However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the sug-gestion that it is the protonated form of the amine substrate that binds to the enzy
Trang 1monoamine oxidase A indicates stabilization of the
neutral amine in the enzyme–substrate complex
Rachel V Dunn1, Ker R Marshall2, Andrew W Munro1and Nigel S Scrutton1
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
N S Scrutton, Faculty of Life Sciences,
Manchester Interdisciplinary Biocentre,
University of Manchester, 131 Princess
Street, Manchester M1 7DN, UK
Fax: +44 161 3068918
Tel: +44 161 3065152
E-mail: nigel.scrutton@manchester.ac.uk
(Received 10 April 2008, revised 25 May
2008, accepted 2 June 2008)
doi:10.1111/j.1742-4658.2008.06532.x
A common feature of all the proposed mechanisms for monoamine oxidase
is the initiation of catalysis with the deprotonated form of the amine sub-strate in the enzyme–subsub-strate complex However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the sug-gestion that it is the protonated form of the amine substrate that binds to the enzyme To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow tech-niques with protiated and deuterated substrates For all substrates used, there is a macroscopic ionization in the enzyme–substrate complex attrib-uted to a deprotonation event required for optimal catalysis with a pKa of 7.4–8.4 In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant (kred⁄ Ks, 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 pKaof the free amine is above 9.3 for all substrates, and is greatly per-turbed (DpKa 2) on binding to the enzyme active site This perturbation
of the substrate amine pKa 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.
Trang 2three 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,
N-methyltryptophan oxidase, supported either a direct
hydride transfer mechanism or, possibly, a discrete
electron transfer mechanism [9] Support for a
modi-fied single electron transfer mechanism came following
the identification of a stable tyrosyl radical in partially
reduced MAO A [10] More recent EPR studies have
questioned this assignment and suggested that the
rad-ical species detected in partially reduced MAO is due
solely to the covalently linked flavin semiquinone,
leading to support for the direct hydride transfer
mechanism [11]
A common feature of all the proposed mechanisms
is the initiation of catalysis with the deprotonated
form of the amine substrate, and it is widely accepted
that it is the deprotonated form of the substrate that
binds in the functional enzyme–substrate (ES)
com-plex [12–14] By contrast, recent kinetic studies on
the pH dependence of the steady-state kinetic
param-eters for MAO A were interpreted to indicate that it
is the protonated form of the substrate that binds to
the enzyme [15] Due to the conflicting evidence from
the relatively few studies on the effects of pH on
MAO catalysis, a more comprehensive analysis is
required
The present study reports on the pH dependence of
recombinant human liver MAO A as characterized by
both steady-state and stopped-flow techniques The
effect of pH on the kinetic isotope effect (KIE) of the
reductive half-reaction is also presented The results
obtained provide insight into how monoamine oxidases
are able to function efficiently at physiological pH with
the deprotonated amine substrate, despite the high pKa
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 kcat 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 pKa value of 7.9 ± 0.1 obtained from curve fitting for both substrates The observed macroscopic ionization corresponds to a group in the ES complex that must be deprotonated for optimal activity The kcat⁄ Km values exhibit a bell-shaped pH profile with corresponding pKa values of 8.5 ± 0.1 and 9.2 ± 0.1 for benzylamine (see supple-mentary Fig S1), and 8.0 ± 0.2 and 8.8 ± 0.2 for kynuramine (Fig 1B) These results indicate that, with increasing pH, a favourable deprotonation step is followed by an unfavourable deprotonation event, either in the free enzyme or free substrate, to produce the bell-shaped pH profile
At pH 7.5 and below, the flavin monitored reductive half-reaction transients from stopped-flow assays were fitted using a single exponential function to determine the apparent rate constants for FAD reduction How-ever, above pH 7.5, the reaction traces were fitted instead with a double-exponential function, as a second, slower process was resolved in the flavin reductive reac-tion This biphasic behaviour has been observed previ-ously with para-substitued phenylethylamines, and the slow phase was attributed to the release of the imine product from the reduced enzyme [17] Because the slow phase was only a minor component of the total ampli-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.
