Mechanistic studies of human spermine oxidase: Kinetic mechanism and pH effects

Article abstract of DOI:10.1021/bi9017945

In mammalian cells, the flavoprotein spermine oxidase (SMO) catalyzes the oxidation of spermine to spermidine and 3-aminopropanal. Mechanistic studies have been conducted with the recombinant human enzyme. The initial velocity pattern in which the ratio between the concentrations of spermine and oxygen is kept constant establishes the steady-state kinetic pattern as ping-pong. Reduction of SMO by spermine in the absence of oxygen is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with a limiting value (k₃) of 49 s^-1 and an apparent K _d value of 48 μMat pH8.3. The rate constant for the slow step is independent of the spermine concentration, with a value of 5.5 s^-1, comparable to the k_cat value of 6.6 s^-1. The kinetics of the oxidative half-reaction depend on the aging time after the spermine and enzyme are mixed in a double-mixing experiment. At an aging time of 6 s, the reaction is monophasic with a second-order rate constant of 4.2 mM^-1 s^-1. At an aging time of 0.3 s, the reaction is biphasic with two second-order constants equal to 4.0 and 40 mM^-1 s^-1. Neither is equal to the k_cat/K_O2 value of 13 mM ^-1 s^-1. These results establish the existence of more than one pathway for the reaction of the reduced flavin intermediate with oxygen. The k_cat/K_M value for spermine exhibits a bell-shaped pH profile, with an average pK_a value of 8.3. This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for k₃ shows a pK_a value of 7.4 for a group that must be unprotonated. The pK_i-pH profiles for the competitive inhibitors N,N′-dibenzylbutane-1,4-diamine and spermidine show that the fully protonated forms of the inhibitors and the unprotonated form of an amino acid residue with a pK_a of ～7.4 in the active site are preferred for binding. 2009 American Chemical Society.

Full text of DOI:10.1021/bi9017945

3
86 Biochemistry 2010, 49, 386–392
DOI: 10.1021/bi9017945
†
Mechanistic Studies of Human Spermine Oxidase: Kinetic Mechanism and pH Effects
‡
§
,‡
Mariya S. Adachi, Paul R. Juarez, and Paul F. Fitzpatrick*
‡
Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center, San Antonio,
Texas 78229 and Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
§
Received October 19, 2009; Revised Manuscript Received December 7, 2009
ABSTRACT: In mammalian cells, the flavoprotein spermine oxidase (SMO) catalyzes the oxidation of spermine
to spermidine and 3-aminopropanal. Mechanistic studies have been conducted with the recombinant human
enzyme. The initial velocity pattern in which the ratio between the concentrations of spermine and oxygen is
kept constant establishes the steady-state kinetic pattern as ping-pong. Reduction of SMO by spermine in the
absence of oxygen is biphasic. The rate constant for the rapid phase varies with the substrate concentration,
-
1
with a limiting value (k ) of 49 s and an apparent K value of 48 μM at pH 8.3. The rate constant for the slow
3
d
-
1
step is independent of the spermine concentration, with a value of 5.5 s , comparable to the kcat value of
.6 s . The kinetics of the oxidative half-reaction depend on the aging time after the spermine and enzyme are
-
1
6
mixed in a double-mixing experiment. At an aging time of 6 s, the reaction is monophasic with a second-order
-
1 -1
rate constant of 4.2 mM
s . At an aging time of 0.3 s, the reaction is biphasic with two second-order
-
1
-1
-1 -1
constants equal to 4.0 and 40 mM
s
. Neither is equal to the kcat/KO2 value of 13 mM
s . These results
establish the existence of more than one pathway for the reaction of the reduced flavin intermediate with
oxygen. The kcat/KM value for spermine exhibits a bell-shaped pH profile, with an average pK value of 8.3.
a
This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for
k shows a pK value of 7.4 for a group that must be unprotonated. The pK -pH profiles for the competitive
3
a
i
0
inhibitors N,N -dibenzylbutane-1,4-diamine and spermidine show that the fully protonated forms of the
inhibitors and the unprotonated form of an amino acid residue with a pK of ∼7.4 in the active site are
a
preferred for binding.
1
The polyamines spermine, spermidine, and putrescine are
N -acetylspermine (9). In contrast, plant polyamine oxidases
1
important in cell proliferation, differentiation, and survival (1).
