
R. V. Dunn et al. Isotope effects and their pH dependence in MAO A
FEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS 3853
trimethylamine dehydrogenase, mouse polyamine oxi-
dase and monomeric sarcosine oxidase exhibit acidic
shifts in substrate pK
a
values of 3.3–3.6, 0.8 and 2.6,
respectively, upon substrate binding to the active site
[18,23,24]. Therefore, the active site of each of these
enzymes is organized to stabilize the neutral form of
the amine substrate by approximately 11 kJÆmol
)1
rela-
tive to the charged, protonated form.
The steady-state oxidation of kynuramine by
MAO A has been studied previously, and the overall
trends of the data are very similar to those reported in
the present study, which suggests that variations in

or K
s
values with pH for all substrates used in the present
study are shown in Table 2. Unlike the values for
kynuramine, the K
m
or K
s
values for all other
substrates tested exhibited a general decrease with
increasing pH in the range from $ 6.5–8.5. When the
pK
m
or pK
s
values are plotted as a function of pH
(results not shown), the initial slope at low pH is < 1,
which may simply reflect that the relevant macroscopic
ionizations are not sufficiently separated to be individ-
ually identified. There are too few points at high pH
to accurately calculate the change of slope that occurs
above pH 9 for all substrates, although it is clear that
the K
m
and K
s
values are increased.
Conclusions
Despite the suggestion that it is the protonated form
of the substrate that binds the enzyme, it is difficult to

˚
resolution [25], allowing a more
detailed knowledge of the active site geometry of
Table 2. pH dependence of K
m
and K
s
values determined for MAO A at 20 °C.
pH
K
m
(mM) K
s
(mM)
Benzylamine Kynuramine Benzylamine PEA
6.5 – 0.042 ± 0.003 0.213 ± 0.012 0.019 ± 0.017
7.0 0.31 ± 0.04 0.047 ± 0.003 0.134 ± 0.032 0.145 ± 0.007
7.2 – – 0.136 ± 0.007 –
7.5 0.20 ± 0.02 0.041 ± 0.002 0.138 ± 0.014 0.122 ± 0.015
8.0 0.10 ± 0.01 0.044 ± 0.003 0.034 ± 0.004 0.078 ± 0.003
8.5 0.077 ± 0.002 0.045 ± 0.002 0.033 ± 0.004 0.048 ± 0.002
9.0 0.087 ± 0.004 0.082 ± 0.005 0.039 ± 0.004 0.028 ± 0.002
9.2 – – 0.079 ± 0.019 0.042 ± 0.016
9.5 0.137 ± 0.003 0.261 ± 0.028 0.424 ± 0.108 0.268 ± 0.125
Isotope effects and their pH dependence in MAO A R. V. Dunn et al.
3854 FEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS
MAO A. Inspection of the active site suggests that
there are several candidates responsible for the unfa-
vourable deprotonation event that occurs at alkaline
pH, including multiple tyrosine residues, the covalently

was first cloned into pGem-T Easy (Promega, Madison,
WI, USA) following A-tailing using standard techniques. A
modified version of the pPICZA plasmid (Invitrogen, Carls-
bad, CA, USA) was used as the final expression vector, in
which the NcoI site upstream of the Zeocin resistance gene
was mutated using the primer 5¢-GGTGAGGAAC
TAAAACATGGCCAAGTTGACCAGTGC-3¢ and its
reverse complement. A unique NcoI site was then intro-
duced at the multiple cloning site generating a Kozak
sequence to allow efficient translation initiation of the
inserted gene, using the primer 5¢-CAACTAATTATTCG
AAACCATGGATTCACGTGGCCC-3¢ and its reverse
complement. The modified pPICZA vector was then
digested with NcoI and XhoI, and similarly digested maoA
inserted following gel purification. The sequence of the
cloned gene was confirmed by DNA sequencing. All site-
directed mutagenesis reactions were performed using the
Stratagene QuikChange site-directed mutagenesis kit (Strat-
gene, La Jolla, CA, USA) with Pfu Turbo DNA polymer-
ase; except that the DNA was transformed into Novablue
competent cells (Novagen, Madison, WI, USA). The
pPICZAmaoA plasmid was linearized with PmeI and trans-
formed into Pichia pastoris strain KM17H by electropora-
tion following standard protocols [26]. Successful
transformants were selected on agar plates containing
100 lgÆmL
)1
of Zeocin. Multiple integrants were selected
by growth on plates with increasing Zeocin concentrations,
and screened for MAO A expression. Typically, 8 L of cul-

calculated by following the initial increase of A
316
due to
production of 4-hydroxyquinone and using an extinction
coefficient of 12 000 m
)1
Æcm
)1
[28]. One unit of enzyme
activity is defined as the amount of enzyme required to
oxidize 1 l mol of kynuramine in 1 min.
Steady-state kinetic measurements
Steady-state kinetic measurements were performed at 20 °C
in 20 mm Bis-Tris propane buffer containing 0.5% (w ⁄ v)
reduced Triton X-100, 50 mm NaCl and 20% glycerol. The
pH of the buffer was set by the addition of small amounts
of concentrated HCl or NaOH, and was in the range 6.5–
9.5. The rate of enzymatic activity was determined by moni-
toring the initial linear increase in absorbance at 250 nm
due to the production of benzaldehyde, employing an
extinction coefficient 12 800 m
)1
Æcm
)1
[29], and using a
Varian Cary 50 Bio spectrophotometer (Varian Inc., Palo
Alto, CA, USA). The concentration of benzylamine was
typically in the range 0.02–2 m m, and the assay was started
by the addition of MAO A to a final concentration of
0.6 lm. Michaelis–Menten kinetic behaviour was observed

