From the School of Biological and Medical Sciences, University of St. Andrews, Irvine Building, North Street, St. Andrews, KY16 9AL, Scotland, United Kingdom
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ABSTRACT |
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Mitochondrial monoamine oxidases A and B (MAO A and MAO B) are ubiquitous homodimeric FAD-containing oxidases that catalyze the oxidation of biogenic amines. Both enzymes play a vital role in the regulation of neurotransmitter levels in brain and are of interest as drug targets. However, little is known about the amino acid residues involved in the catalysis. The experiments reported here show that both MAO A and MAO B contain a redox-active disulfide at the catalytic center. The results imply that MAO may be a novel type of disulfide oxidoreductase and open the way to characterizing the catalytic and chemical mechanism of the enzyme.
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INTRODUCTION |
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Flavin-containing mitochondrial monoamine oxidases A and B (MAO A and MAO B)1 catalyze the oxidative deamination of neurotransmitters, such as dopamine, serotonin, and noradrenaline in the central nervous system and peripheral tissues. The enzymes share 73% sequence homology and follow the same kinetic and chemical mechanism but have different substrate and inhibitor specificities (1). Inhibitors of these enzymes are medically important antidepressants, but the rational design of new inhibitor drugs is hampered by the lack of the active site structure and by remaining controversies in the catalytic mechanism.
Chemical modification experiments provide evidence that a histidine
residue (2, 3) is essential for the catalysis. There is also strong
evidence that two cysteine residues are present in the active site of
MAO (3-9). The inhibition of MAO by sulfhydryl reagents, first
observed in 1945 (10), is well established (3-6), but a role for
essential cysteine residues in the catalytic mechanism has not been
identified. In the chemical mechanism, there is still controversy about
whether MAO-catalyzed oxidative deamination proceeds via a radical
mechanism, hydride transfer, or oxidation of a carbanion intermediate.
In the most extensively tested hypothesis (11), the transfer of one
electron from the amine to the enzyme (presumably to the flavin) is
followed by the cleavage of the -carbon-hydrogen bond to produce the
amino radical. The substrate radical either passes a second electron to
the flavin or combines with an unknown active site radical to give a
covalent adduct that decomposes to the imine. However, no
flavosemiquinone has ever been detected in the catalytic cycle
(12-15). When flavosemiquinone is generated by reduction with
dithionite, the weak epr signal observed (about half that expected)
suggested that there might be coupling of FAD radical with an unknown
protein radical (14). If such a putative non-FAD radical can be
generated by reduction by dithionite, the number of electrons required
to reduce the enzyme should be more than the two necessary for the FAD
alone. The data presented in this paper demonstrate the presence of a second redox-active group in addition to the flavin in the active site
of MAO.
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EXPERIMENTAL PROCEDURES |
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Enzymes and Reagents--
Human liver MAO A heterologously
expressed in yeast and MAO B from beef liver were purified as
previously reported (16, 17). The concentration of each sample was
determined from the oxidized minus reduced extinction coefficient at
456 nm of 10,800 M1 cm
1 for MAO
A and 10,300 M
1 cm
1 for MAO B. Enzyme activity was determined spectrophotometrically using kynuramine
(1 mM) for MAO A and benzylamine (3 mM) for MAO B.
Anaerobic Titrations-- The titrations with sodium dithionite were carried out in a custom-made quartz anaerobic cuvette with a side arm, in an atmosphere of high purity argon. Dithionite standardized by titration of riboflavin was added via a gas-tight syringe attached to the cuvette. The spectra were recorded in a Shimadzu UV-2101PC spectrophotometer.
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RESULTS AND DISCUSSION |
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Reductive Titration of MAO A--
Human liver MAO A was made
anaerobic by equilibration with argon and titrated with standardized
dithionite to count the electrons required for full reduction of flavin
cofactor. Fig. 1 shows the spectral
changes occurring during the anaerobic titration with dithionite. The
inset to Fig. 1 shows the changes in absorbance at 412 nm
where the flavosemiquinone absorbs strongly and at 456 nm, the
wavelength where bleaching is observed for both the one-electron reduction steps of a typical flavin. Full reduction required 2.0 ± 0.12 mol (n = 5) of dithionite per mol of flavin
which is four electron equivalents (Fig. 1, inset), two more
than expected for a flavin alone. The final spectrum was typical of the
fully reduced enzyme (5). These data suggest that there is a
redox-active functional group in the active site of MAO in close
proximity to flavin, which (i) interacts with the flavin resulting in
some stabilization of the flavosemiquinone, (ii) has a redox potential close to that for the flavin, so that both the flavin and this group
are reduced simultaneously, and (iii) is able to accept two extra
electrons upon the dithionite titration. The likely candidate for this
group is a disulfide, because cysteines are known to be essential for
the activity of MAO. The potential for the cysteine-cystine redox
couple is 220 mV (18), close to that for FAD (
208 mV (19)) and to
that for MAO A (
210 mV) and MAO B (
220 mV) (20), and disulfide can
be reduced by dithionite. Finally, there is an established precedent
for flavin-dithiol interaction in the active sites of enzymes in the
disulfide oxidoreductase family (21).