Because of the absolute requirement of these compounds for cell
growth, the polyamine metabolic pathway has been extensively
studied. Polyamine catabolism is mediated by the activity of three
enzymes (2). Spermidine/spermine N-acetyltransferase acetylates
spermine and spermidine to produce the N-acetylated com-
pounds (3). These can be exported from the cell or oxidized by
preferentially oxidize spermine rather than N -acetylspermine.
In addition, the plant enzymes oxidize the endo C-N bond of
the substrate, while the mammalian enzymes oxidize the exo
bond (10). The yeast enzyme Fms1 is also classified as a spermine
oxidase, but the kcat/KM values for spermine, N -acetylspermi-
dine, and N -acetylspermine are comparable (11). The structural
1
1
bases for these differences in specificity and reactivity are not
known.
1
the peroxisomal enzyme polyamine oxidase (PAO) to yield
spermidine or putrescine, hydrogen peroxide, and 3-acetamido-
propanol (4). Alternatively, the cytosolic enzyme spermine
oxidase (SMO) can catalyze the oxidation of spermine directly
to spermidine (Scheme 1), bypassing the necessity for acetyla-
tion (5, 6). The relative contribution of these two enzymes to
polyamine catabolism has not been established. While PAO is
constitutive, SMO is induced by polyamine analogues (7). In-
deed, it has been suggested (8) that SMO is responsible for the
antitumor effects of polyamine analogues.
Both SMO and PAO are flavoproteins and thus members of
the family of flavin amine oxidases. Structurally, most flavin
amine oxidases can be classified as members of the monoamine
oxidase (MAO) family that includes MAO A and B, lysine-
specific demethylase (LSD1), and
L-amino acid oxidase or as
members of the -amino acid oxidase (DAAO) family that
D
includes DAAO, sarcosine oxidase, and glycine oxidase, among
others (12). The three-dimensional structures of maize PAO and
yeast Fms1 have been determined (13, 14), establishing that they
have the same overall structure as MAO (15) and that the
mammalian SMO and PAO also belong to the MAO structural
family. While amine oxidation by members of the DAAO family
is more generally accepted to involve direct hydride transfer
(16-20), the mechanism of amine oxidation by MAO has been a
matter of controversy (12, 21-23).
Because of its relatively recent discovery (5, 7), the mechanism
of SMO has not been established. We describe here analysis of the
kinetic mechanism of human SMO using both steady-state and
rapid reaction kinetic methods and analysis of the effects of pH
on the steady-state and reductive half-reaction kinetics. The
Human SMO has a strong preference for spermine as a
1
substrate (6), while PAO prefers N -acetylspermidine and
†
This work was supported in part by National Institutes of Health
Grant GM058698.
*To whom correspondence should be addressed: Department of
Biochemistry, University of Texas Health Science Center, San Antonio,
TX 78229. Phone: (210) 567-8264. Fax: (210) 567-8778. E-mail:
fitzpatrick@biochem.uthscsa.edu.
1
0
Abbreviations: SMO, spermine oxidase; DBDB, N,N -dibenzyl-1,4-
diaminobutane; PAO, polyamine oxidase; Ni-NTA, nickel nitrilotria-
cetic acid; MAO, liver monoamine oxidase; LSD1, lysine-specific
demethylase; DAAO, D-amino acid oxidase.
pubs.acs.org/Biochemistry
Published on Web 12/11/2009
r 2009 American Chemical Society

Article
Biochemistry, Vol. 49, No. 2, 2010 387
Scheme 1
HEPES and 10% glycerol (pH 8.0). Any precipitated protein was
removed by centrifugation for 10 min at 14000g, and the visible
absorbance spectrum of the supernatant was recorded. The
change in absorbance between the enzyme-bound FAD and
the free FAD after denaturation gave an extinction coefficient of
-
1
-1
1
1.7 mM cm at 458 nm for the flavin in SMO based on the
-
1
-1
extinction coefficient of FAD of 11.3 mM cm (24). This
value was used to determine the enzyme concentration.
Assays. Enzyme activity was determined in a buffer contain-
ing 10% glycerol by following oxygen consumption with a
Yellow Springs Instrument model 5300 biological oxygen moni-
tor. All assays were conducted at 25 ꢀC. The buffers were 200 mM
Tris-HCl from pH 7.0 to 8.75 and 200 mM CHES from pH 9.0 to
9
.35. For assays not run in air-saturated buffer, the appropriate
mixture of O and N was bubbled into the assay mixture for
2
2
1
0 min prior to starting the reaction.