Trang 3similar pH profiles to those obtained for the equivalent
steady-state parameters (see supplementary Fig S2) At
each pH, the value of kred was found to be less than
that of kcat, 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 kred exhibited a single ionization with a
corresponding pKaof 7.4 ± 0.1, and the kred⁄ Ks
exhib-ited a bell-shaped profile with pKa values of 8.6 ± 0.7
and 8.3 ± 0.7 obtained from curve fitting
By contrast to all other substrates, the pH
depen-dence of kred for MAO A-catalysed phenylethylamine
(PEA) oxidation displayed a bell-shaped profile, with
corresponding pKa values of 8.4 ± 0.2 and 8.7 ± 0.2
(Fig 1C) The cause of the additional macroscopic
ioni-zation on the alkaline side of the pH profile for PEA is
unknown For benzylamine and PEA, it has been
estab-lished that the rate-limiting step of flavin reduction is
due to aC-H bond cleavage, and it is unlikely that a
dis-tinct catalytic step affects flavin reduction to produce
the different pH dependence From quantitative struc-ture activity studies with MAO A, it has been shown that different factors influence the correct positioning
of para-substituted phenylethylamines compared to para-substituted benzylamines, and that these are required for efficient catalysis [12,17] It was suggested that the greater steric flexibility of the ethylamine side chain allows efficient aC-H bond cleavage without con-fining the phenyl ring to a specific orientation There-fore, the greater flexibility of the substrate when bound
to the active site may allow it to contact additional ionizable residues that influence the correct orientation for catalysis and affect the resulting pH profile The
kred⁄ Ks data also exhibit a bell-shaped pH profile, but meaningful pKavalues cannot be determined due to the large errors associated with these data (Fig 1D) A summary of all pKavalues 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
kcat
pH
0 10 20 30 40 50
kcat
pH
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
kred
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
kred
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-tion of MAO A-catalysed oxidahalf-reac-tion of phen-ylethylamine at 20 C.
Table 1 pK a values obtained from curve fitting for MAO A at 20 C ND, not determined.
Trang 4ionizations may be due to the substrate rather than to
groups on the enzyme A potential way to identify
sub-strate ionizations is to perturb the subsub-strate pKa (e.g
by deuteration) and to observe a corresponding shift
in the macroscopic ionization Deuteration of the
atoms bonded to the amine nitrogen is known to cause
an increase in the amine pKa; 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 kredfor 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-cant for TMADH, and may be explained by the
greater increase in substrate pKa upon perdeuteration
of trimethylamine (DpKa= 0.3) [18] compared to a-C deuteration of benzylamine (expected DpKa= 0.032) [19] A mechanism describing the ionization of the sub-strate and its effect on flavin reduction is shown in Scheme 2, where KAS and KAES are the dissociation constants for the free substrate and the enzyme-bound substrate, respectively [22] It is assumed that the rate
of flavin reduction (kred) is slow relative to the dissoci-ation steps, so that they remain in thermodynamic equilibrium It can be seen that if the pKaof the amine substrate is increased (e.g in the deuterated analogue), this will lead to a greater proportion of the unreactive 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 pKamechanism of monoamine oxidase
The accuracy of the derived pKa values from the bell-shaped pH profiles for kred⁄ Ksor kcat⁄ Kmis 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 pKa values when pKa1)pKa2 is < 0.6 [22] Despite this drawback, the pH profiles are still of qual-itative value Based upon the assignment of the ioniza-tion in the ES complex to that of the bound substrate,
it follows that the acid limb of the bell-shaped kred⁄ Ks
or kcat⁄ Km profile is due to deprotonation of the free substrate and that the alkaline limb is due to the unfa-vourable deprotonation of an unknown group on the enzyme at high pH The stated pKa values of the free substrates (9.3–9.9) are higher than those obtained from curve fitting (8.0–8.6) and may simply reflect the error in curve fitting as mentioned above The main effect of substrate binding is to perturb the amine pKa
to more acidic values; as the bound substrate has a
pKa of 7.4–8.4, this corresponds to a DpKa of approxi-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
pH
kred
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
10
12
16
18
pH
Fig 2 pH dependence of the reductive half-reaction of MAO
A-ca-talysed oxidation at 20 C with 5 m M benzylamine (filled circles, left
axis) or 5 m M deuterated benzylamine (open circles, right axis).