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Effect of Sulfhydryl Reagents--
The sulfhydryl reagent,
4,4'-dipyridyl disulfide, is known to inactivate MAO A and B with the
liberation of 2 mol of 2-thiopyridone per mol of enzyme inactivated,
indicating that 2 thiols have been modified (5).
D-Amphetamine, a competitive inhibitor, protects against
the inactivation, suggesting that the groups modified are located in
the active site of the enzyme. We confirmed these observations using
DPDS. Inactivation of MAO A was biphasic (data not shown), with second
order rate constants of 34 ± 3 M1
s
1 and 19 ± 3 M
1
s
1, for the fast and the slow phases, respectively,
similar to those reported earlier for MAO A from human placenta (5).
The inactivation of MAO B was monophasic, with a rate constant of
32 ± 2 M
1 s
1. However,
other sulfhydryl reagents, such as p-iodoacetamide incubated with MAO A for 2 h at 1 mM concentration or
arsenite (which reacts with two free thiols in close proximity),
neither inhibited the activity nor decreased the amount of dithionite required to fully reduce MAO, indicating that there is no thiol in
close proximity to the flavin cofactor susceptible to arsenite or
iodoacetamide in MAO.
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Effect of -Mercaptoethanol--
-Mercaptoethanol, which
reduces disulfides, inhibited MAO A (in agreement with Ref. 22) in a
concentration- and time-dependent manner. The concentration
required for 50% inhibition after 15 min in aerobic solution was 0.25 M. The inhibition was fully reversible by dilution.
Incubation of MAO A with
-mercaptoethanol (5 mM) under
anaerobic conditions resulted in the reduction of the flavin cofactor.
When oxygen is added to the reaction mixture containing reduced enzyme
and
-mercaptoethanol, the flavin is reoxidized instantly. No
reduction of free riboflavin was observed under the same conditions.
This cannot be attributed to the difference in the redox potential
between the free flavin in solution and the flavin in MAO because these
are similar (19, 20). We speculate that
-mercaptoethanol reduces the
active site disulfide, which, in turn, reduces the cofactor.
Effect of Ligand-- Reductive titration of MAO A in the presence of the inhibitor (or pseudo-substrate), D-amphetamine (Fig. 3), gave completely different results from those obtained for the free native enzyme. It took two electron equivalents to reach the maximum absorbance at 412 nm, indicating the maximum formation of the semiquinone. No further significant spectral changes were observed even after the addition of more than eight electron equivalents and the incubation of the enzyme with dithionite for 5 h. There is no obvious explanation for why only the semiquinone of MAO is formed in the presence of D-amphetamine. Amphetamine does not alter significantly the redox potential for the one-electron reduction of MAO A (20), yet the presence of the inhibitor in the active site prevented further reduction of flavin. Consistent with the inhibition of reduction is the previous observation that D-amphetamine prevented reoxidation of pre-reduced MAO A (23).
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Reductive Titration of MAO B-- The reductive titration of MAO B (not shown) gave results identical with those for MAO A (Figs. 1 and 2). Four electron equivalents were required to reduce the native enzyme and two after treatment with DPDS. The redox stoichiometry for both MAO A and B is confirmed by the slopes of plots relating equilibrium concentrations of the enzyme and the reporter dye in redox potential measurements (20). The slopes for the enzyme alone were twice that expected (implying that 4 eq were required for full reduction, as demonstrated directly here) whereas those for the enzyme-substrate complex indicated the expected 2 equivalents.
Conclusions-- The data reported here suggest that MAO contains a redox-active disulfide in the active site and, therefore, may be a new type of disulfide oxidoreductase. We speculate that when an amine is oxidized, electrons pass from the amine to the disulfide and then to the flavin. The formation in MAO of a carboxyl-imidazole-disulfide triad, similar to that demonstrated for disulfide oxidoreductases, would result in an increased positive charge on the disulfide which would facilitate electron transfer from the amine substrate. Similarities with the disulfide oxidoreductase family of enzymes, which includes glutathione oxidoreductase, lipoamide dehydrogenase, and thioredoxin reductase, may be useful in elucidating the mechanism. However, although there are structural analogies between the N-terminal region of MAO and the nucleotide binding region of lipoamide dehydrogenase (24), the dithiol sequence motif common to the disulfide oxidoreductase family (25) is not found in MAO.
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FOOTNOTES |
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* This work was supported by the University of St. Andrews.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to Professor Thomas P. Singer in recognition of his 50 years of research on MAO.
Permanent address: Dept. of Biochemistry and Biophysics,
University of California at San Francisco, Molecular Biology Division, 4150 Clement St., San Francisco, CA 94121.
§ To whom correspondence should be addressed: University of St. Andrews, Irvine Bldg., North St., St. Andrews, KY16 9AL, Scotland, UK. Tel.: 44-1334-463411; Fax: 44-1334-463400; E-mail: rrr{at}standrews.ac.uk.
1 The abbreviations used are: MAO, monoamine oxidase; DPDS, 4,4'-dipyridyl disulfide.
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REFERENCES |
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