Rapid Reaction Kinetics. Rapid reaction kinetic measure-
results provide insight into the mechanism of amine oxidation by
SMO and the basis for substrate specificity.
ments were performed using an Applied Photophysics SX-20
stopped-flow spectrophotometer. We made the instrument anae-
robic by filling the system with a solution of 30 nM glucose
oxidase and 1 mM glucose in anaerobic buffer the night before
the experiment was conducted. Anaerobic conditions were
reached via application of several cycles of vacuum and oxy-
gen-scrubbed argon to enzyme solutions, while substrate solu-
tions were bubbled with argon. Glucose and glucose oxidase at
final concentrations of 5 mM and 36 nM, respectively, were
added to all solutions to maintain anaerobic conditions. For pH
profiles, the buffers were 200 mM Tris-HCl and 200 mM CHES
from pH 7.0 to 8.75 and from pH 9.0 to 9.4, respectively. For the
sequential mixing stopped-flow experiment used to study the
reaction of the reduced enzyme with oxygen, 60 μM enzyme was
first mixed anaerobically with 60 μM spermine. After aging times
of 6 and 0.3 s, this was mixed with the oxygenated buffer
equilibrated with different oxygen concentrations. All experi-
ments were conducted at 25 ꢀC.
MATERIALS AND METHODS
Materials. Spermine and spermidine trihydrochloride were
purchased from Acros Organics (Geel, Belgium). 1,12-Diamino-
dodecane was from Sigma-Aldrich (Milwaukee, WI). N,N -
Dibenzyl-1,4-diaminobutane (DBDB) was from Prime Organics
0
(
(
Woburn, MA). The pET28b(þ) vector was from Novagen
Madison, WI). The pJ10:G04331 vector encoding human sper-
mine oxidase was obtained from DNA 2.0 (Menlo Park, CA).
Plasmid pGro7 was from Fisher Scientific. The nickel nitrilo-
triacetic acid (Ni-NTA) agarose resin was purchased from
Invitrogen (Carlsbad, CA).
Expression and Purification of Human Spermine Oxi-
dase. The DNA encoding the human SMO in a pDrive vector
with codons optimized for expression in Escherichia coli was
subcloned into the pET28b(þ) vector using the NdeI and EcoRI
0
0
sites at the 5 and 3 ends, respectively, for expression of the His-
tagged enzyme. The plasmid was transformed into BL21(DE3)
E. coli together with plasmid pGro7 to assist protein folding
Data Analysis. The kinetic data were analyzed using Kalei-
daGraph (Synergy Software, Reading, PA). To determine kcat
and K values, initial rate data were fitted to the Michae-
lis-Menten equation. Equation 1 was used to analyze data for
inhibition by competitive inhibitors and obtain the K values.
/
during expression. After induction with 0.15 mM isopropyl β-
D
-
K
M
M
thioglucanopyranoside, the cells were grown overnight at 18 ꢀC
in LB broth containing kanamycin and chloramphenicol. The
cells were harvested by centrifugation at 5000g for 30 min at 4 ꢀC.
The cell paste was resuspended in 50 mM HEPES (pH 8.0), 10%
glycerol, 2 μM pepstatin, 2 μM leupeptin, 0.1 mM NaCl, 100 μg/
mL phenylmethanesulfonyl fluoride, and 100 μg/mL lysozyme
and lysed by sonication. After the lysate was centrifuged at
i
Equation 2 was used to analyze the pH dependence of kinetic
parameters that decrease at both low and high pH. Equation 3
was used to analyze the pH dependence of kinetic parameters that
decrease at low pH.
E
k
cat
S
I
ꢀ
ꢁ
ð1Þ
22400g, solid ammonium sulfate was added to the supernatant to
¼
v
0
reach 35% saturation. After centrifugation at 22400g for 30 min
at 4 ꢀC, the supernatant was brought to 50% ammonium sulfate
saturation. The pellet was resuspended in 50 mM HEPES (pH
KM 1 þ
þ S
K
i
0
1
8.0), 10% glycerol, 0.1 mM NaCl, 2 μM pepstatin, and 2 μM
B
B
C
C
C
A
leupeptin and loaded onto a 10 mL Ni-NTA column equilibrated
with this buffer. The protein was eluted with a linear gradient
from 0 to 90 mM imidazole in 20 column volumes of the same
buffer. Fractions containing SMO were pooled and concentrated
by the addition of solid ammonium sulfate to 50% saturation.