Inset: calculated KIE as a function of pH.
ES + SH
k1
k1
k2
k2
' '
KA
kred
+ ESH+
E + S ES E + P
Scheme 2 Control of flavin reduction by substrate ionization.
Trang 5trimethylamine dehydrogenase, mouse polyamine
oxi-dase and monomeric sarcosine oxioxi-dase exhibit acidic
shifts in substrate pKa 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
buffer composition have minimal effect [15] However,
the interpretation of the results was different in the
previous study It was suggested that, due to the initial
increase in rate with increasing pH and the relative
invariance of the Km values over the same pH range
(in which the concentration of the neutral form of the
substrate would be insignificant compared to the
pro-tonated form), it must be the propro-tonated form of the
substrate that binds to the enzyme, with subsequent
substrate deprotonation required for catalysis to
pro-ceed Therefore, the macroscopic ionization in the ES
complex was assigned to a group on the enzyme,
rather than to the ionization of bound substrate, as
indicated by data reported in the present study The
invariance of the Km values at low pH makes this a
plausible explanation, although it may be over
simplis-tic to assume that an exponential dependence of Km
with pH is required to indicate the binding of the
deprotonated form because the pH dependence of Km
or Ks is affected by all macroscopic ionizations
occur-ring in the system [22] The variations in the Kmor Ks
values with pH for all substrates used in the present
study are shown in Table 2 Unlike the values for
kynuramine, the Km or Ks values for all other
substrates tested exhibited a general decrease with
increasing pH in the range from 6.5–8.5 When the
pKm or pKs 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 Kmand Ksvalues are increased
Conclusions
Despite the suggestion that it is the protonated form
of the substrate that binds the enzyme, it is difficult to envisage specific binding of the charged substrate when the active site is organized for binding and activation
of the neutral form In addition, the pH dependence of the KIE and the observation of similar perturbation effects on substrate pKa values with other amine oxid-ases further support the catalytic significance of the deprotonated form Thus, we propose that binding of the substrate to the active site leads to a perturbation
of the pKa, effectively increasing the concentration of the neutral amine species We do not propose that it is only the protonated form that initially binds, but rather that preferential binding of the deprotonated form to the active site leads to a shift in the equilib-rium of the substrate ionization The present study emphasizes the benefits of using deuteration of com-pounds in conjunction with standard stopped-flow and steady-state analyses to provide deeper insight into reaction mechanism In the case of the amine oxidases, the perturbation of the substrate pKa upon binding to the active site appears to be a general feature, allowing efficient function of the enzyme at physiologically relevant pH values
The crystal structure of MAO A has recently been solved to 2.2 A˚ 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
Trang 6MAO 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
linked FAD, and possibly Lys305 that is co-ordinated
to the flavin via a water molecule To be confident
about any assignment, future work combining
muta-genesis studies with stopped-flow kinetic analysis is
required
Experimental procedures
Materials
Bis-Tris propane buffer, reduced Triton X-100,
kynur-amine, benzylkynur-amine, and b-phenylethylamine were obtained
from Sigma (St Louis, MO, USA) Deuterated benzylamine
CDN Isotopes (Quebec, Canada)
Expression and purification of MAO A
The gene encoding human liver MAO A was amplified
from a cDNA clone obtained from MRC Geneservices
(Cambridge, UK) using the primers 5¢-GTCTTCGAA
ACCATGGAGAATCAAGAGAAGGCGAGTATCGCGG
G-3¢ and 5¢-GAGAGCTCGAGAACAGAACTTCAAGAC
CGTGGCAGGAGC-3¢ The NcoI and XhoI sites used for
further cloning are shown underlined The amplified DNA
