Vol. 91, No. 3, 1979 December
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14, lW9
Pages
991-996
THE ENZYME "ALDEHYDE OXIDASE"
IS AN IMINIDM OXIDASE. l'(5') REACTION WITH NICOTINE A IMINIUM ION Svante
Department Receiyed
Brandlnge
and Lars
of Organic Chemistry, Stockholm, S-106
October
22,
Lindblom
Arrhenius Laboratory, 91 Stockholm, Sweden
University
of
1979
SUMMARY: The second of the two reaction steps involved in the metabolic transformation of (-)-nicotine to (-)-cotinine (2) (i.e., the oxidation of the intermediate 2) is mediated mainly, if not solely, by the enzyme aldehyde oxidase (EC 1.2.3.1). Of the molecular species that constitute 2, nicotine A1'(5') iminium ion (Za) appears to serve as the substrate. The enzyme has a strong affinity for Yj, as shown in a study on-the inhibition of the oxidation of 3-(aminocarbonyl)-1-methylpyridinium chloride. This study gave a value of Ki = = 6 uM ; Km = 2 pM (pH 7.4). Mainly in view of this finding, "iminium oxidase" seems to be a more adequate name than "aldehyde oxidase" for this enzyme. The metabolic bolite the
transformation
(-)-cotinine
(2)
intermediate
catalyzed
(-)-nicotine
in mammals is
2 (1,2).
by the
of
The first
cytochrome
a two-step reaction
P-450
(1)
system
into
its
reaction
step
seems
and the
principal
which
meta-
proceeds
via
to be hydroxylation
product
formed
initially
should
thus
be -2b (1,3). An equilibrium mixture (z), the main components of which are iminium ion -2a and the carbinolamine 2&, is then rapidly generated from -2b (3,4). Other species hypothetically present are -2c and 2cJ, but the amounts of these are so low that they could not be detected by NMR (4). The composition of
the
-2 is dependent librium level solution
on pH and on other of 2
but
approximately
The second Hucker aldehyde
was found
reaction
variables
to be approximately
90 % in 0.2 step
as well.
in the
M sodium
Thus,
at pH 7.4
75 % in unbuffered phosphate
transformation
buffer
of 1 into
mouse
by purified
liver
rabbit
A recently
microsomes, liver
developed
diperchlorate
(A)
the
oxidation
enzymatic
of
the
aldehyde method
02,
and NADPH, could oxidase (EC 1.2.3.1)
for
the
synthesis
indeed (5). (4)
(4). 2 was found
ion -2a has made it possible of -2 to -3 more closely. The results
by
to be "an --in situ
be oxidized
to -3
of a crystalline
iminium
equi-
aqueous
by a soluble enzyme which was suggested --et al. to be mediated oxidase" (1). In 1972, Hill --et al. showed that 2, generated
from 1,
the
hydro-
to investigate are
reported
below.
MATERIALS AND METHODS The 2a hydrodiperchlorate (2) was synthesized A controlTxperiment demonstrated that autooxidation a yield of 2 of less than 0.5 % was obtained after through a solution of 2 in sodium phosphate buffer, NMNA = 3-(aminocarbonyl)-l-methylpyridinium
as described previously (4). of 4 to 3 proceeded slowly; occa.sTonal-bubbling of O2 pH 7.4, for 30 min at
chloride 0006-291 991
Copyright All rights
X/79/230991
-06$01.00/O
@ I979 by Academic Press. Inc. of reproduction in anyform reserved.
Vol. 91, No. 3, 1979
BIOCHEMICAL
PY “’
2a
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS
0+/ y
1;“’
py\\’ c-J--N
-
PY “’
OH
0 c-s N dH3 3
AH, 2b
CHO py = 3-pyridyl
PY “’
Fig.
1. Metabolic
transformation
PY”’
of
0\ y
(-)-nicotine.