The pellet resulting from the final precipitation was resuspended
in 50 mM HEPES (pH 8.0) and 10% glycerol and dialyzed
against three changes of the same buffer. The resulting protein
sample was centrifuged at 22400g for 30 min at 4 ꢀC to remove
precipitated protein and stored at -80 ꢀC.
log Y ¼ log
ð2Þ
ð3Þ
@
H
K
2
1
þ
þ
K1
H
0
1
B
B
@
C
C
C
A
log Y ¼ log
H
1
þ
K1
In these equations, Y is the experimental value of the kinetic
parameter of interest, K and K are the dissociation constants for
Extinction Coefficient of Human Spermine Oxidase. An
equal volume of 10 M urea was added to enzyme in 50 mM
1
2
the ionizable groups, and C is the pH-independent value of the

3
88 Biochemistry, Vol. 49, No. 2, 2010
Adachi et al.
parameter. To determine the kinetic parameters for the reduction
of SMO by spermine, stopped-flow traces were fitted to eq 4,
which describes a biphasic exponential decay
-
λ
1
t
2
-λ t
A ¼ A
¥
þ A
1
e
þ A
2
e
ð4Þ
where λ and λ are the first-order rate constants for each phase,
1
2
A and A are the absorbances of each species at time t, and A is
1
2
¥
the final absorbance. The resulting pseudo-first-order rate con-
stants were analyzed using eq 5
3
k S
k
obs
¼
ð5Þ
K þ S
FIGURE 1: Double-reciprocal plot of the initial rate vs the spermine
concentration at a fixed ratio of spermine to oxygen concentration of
d
where k is the rate constant for flavin reduction and K is the
2 at pH 8.3 and 25 ꢀC.
3
d
apparent dissociation constant for the substrate. The rate con-
stant for flavin oxidation was obtained by fitting the change in
absorbance of the flavin with time to eqs 4 and 6 at delay times of
a
Table 1: Steady-State Kinetic Parameters for Human Spermine Oxidase
-1 b
k
cat(spermine) (s
)
6.6 ( 0.2
37 ( 7
K
k
O (spermine) (μM)
565 ( 66
2
0
.3 and 6 s, respectively.
A ¼ A
The value of k in Scheme 4 was calculated using Microsoft Excel
kcat/K
0.40 ( 0.06
spermine
-1 c
cat(N-acetylspermine)
-1 c
-1
(mM
s
)
(s )
1
-λ t
¥
þ A
1
e
ð6Þ
K
spermine (μM)
190 ( 32
k
cat/KN-acetylspermine
-1 c
0.8 ( 0.2
492 ( 97
-
1
(
mM
s )
c
7
kcat/KO2
12.6 ( 1.6
KN-acetylspermine (μM)
(spermine)
-1 d
-1
2004.
(mM
s )
a
b
Conditions: pH 8.3 and 25 ꢀC. Determined by varying the concentra-
c
RESULTS
tions of both oxygen and spermine. Determined at 250 μM oxygen.
Determined at 500 μM spermine.
d
Kinetic Mechanism of Human Spermine Oxidase.
Flavoprotein oxidases can exhibit either a parallel or an inter-
secting line pattern in double-reciprocal plots when both sub-
strates are varied. The former results when flavin reduction is
effectively irreversible, even though these enzymes do not follow
the standard ping-pong kinetic mechanism (25). For a bisubstrate
enzyme reaction, the proper steady-state kinetic equation can
readily be identified using the fixed ratio approach (26). In this
method, the ratio between the concentrations of both substrates
is kept constant and the initial rate is determined as a function of
the concentration of one of the substrates. Curvature in a double-
reciprocal plot suggests a sequential pattern, since there will be a
term containing the square of the concentrations of one of the
F
as the substrate. The line is from a fit of the data to eq 2.
IGURE 2: kcat/K -pH profile for spermine oxidase with spermine
M
substrates in the rate equation (eq 7 with a = [O ]/[spermine]).