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
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
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
transformants were selected on agar plates containing
by growth on plates with increasing Zeocin concentrations, and screened for MAO A expression Typically, 8 L of cul-ture were grown in baffled flasks in an orbital incubator at
induction by centrifugation at 2000 g for 10 min The cells were resuspended in Pichia breakage buffer to
cell disruptor at 40 000 psi (TS-series 1.1 kW model; Con-stant Systems Ltd, Daventry, UK) followed by cooling on ice MAO A was then purified essentially as described pre-viously [27] Active fractions eluted from the DEAE-Sepha-rose column were concentrated by ultrafiltration and stored
against 20 mm potassium phosphate (pH 7.0), containing 20% glycerol, to remove the competitive inhibitor d-amp-hetamine that is present during the later stages of purifica-tion Typical yields from an 8 L growth were between 80–120 mg of purified MAO A Enzyme concentration
Enzyme assays
Routine activity measurements were conducted using a continuous spectrophotometric assay with kynuramine as
Triton X-100 and 0.2 mm kynuramine The activity was
production of 4-hydroxyquinone and using an extinction
activity is defined as the amount of enzyme required to oxidize 1 lmol of kynuramine in 1 min
Steady-state kinetic measurements
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
Varian Cary 50 Bio spectrophotometer (Varian Inc., Palo Alto, CA, USA) The concentration of benzylamine was typically in the range 0.02–2 mm, 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
at each pH studied; the only exception was at pH 6.5, where the rate of enzymatic activity was only assayed at saturating substrate concentrations due to the slow rate
Trang 7observed Identical experiments were performed with
as described above, and the final concentration of MAO A
in the assay was 0.1 lm
Single wavelength anaerobic stopped-flow
kinetic experiments
The reductive half-reaction of MAO A was studied using
an Applied Photophysics SX.18MV stopped-flow
spectro-photometer (Applied Photophysics Ltd, Leatherhead, UK)
housed in a Belle Technology glove box (< 5 p.p.m
oxygen) (Belle Technology, Portesham, UK) Studies were
performed in 20 mm Bis-Tris propane buffer containing
Buffer solutions were purged with nitrogen for 1 h and
then left to equilibrate overnight in the glove box
Dialy-sed MAO A was exchanged into the appropriate anaerobic
solutions of the substrates were also diluted into the
appropriate buffer To remove any final traces of oxygen,
10 units of glucose oxidase (Sigma) and 10 mm glucose
were added per mL of solution and left to incubate for
30 min once loaded into the stopped-flow syringes The
reactions were started by rapid mixing of 10 lm oxidized
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
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
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
[30] using the origin software package The pH dependence
of the kinetic parameters were fitted to an equation
descri-bing either a single (Eqn 1) or double (Eqn 2) ionization, as
values
10ðpHÞþ 10ðpK a Þ ð1Þ
1þ 10ðpK a1 pHÞþ 10ðpHpK a2 Þ ð2Þ
Where EH and E are the limiting catalytic activities of the protonated and deprotonated forms of the ionization
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
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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 20C
Fig S2 pH dependence of the reductive half-reaction
of MAO A-catalysed oxidation of benzylamine at
20C
Fig S3 Reaction transient for MAO A-catalysed oxidation of 0.5 mm PEA at pH 9.0 and 20C Fig S4 Reaction transient for MAO A-catalysed oxidation of 0.4 mm benzylamine at pH 8.5 and
20C
Trang 9Fig S5 Substrate dependence of the reductive
half-reaction of MAO A-catalysed oxidation of PEA at
pH 8.5 and 20C
This material is available as part of the online article
from http://www.blackwell-synergy.com
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