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Nitrogen Kinetic Isotope Effects for the Monoamine Oxidase
B-Catalyzed Oxidation of Benzylamine and (1,1-2H2)Benzylamine:
Nitrogen Rehybridization and CH Bond Cleavage Are Not Concerted
Susanna MacMillar,† Dale E. Edmondson,‡ and Olle Matsson†,*
†Department of Biochemistry and Organic Chemistry, Uppsala University, P.O. Box 576, SE-75123 Uppsala, Sweden
‡Department of Biochemistry and Chemistry, Emory University, 1510 Clifton Road, Atlanta, Georgia 30322, United States
S Supporting Information
b
right side) or the product formation (route on the left side).
ABSTRACT: Nitrogen kinetic isotope effects for the
oxidation of benzylamine and (1,1-2H2)benzylamine by
recombinant human monoamine oxidase B show that
cleavage of the CH bond is not concerted with rehybridiza-
tion of the nitrogen atom.
Recently performed semiempirical calculations on the isoallox-
azine ring system and some para-substituted benzylamines
undergoing the polar nucleophilic mechanism suggest that
adduct formation is the rate-limiting step of the reaction.5
Fitzpatrick and co-workers have reported kinetic isotope effect
(KIE) investigations for several flavoprotein oxidases, including
D-amino acid oxidase,6 L-tryptophan monooxygenase,7 and
N-methyltryptophan oxidase.8 Interpretation of the nitrogen
KIEs for flavoprotein amine oxidation reactions were aided by
quantum-chemical calculations using alloxazine and dimethyla-
mine as a model system.8 Their overall conclusion was in support
of a hydride transfer or a single electron transfer, thus dismissing
the nucleophilic addition mechanism. Moreover, it was suggested
that MAOs operate by the same mechanistic pathway, given their
structural similarities to flavoprotein amino acid oxidases.9
Many of the mechanistic studies performed have included the
use of KIEs. The substantial magnitudes of the KIEs observed
onoamine oxidases (MAOs) are flavin-dependent enzymes
M
that catalyze the oxidation of many amines to their
corresponding protonated imines. The protonated imines are
then nonenzymatically hydrolyzed to afford the final carbonyl
compounds. The enzyme itself is regenerated to its active form by
molecular oxygen, which in turn is reduced to hydrogen per-
oxide. In mammals, MAO exists in two different isozymes, A and
B, which differ partly in their substrate and inhibitor specificities.1
Many different approaches have been employed to gain insights
into how MAO effects catalysis, but the mechanism has yet to be
unambiguously established.
D
for MAO B turnover (Dkcat = 4.7; kcat/Km = 4.5) and flavin
reduction (Dkred) are in keeping with the cleavage of the R-CꢀH
bond being the rate-limiting step, fully or to a large degree.10
Further mechanistic insight regarding bond changes in the
substrate during catalysis may be gained by employing heavy-
atom KIEs. A normal 15N KIE signals decreased bonding to the
nitrogen atom, whereas an inverse (less than unity) KIE shows
that increased bonding to the nitrogen atom is partially rate-
limiting.
It is assumed that MAO A and B act by the same mechanism
and that the differences in substrate and inhibitor specificities
stem from structural features of the active sites of the respective
isozymes. In light of the three-dimensional structures determined
for human liver MAO A and B, this seems to be a reasonable
assumption. Several mechanisms have been proposed for amine
oxidation by flavoenzymes over the years. These mechanisms
include as key steps either single electron transfer reactions,
direct hydride transfer from the amine to the flavin, or nucleo-
philic addition of the amine to the flavin.1
For MAO, the main attention has been given to the aminium
cation radical mechanism according to Silverman2 and the polar
nucleophilic mechanism as put forth by Edmondson.3 The
radical mechanism (Scheme 1) is initiated by a reversible single
electron transfer from the unprotonated amine to the flavin to
produce a flavin radical and an aminium cation radical inter-
mediate. The radical intermediate undergoes proton abstraction,
and then further oxidation takes place directly by electron
transfer or via radical recombination and adduct formation.
Alternatively, the initial electron transfer and proton abstraction
steps might be concerted.2,4
The nitrogen KIE of recombinant human liver MAO B acting
on benzylamine was determined under steady-state conditions
with respect to substrate concentration. Competitive kinetics
were applied, and a chromatographic procedure in combination
with isotope-ratio mass spectrometry at natural abundance
provided the fraction of reaction and isotopic composition of
the isolated substrate.
The finding of a significant nitrogen KIE (Table 1) demon-
strates that a step involving nitrogen rehybridization is at least
partially rate-limiting. As the kinetic experiments were performed
at pH 7.5, the observed KIE was corrected for the degree of
protonation of the substrate, which in itself is dependent on the
isotopic composition.8 The corrected 15(V/K) value was found
to be 0.9846 ( 0.0005 (Table 1). The inverse nitrogen KIE
supports mechanistic scenarios involving an increase in the
According to the polar nucleophilic mechanism, the initial step
is a nucleophilic attack by the unprotonated amine on the flavin
C4a (Scheme 2). The resulting adduct then decomposes to form
reduced flavin and protonated imine. The proton abstraction
may be concerted with either the adduct formation (route on the
Received: June 17, 2011
Published: July 25, 2011
r
2011 American Chemical Society
12319
dx.doi.org/10.1021/ja205629b J. Am. Chem. Soc. 2011, 133, 12319–12321
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