2
1
value for N -acetylspermine was determined at a constant con-
E
1
K
O2
K
spermine
K
iðspermineÞ
K
O2
centration of oxygen. The value establishes that human SMO
¼
¼
þ
þ
þ
þ
k
1
v
0
k
cat
k
cat½O
2
ꢀ
k
cat½spermineꢀ
cat½spermineꢀ½O
2
ꢀ
prefers spermine to N -acetylspermine as a substrate by ∼50-fold
at pH 8.3 (Table 1). We could not detect any activity with
spermidine as the substrate.
1
KO2
Kspermine
K
iðspermineÞK 2
O
þ
þ
2
kcat kcata½spermineꢀ kcat½spermineꢀ
kcata½spermineꢀ
ð7Þ
pH Dependence of kcat/Kspermine. To gain insight into the
basis of substrate specificity and the roles of amino acid residues
in binding and catalysis, the effect of pH on the kcat/KM value for
spermine was determined. A plot of the data (Figure 2) shows a
bell-shaped dependence with a maximum at pH 8.3. This pH
profile with limiting slopes of unity on either side of the pH
maximum indicates the importance of two ionizable groups in the
free enzyme or free substrate. The data can be fitted to eq 2 to
extract the pK values. However, the two pK values are too close
A linear plot will arise if there is no term in the rate equation
containing the concentrations of both substrates, as in a ping-
pong mechanism (eq 8).
E
1
K
O2
K
spermine
¼
¼
þ
þ
þ
2
k
v
0
k
cat
k
cat½O
ꢀ
cat½spermineꢀ
a
a
1
K
O2
K
spermine
together to resolve, so that only the average of the two pK
8.3 ( 0.03) can be determined with confidence (Table 2).
pH Dependence of pK Profiles. To corroborate the re-
a
values
þ
ð8Þ
(
k
cat
k
cata½spermineꢀ
k
cat½spermineꢀ
i
For SMO, a double-reciprocal plot of the initial rate versus the
spermine concentration at a fixed ratio of spermine to oxygen is
linear (Figure 1). A fit of these data to the Michaelis-Menten
quired protonation states of individual nitrogens in the substrate
and aid in further distinguishing between groups that are
responsible for binding or catalysis, we examined several sub-
equation gives a kcat value of 6.6 ( 0.2 s- . The individual kcat
1
/
strate analogues (Scheme 2) as inhibitors. Spermidine and N,N -
0
KM values for spermine and oxygen were determined in separate
analyses by varying each at a fixed concentration of the other.
The resulting values are listed in Table 1. In addition, the kcat/KM
dibenzyl-1,4-diaminobutane (DBDB) are inhibitors for SMO,
while concentrations of 1,12-diaminododecane up to 2 mM do
not result in any detectable inhibition even at concentrations of

Article
Biochemistry, Vol. 49, No. 2, 2010 389
Table 2: pK
a
Values for Spermine Oxidase
pK
kinetic parameter
1
pK
2
k
cat/Kspermine
8.3 ( 0.03
7.9 ( 0.03
7.3 ( 0.2
7.4 ( 0.02
8.3 ( 0.03
7.9 ( 0.03
8.6 ( 0.1
-
K
K
i(spermidine)
i(DBDB)
k
3(spermine)
F
IGURE 4: Absorbance spectra of flavin intermediates observed in
the reductive half-reaction of spermine oxidase (16.8 μM) by sper-
mine at pH 8.3: (1) spectrum of spermine oxidase before reaction, (2)
spectrum at the end of the first phase, and (3) final spectrum.
FIGURE 3: pK -pH profiles for spermidine (b) and DBDB (2) as
i
inhibitors of spermine oxidase. The lines are from fits to eq 2.
Scheme 2
F
IGURE 5: pH dependence of the rate constant for reduction of
spermine oxidase by spermine at 25 ꢀC. The line is from a fit of the
data to eq 3.
Scheme 3
rate constant for the slower phase is independent of the spermine
-1
concentration, with a value of 5.5 ( 0.3 s at pH 8.3. These
results are consistent with the mechanism in Scheme 3. The first
phase is due to binding of spermine with no significant absor-
bance change, followed by amine oxidation and flavin reduction.