MAO A with various concentrations of either benzylamine
or phenylethylamine. A minimum of six substrate concen-
trations were used at each pH that spanned almost two
orders of magnitude. The rate of flavin reduction was
monitored under pseudo first-order conditions by follow-
ing the decrease in A
456
.
Data analysis
Steady-state kinetic data were fitted with the Michaelis–
Menten equation using nonlinear least-squares analysis
incorporated into the origin software package (OriginLab
Corp., Northampton, MA, USA), and the maximal cata-
lytic centre activity (k
cat
) and the Michaelis constant (K
m
)
determined. The observed rates from stopped-flow data
were obtained by fitting the reaction traces to an equation
for either single- or double-exponential decay with offset,
as appropriate. Analysis was performed by nonlinear least-
squares regression on an Acorn RISC PC (Acorn Com-
puters, Cambridge, UK) using spectrakinetics software
(Applied Photophysics). The observed rate of enzyme
reduction was found to have a hyperbolic dependence with
respect to substrate concentration at each pH. The limiting
rate of flavin reduction (k
red
) and the substrate binding con-

ðpHÀpK
a2
Þ
ð2Þ
Where EH and E are the limiting catalytic activities of the
protonated and deprotonated forms of the ionization
group, respectively; and T
max
is the theoretical maximal
value. For the pH profile in which a double ionization is
observed, it is assumed that the observed parameter is
dependent upon the singly protonated species, therefore
producing a bell-shaped profile tending towards zero at the
extremes of pH. Examples of the reaction transients and
further details regarding treatment of the data are given in
supplementary Figs S3–S5.
Acknowledgements
This work was funded by the UK Biotechnology and
Biological Sciences Research Council. N.S.S. is a
BBSRC Professorial Research Fellow.
References
1 Binda C, Newton-Vinson P, Hubalek F, Edmondson
DE & Mattevi A (2002) Structure of human mono-
amine oxidase B, a drug target for the treatment of
neurological disorders. Nat Struct Biol 9, 22–26.
2 Cesura AM & Pletscher A (1992) The new generation
of monoamine oxidase inhibitors. Prog Drug Res 38,
171–297.
3 Scrutton NS (2004) Chemical aspects of amine
oxidation by flavoprotein enzymes. Nat Prod Rep 21,

amine oxidase A. J Biol Chem 280, 4627–4631.
11 Kay CW, El Mkami H, Molla G, Pollegioni L & Ram-
say RR (2007) Characterization of the covalently bound
anionic flavin radical in monoamine oxidase a by elec-
tron paramagnetic resonance. J Am Chem Soc 129,
16091–16097.
12 Miller JR & Edmondson DE (1999) Structure-activity
relationships in the oxidation of para-substituted
benzylamine analogues by recombinant human liver
monoamine oxidase A. Biochemistry 38, 13670–13683.
13 McEwen CM, Sasaki G & Lenz WR (1968) Human
liver mitochondrial monoamine oxidase. I. Kinetic stud-
ies of model interactions. J Biol Chem 243, 5217–5225.
14 Edmondson DE, Bhattacharrya AK & Xu J (2000) Evi-
dence for alternative binding modes in the interaction
of benzylamine analogues with bovine liver monoamine
oxidase B. Biochim Biophys Acta 1479, 52–58.
15 Jones TZE, Balsa D, Unzeta M & Ramsay RR (2007)
Variations in activity and inhibition with pH: the pro-
tonated amine is the substrate for monoamine oxidase,
but uncharged inhibitors bind better. J Neural Transm
114, 707–712.
16 Ramsay RR (1991) Kinetic mechanism of monoamine
oxidase A. Biochemistry 30, 4624–4629.
17 Nandigama RK & Edmondson DE (2000) Structure-
activity relations in the oxidation of phenethylamine
analogues by recombinant human liver monoamine
oxidase A. Biochemistry 39, 15258–15265.
18 Basran J, Sutcliffe MJ & Scrutton NS (2001) Optimiz-
ing the Michaelis complex of trimethylamine dehydroge-

opening the entry for substrates ⁄ inhibitors. Proc Natl
Acad Sci USA 105, 5739–5744.
26 Invitrogen Corporation (1998) EasySelect Pichia
Expression Kit: A Manual of Methods For Expression
of Recombinant Proteins Using pPICZ and pPICZa in
Pichia pastoris. Invitrogen Corporation, San Diego,
CA.
27 Li M, Hubalek F, Newton-Vinson P & Edmondson DE
(2002) High-level expression of human liver monoamine
oxidase a in Pichia pastoris: comparison with the
enzyme expressed in Saccharomyces cerevisiae. Protein
Expr Purif 24, 152–162.
28 Weyler W & Salach JI (1985) Purification and proper-
ties of mitochondrial monoamine-oxidase type-A from
human-placenta. J Biol Chem 260, 13199–13207.
29 Walker MC & Edmondson DE (1994) Structure-activ-
ity-relationships in the oxidation of benzylamine
analogs by bovine liver mitochondrial monoamine-
oxidase-B. Biochemistry 33, 7088–7098.
30 Strickland S, Palmer G & Massey V (1975) Determina-
tion of dissociation-constants and specific rate of
constants of enzyme-substrate (or protein-ligand) inter-
actions from rapid reaction kinetic data. J Biol Chem
250, 4048–4052.
Supplementary material
The following supplementary material is available
online:
Fig. S1. pH dependence of the steady-state kinetic
parameters of MAO A-catalysed oxidation of benzyl-
amine at 20 °C.