35 OC. For the enzymic reactions the hydrodiperchlorate of 2a and the corresponding dichloride served equally well as starting material. A?airiy concentrated solution of 2a hydrodiperchlorate (4) (> 10 ml4) decolorized 2,6-dichloroindophenol (i.e.~,6-dichloro-4[(4'-hyd~oxyphenyl)iminol-2,5-cyclohexadien-l-one) within 2Gin at 22 oC but, as seen spectropolarimetrically at 600 nm (6), no reduction of the indophenol by 4 took place during 10 min under the conditions used for the determination of Km (Fig. 3). Reduction of potassium hexacyanothis compound was not suitable as an artificial electron ferrate(II1) was faster; acceptor. Due to the instability of aqueous solutions of 2a hydrodiperchlorate, these were kept on ice and used within 1 h. Determinations of the relative amounts of 2a and 2b at equilibrium in 0.2 M sodium phosphate buffer at pH 7.45, 8.45, and 9.20 wereperformed using the polarimetric method (4). The results are oxidase (EC 1.2.3.2) was purchased from presented in Table 1. Milk xanthine Sigma and assayed as previously described (7). The activity towards -2a hydrodiperchlorate was measured as for aldehyde oxidase (see below). Purification of aldehyde oxidase. Rabbit liver aldehyde oxidase was partially purified (90 times with a 30 % yield) by carrying out four of the six purification steps described by Felsted et a1.(8), with the exception (9) that the enzyme was eluted from the calcium phosphate gel with 0.05 M instead of 0.01 M phosphate buffer, pH 7.8. Protein determinations were performed according to Kalckar (10). The enzyme was assayed using 3-(aminocarbonyl)-l-methylpyridinium chloride (N1Inethylnicotinamide, NMNA), which is oxidized to a mixture of pyridones (8), and by following the increase in optical density at 300 nm as previously described Changing the buffer from 0.05 to 0.2 M sodium phosphate did not affect (11). the assay result. This assay procedure could not be applied, however, to the crude liver preparation (6,000 x g supernatant); so in this case the hexacyanoferrate(II1) method (11) was used instead. To check this latter procedure it was also used together with the former in assays after the first purification step. Activity with 2a hydrodiperchlorate as substrate was determined by incubating a 0.42 mM solzion with the enzyme preparations in 0.2 M sodium phosphate buffer, pH 7.4, for 4 min at 20-22 'C. Under these conditions the reaction was linear with time for at least 6 min. After 4 min, 0.5 ml 5 M NaOH and 0.62 I.rmol lidocaine were added and then NaCl(s) to saturation. The solution was extracted
992
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
with methylene chloride (4 x 2 ml) and The yield of cotinine 2 was determined loo-120 mesh, 0.2 x 180 cm) using the experiments described below the partially otherwise stated, the experiments were buffer.
the combined by GLC (3 % lidocaine as purified run at 20-22
RESEARCH COMMUNICATIONS
extracts dried with Na2S04. JXR on Gas Chrom Q, an internal standard. In all enzyme was used and, unless oC in 0.2 Y sodium phosphate
RESULTS AND DISCUSSION It has been demonstrated of catalyzing the oxidation determine the
whether
specific
any other
activities
(NMNA) as well
by Hill et al. that aldehyde oxidase is capable of -2 to -3 (5). However, it is also of interest to soluble enzyme is involved. To this end we measured
towards
as towards
of aldehyde
oxidase.
Since
practically
constant
(1.0,
-2a hydrodiperchlorate, the ratio between 1.0,
respectively) and steps l-4, cluded that aldehyde oxidase of -2. Inhibitors (12), had a marked
oxidation quinone
is
mM -4. The effectiveness
parable.
Milk
/min
(EC 1.2.3.2)
oxidized
0.14
question
for
the
be correct,
enzyme
clearly
experiment.
Another
rate
remained preparation it
estradiol
the
and E-benzo-
of -2a hydrodipermM estradiol
with
0.06
as an inhibitor like
can be con-
enzyme mediating
of oxidation
which,
was com-
aldehyde
was found
oxidase,
is
to be much less
(2)
to -3. An amount of this oxidized approximately 1 nmol/ from
contrast,
purification
starting
sole,
e.g.,
(13),
distinguishable
In striking
nmol/min
the
was obtained
-2a hydrodiperchlorate 2.4 pmol/min of xanthine not
in the
partial
activities
procedure,
not
of p-benzoquinone
oxidase
oxidized an amount
autooxidation
substrate
on the
molybdenum-containing
of 4,
hardly
xanthine
if
the
specific
purification oxidase,
An 85 % inhibition
in oxidizing
which
the
chloride
during the
and 1.2
the main,
affect
and 0.42
enzyme which
1.2,
of aldehyde
(4)
effective
1.4,
during
chlorate
an unspecific
to 2.