The slower second phase can be attributed to dissociation of the
oxidized amine from the enzyme. The spectrum of the inter-
mediate does not show any evidence of the charge-transfer
absorbance band often seen with flavoprotein oxidases; no such
band has been reported for the related enzymes PAO (28, 29) and
MAO (30).
spermine well below the KM value. Both spermidine and DBDB
exhibit competitive inhibition versus spermine (data not shown)
with K values at pH 8.3 of 0.55 ( 0.02 and 0.32 ( 0.08 mM,
i
respectively. The effects of pH on these K values were deter-
The effect of pH on the value of k was determined. As shown
i
3
mined. As shown in Figure 3, both pH-pK profiles are bell-
in Figure 5, the value of k decreases at low pH but is independent
i
3
shaped, so that the data were fit to eq 2 to extract the pK values
of pH at high pH. These data were fit to eq 3 to yield a pK value
a
a
(
Table 2). For spermidine, this gives pK values too close together
of 7.4 ( 0.02 for the group that must be unprotonated for
reduction.
a
for accurate resolution (27); consequently, only the average of the
two pK values can be estimated reliably with this inhibitor. For
Oxidative Half-Reaction. Stopped-flow spectrophotometry
was also used to determine the kinetics of the reaction of the
reduced enzyme with oxygen. This was done in a double-mixing
experiment. SMO was first mixed with spermine in the absence
of oxygen to form the reduced complex. After the enzyme
was reduced, it was mixed with buffer equilibrated with different
concentrations of oxygen. When SMO and spermine are allowed
to react for 6 s before being mixed with oxygen, the increase
in absorbance due to flavin oxidation is monophasic (Figure 6).
The observed first-order rate constant for the reaction varies
directly with the concentration of oxygen (Figure 7), giving
a
DBDB, the two pK values obtained by fitting the data to eq 2 are
a
further apart and can be determined.
Reductive Half-Reaction. Stopped-flow spectroscopy was
used to determine the kinetics of flavin reduction. When SMO
and spermine are mixed rapidly in the absence of oxygen, the
spectrum of oxidized flavin rapidly changes to the one of reduced
flavin, followed by a slower phase with a much smaller absor-
bance change (Figure 4). Only the first-order rate constant for the
fast phase is substrate-dependent. The observed rate constants at
different spermine concentrations for this phase can be fit to eq 5.
At pH 8.3, this gives values of 48 ( 8.2 μM for the apparent K
a second-order rate constant for the reaction of the reduced
d
value for spermine and 49 ( 1.3 s- for k , the rate constant for
1
enzyme with oxygen of 4.2 ( 0.03 mM
-1 -1
s . This kinetic
3
flavin reduction at saturating concentrations of spermine. The
behavior is consistent with an irreversible second-order reaction

3
90 Biochemistry, Vol. 49, No. 2, 2010
Adachi et al.
Table 3: Intrinsic Kinetic Parameters for Spermine Oxidase with Spermine
as the Substrate at pH 8.3
-
1
K
d
(μM)
48 ( 8
49 ( 1
40 ( 5
k
k
k
7
9
(s
(s
)
)
5.9 ( 0.6
5.5 ( 0.3
4.0 ( 0.04
-
1
-1
k
k
3
(s
)
-
1
-1
-1 -1
5
(mM
s
)
11 (mM
s )
F
IGURE 6: Absorbance changes during the reaction of reduced sper-
mine oxidase with 607 μM oxygen at pH 8.3 after first allowing the
enzyme to react with spermine for 6 s (1) or 0.3 s (2). For the sake of
clarity, only every fifth point is shown. The lines are from fits of the
data to eq 4 or 6.
F
IGURE 8: Comparison of the observed rates of spermine oxidation
by spermine oxidase as a function of oxygen concentration (O) with
values calculated from the mechanism of Scheme 4 and the kinetic
parameters listed in Table 3 (4), at pH 8.3 and 25 ꢀC. The line is from
a fit of the calculated data to the Michaelis-Menten equation.
The oxidative reaction cannot be described as a simple reaction
of only one of the SMO intermediates with oxygen since the
absorbance change during oxidation is either monophasic or
biphasic depending on the aging time. At an aging time of 6 s, all
of the oxidized product would have dissociated from the reduced
enzyme, so that oxygen is reacting with the free reduced enzyme.