3-(aminocarbonyl)-l-methylpyridinium
that
a solution
obtained
in an
of aldehyde
oxidase
of NMNA converted
0.17 nmol/min of -4 into -3. 2a 2d is actually serving as -enzyme. Hill et al. (5) favored can -2c but this hypothesis since it would be expected to lead to the formation of 4is which
-methylamino-4-(3'-pyridyl)-butanoic
of
the
forms
acid,
a compound
which
is
not
converted
(14) into -3 under the conditions used. Instead, we have obtained evidence that 2a is the substrate. The evidence for this comes from a study on the pH dependence of the inhibitory action of -2a hydrodiperchlorate (4) on the oxidation of NMNA catalyzed by aldehyde oxidase (Fig. 2). It was initially observed that with 1.0 mM NMNA, 0.83 mM 2, and aldehyde oxidase the oxidation of NMNA was inhibited the
virtually
the
values for inhibition
the
oxidation
K,,,
completely,
same as when NNNA was omitted.
whereas the rate of cotinine In the investigation of
the
formation was pH dependence,
NMNA were determined at pH 7.45, 8.45, and 9.20 (Table 1) and exerted by a constant amount of -2a hydrodiperchlorate (4) - on of NMNA was studied at various concentrations of the latter and
993
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.0
0
Fig. 2. Inhibition by 2a hydrodiperchlorate (4) (0.083 mM) of the oxidation of NMNA catalyzed by aldehyde oxidase at three-different pH’s. The measured results were analyzed using a program @MDP3R, University of California, Los Angeles, USA) for unweighted non-linear regression analysis. The model fitted was that of a linear competitive inhibition. Constants Km for NMNA and Ki for i are presented in Table 1.
at the
same three
constants
Ki
tive
amounts
pH's
have Table 6.7
were
pH values.
The inhibition
calculated
for
from
of -2a and -2b at equilibrium estimated by measuring
been 1.
Calculation
x 0.85
= 5.7;
former set only is i = -2a hydrodiperchlorate. PR
5
the
kinetic
in 0.2 the
to be competitive data
M phosphate
optical
rotation
(Fig.
2).
buffer
and rela-
of various
as a function
of sets of Ki for & and 2. The.sets are obtained thus: x 0.15 - 1.0, etc. The approximate constancy of the evidence that 2a rather than2 is the true inhibitor. Ki i
for
Molar ratio --2a/2b
(N-0
Calculated for 2a (PM) -
Ki
Calculated K. for 2b ‘(PM) -
0.23
+ 0.05
6.7
-+ 1.0
85115
5.7
1.0
8.45
0.34
-+ 0.08
10.0
-+ 2.0
35/65
3.5
6.5
9.20
0.32
-+ 0.03
16.0
+ 2.0
15185
2.4
13.6
7.45
The
6.7
Km for
NMNA (a)
was found
994
of
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 3. Lineweaver-Burk plot of the oxidation of 2a hydrodiperchlorate (4) to 2 catalyzed by aldehyde oxidase at pH 7.4 (22 'C). The reaction rate was measured by following (at 600 nm) the aldehyde oxidase catalyzed reduction of 2,6-dichloroindophenol (6). A Km of 2 PM was calculated from the intercept on the abscissa.
pH (Table
l)(4).
Values
thus
obtained
can be used
of Ki for
1). Since apparent -2a and -2b (Table undergo a 50 % and a two-fold increase,
only
studied,
it
undergo inhibitory
form,
then
its
low Km value
actually
the
lowest 15). buffer
If set
it
is assumed
Cotinine weakly
the
substrate
in the
oxidase
that
the
for
for
same is
NMNA
pH interval
NMNA does true
for
for
calculated for -2a (Table 2b. Thus, be the -2a should for the enzyme.
not
the
1) must be inhibitory
feature
of -2a in its interaction with aldehyde oxidase of 2 uM (pH 7.4), obtained by measuring the dichloroindoof
the
reduced
mM has previously using
(A)
max
of Ki values the set
activity
0.22
two new sets
values
respectively,
of aldehyde
form
of
the
K recorded for any substrate for m previously reported are about one order
, pH 7.8,
and only
affinity
lowest
values A K, of
the
than
also
striking
reductase
the
likely
and therefore,
phenol
that change.
as more
The most is
likely
any drastic
regarded form
is
to calculate
K, and v
does
inhibits
been obtained
the dichloroindophenol not
inhibit the
oxidation
the
for
method oxidation
enzyme aldehyde
This
oxidase;
is
the
of magnitude
higher
M phosphate
(9,
(16).
of -2a hydrodiperchlorate (d) 1.0 mM NMNA and 2.1 mM 3
of NMXA. At
the
shows an optical
and 9.5.
approximately
3).