At an aging time of 0.3 s, a significant fraction of the enzyme still
has the oxidized product bond, resulting in a biphasic reaction in
which the rate constants for both phases depend on the oxygen
concentration. The rapid phase can be attributed to the reaction
of the reduced enzyme-product complex with oxygen. The
excellent agreement of the rate constant for the slow phase at
the shorter aging time with the rate constant for the reaction at
the longer aging time supports the conclusion that the slow phase
is due to the reaction of the free reduced enzyme. An increase in
the rate constant for oxidation when product is bound to a
reduced flavoprotein oxidase has been observed with other flavin
amine oxidases (31).
F
IGURE 7: Dependence of the rate constants for flavin oxidation on
the oxygen concentration at pH 8.3 and 25 ꢀC when the enzyme and
spermine are first allowed to react for 6 s (A) or 0.3 s (B).
Scheme 4
between the reduced enzyme and oxygen, with no evidence of an
oxygen-enzyme complex. In contrast, when the aging time is
shortened to 0.3 s, the absorbance increase is faster and biphasic
Mechanisms including only one path for reaction of the
reduced flavin intermediate with oxygen result in the rate
constant for the oxidative reaction being equal to the kcat/KO2
value (32). However, the values of the rate constants for both
(
Figure 6). The rate constants for both phases vary directly with
the concentration of oxygen (Figure 7), again consistent with
irreversible bimolecular reactions. For the fast phase, the second-
order rate constant is 40 ( 5 mM
it is 4.0 ( 0.04 mM
to the rate constant when the delay time is 6 s.
phases in the present case are significantly different from the kcat/
-
1 -1
s
, while for the slow phase,
-1 -1
KO2 value of 12.6 ( 1.6 mM
s . When flavin oxidation occurs
-
1 -1
s . The latter value is essentially identical
by both pathways in Scheme 4, the kcat/KO2 value is not a simple
function of the individual rate constants. The kinetic equation for
this mechanism is eq 9. This is given in double-reciprocal form to
more readily illustrate the oxygen dependency. The only rate
DISCUSSION
The results of the steady-state and rapid-reaction kinetic
analyses of human SMO described here are consistent with the
kinetic mechanism of Scheme 4 and the values for the individual
rate constants given in Table 3. The ping-pong kinetic pattern
established by the data in Figure 1 establishes that reduction is
effectively irreversible (25). The Kd value, the first-order rate
constant in Scheme 4 that was not measured directly is k , the rate
constant for dissociation of the product from the oxidized
7
enzyme. To obtain the value of k , the initial rate of spermine
7
oxidation was calculated using eq 9 and the values of the other
kinetic parameters listed in Table 3. The differences between the
observed and calculated rates at the concentrations of oxygen
constant for flavin reduction (k ), and the constant for release of
in the experiment shown in Figure 8 are minimized when k is
5.94 s . Fitting these calculated rates to the Michaelis-Menten
3
7
-
1 2
product from the reduced EFLredP complex (k ) can all be
9
obtained from the stopped-flow analyses of the reductive half-
2
reaction in the absence of oxygen. The value of k is comparable
The best way of estimating the confidence interval for the value of k
7
9
is not obvious. Varying the value by (0.1 from a value of 5.94 results in a
visibly poorer fit to the data. However, the precision of the individual
to kcat, so that this step could be along the catalytic pathway for
SMO. The values of k5 and k11 are from the stopped-flow
analyses of the oxidative half-reaction.
rates is only 5-10%, and the values of k and k9 have precisions
5
of ∼10%. Thus, a reasonable estimate for the precision of k
7
is 10%.

Article
Biochemistry, Vol. 49, No. 2, 2010 391
equation yields a kcat/K value of 11.5 ( 1.5 mM-
1
s
-1
and a kcat
due to an amino acid residue in the active site of SMO that must
be unprotonated for binding of either inhibitor, while the basic
limb can be attributed to the need for all of the nitrogens in the
inhibitors to be protonated. On the basis of the sequence of SMO
and the structures of maize PAO and Fms1, likely candidates for
the amino acid residue are His82 and His212.
O2
value of 7.6 ( 0.8 s- . The agreement of these values with the
experimentally determined values (Table 1) establishes that the
branched mechanism of Scheme 4 provides a better model for the
kinetics than simpler models involving only the upper or lower
pathway for the oxidative half-reaction. The possibility of such a
bifurcated mechanism for the oxidative half-reaction has been
suggested previously for flavoprotein oxidases (33), but the
ability of the calculated rates to be fit by the Michaelis-Menten
equation establishes that such behavior would be difficult to
detect in steady-state kinetic assays.
1
The requirements for productive binding of spermine and for
binding of inhibitors are clearly different. This suggests that these
two inhibitors bind to the protein in a slightly different fashion
than does spermine. In the case of DBDB, it is certainly possible
that the inhibitor binds such that neither nitrogen is positioned
where the reactive nitrogen of spermine would be located in the
active site. Spermidine could bind with a primary nitrogen in
place of the reactive secondary nitrogen of spermine. In both
cases, the out-of-register binding would be assisted by an
unprotonated amino acid residue. Structures have been described
for Fms1, a yeast spermine oxidase, with spermidine and 1,8-
diaminooctane bound [Protein Data Bank (PDB) entries 3cn8
and 3bi5]. These show that both inhibitors bind in a manner
different from that of spermine (14), in that neither has an atom
near the N5 position of the flavin, the site to which hydride
transfer would occur.
1
k
2
þ k
3
1
k
5
O
2
k
9
¼
þ
þ
þ
ð9Þ
2
v
0
k
1
k
3
S
k
3
k
11
O
2
ðk
5
O
2
þ k Þ
9
k
7
ðk
5
O
2
þk
9
Þ
The kcat/K -pH profile for spermine is most consistent with
M
the active form of the substrate having three of the four nitrogens
protonated. The concentrations of the mono-, di-, and triproto-
nated forms of spermine are maximal at pH 10.5, 9.5, and 8.4,
respectively (28, 34-37). The expected pH distribution of the
triprotonated form agrees well with the pH optimum in the kcat
/
K -pH profile for spermine of 8.3. The data do not allow us to
SMO differs from mammalian PAOs in the relative preference
M
identify the unprotonated nitrogen, but it is most likely to be the
secondary nitrogen at the site of oxidation. Earlier studies with
mouse PAO have established that the reacting nitrogen must be
neutral for amine oxidation (28), and a neutral nitrogen would be
required for the transfer of hydride to the flavin (12). The need for
this nitrogen to be neutral provides a ready explanation for the
pK of 7.4 seen in the k -pH profile. If the fully protonated form
for spermine versus N-acetylspermine. The kcat/K -pH profiles
M
for the two enzymes suggest that the two enzymes discriminate
between these two substrates on the basis of their protonation
states. PAO requires a form of the substrate with only one
protonated nitrogen (28), while SMO requires the triprotonated
form of spermine. N-Acetylspermine would then be a poor
substrate for SMO because acetylation of N1 prevents it from
being protonated. Conversely, acetylation increases the amount
of substrate with only a single positive charge, the required form
for PAO. The different pH optima are also consistent with the
different locations of the enzymes in the cell. The pH of the
peroxisome where PAO is located is 8-8.5 (41, 42), consistent
with its higher pH optimum (28) compared to the cytosolic SMO.
In conclusion, the complete kinetic mechanism of human
SMO has been determined using both steady-state and rapid
reaction kinetic methods, and the individual rate constants have
been measured. With spermine as the substrate, product release
a
3
of spermine can bind but not react, its pK when bound to the
a
enzyme will be seen in this profile. The alternate possibility that
this pK is due to an amino acid residue that must be unproto-
a
nated cannot be ruled out, especially in light of the results with
inhibitors (vide infra).
The ability of the incorrectly protonated form of spermine to
bind the enzyme is supported by the pK -pH profiles for
i
spermidine and DBDB. The mono- and diprotonated forms of
spermidine have their maximum concentrations at pH 10.4 and
9.1, respectively, too high for this profile to be due only to the
protonation state of spermidine. However, the triprotonated
occurs from both the EFlredP and EFl P complexes. In addition,
ox
form of this inhibitor has a pK value of 8.3 ( 0.1, primarily
the triprotonated form of spermine is the active form of the
substrate, whereas the inhibitors spermine and DBDB bind most
tightly when fully protonated.
a
due to the secondary nitrogen (28, 35-38). This suggests that the
average value of 7.9 obtained from the pK -pH profile is due to
i
binding of the triprotonated form of this inhibitor to a form of the
enzyme in which an active site residue is unprotonated. The
average value of the pK of 7.9 and the pK value of 8.3 for the
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