NMNA in 0.05
a 20 % inhibition was observed. Substrate inhibition 0.3 mM -2a hydrodiperchlorate (A). The pH profile for region
(Fig.
between
pH 6.8
seems to begin oxidation
at about of -4 to -3
BIOCHEMICAL
Vol. 91, No. 3, 1979
Compound has been
i
which
of an aromatic expected
that
of several and that
(2a - hydrodiperchlorate) with respect to its
studied
al compounds
AND BIOPHYSICAL
contain
system iminium
saturated
seems to be the interaction
an oxidizable
have, ions
however, are
with
first
studied
principal
iminium
aldehyde
carbon-nitrogen been
the
RESEARCH COMMlJNICATlONS
before
oxidase.
double
bond
(9,15).
It
intermediates
in
that Sever-
as part is
to be
the metabolism
xenobiotics to the corresponding -N-heterocyclic oxidase plays an important role in the oxidation
aldehyde
ion
lactams of
these
intermediates. As already hyde
oxidase"
pointed is
good alternatives
out
inadequate have
by other for
been
for
Acknowledgements. cation
of
the
and Mr Johan supported
enzyme,
Dr Bengt
Montelius
by the
compounds
Swedish
for
Dr Anders Mannervik
advice Tobacco
but
of the
containing should
oxidase"
We thank
(9,17-19)
reasons, In view
lacking.
aromatic -2a and for several bond moiety, the name "iminium
investigators
various
for
in the
high
aid
affinity
name "aldebecause of the
a carbon-nitrogen
be more
Mzhl&r
the
has been kept
for
descriptive.
carrying
in using
spectrophotometric
enzyme double
out the
the purifi-
computer
work.
This
program, work
was
Company.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Hucker, H.B., Gillette, J.R., and Brodie, B.B. (1960) J. Pharmacol. Exp. Ther. 129, 94-100. Gorrod, J.W., and Jenner, P. (1975) Essays Toxicol. 6, 35-78. Murphy, P.J. (1973) J. Biol. Chem. 248, 2796-2800. L. (1979) Acta Chem. Stand. B33, 187-191. Brandlnge, S., and Lindblom, Hill, D.L., Laster, W.R., Jr, and Struck, R.F. (1972) Cancer Res. 32, 658-665. Rajagopalan, K.V., and Handler, P. (1964) J. Biol. Chem. 239, 2022-2026. Kalckar, H.M. (1947) J. Biol. Chem. 167, 429-443. Felsted, R.L., Chu, A.E.-Y., and Chaykin, S. (1973) J. Biol. Chem. 248, 2580-2587. Krenitsky, T.A., Neil, S.M., Elion, G.B., and Hitchings, G.H. (1972) Arch. Biochem. Biophys. 150, 585-599. Kalckar, H.M. (1947) J. Biol. Chem. 167, 461-475. Rajagopalan, K.V., and Handler, P. (1966) Methods Enzymol. 9, 364-368. Rajagopalan, K.V., Fridovich, I., and Handler, P. (1962) J. Biol. Chem. 237, 922-928. Bray, R.C. (1975) The Enzymes 12 B, 299-419. Hucker, H.B., and Gillette, J.R. (1960) Fed. Proc., Fed. Amer. Sot. Exp. Biol. 19, 30. Stubley, C., Subryan, L., Stell, J.G.P., Perrett, R.H., Ingle, P.B.H., D.W. (1977) J. Pharm. Pharmacol. 29, 77 P. and Mathieson, Branzoli, U., and Massey, V. (1974) J. Biol. Chem. 249, 4339-4345. Ref. 13, p. 352. H. (1978) Wiss. Z., Karl-Marx-Univ., Leipzig, Math.Kurth, J., and Aurich, -Naturwiss. Keihe 27, 47-54. @hkubo, M., and Fujimura, S. (1978) Cancer Res. 38, 697-702.
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