Structure-to-Function Relationship of
Mini-Lipoxygenase, a 60-kDa Fragment of Soybean Lipoxygenase-1 with
Lower Stability but Higher Enzymatic Activity*
Almerinda
Di Venere
§,
Maria Luisa
Salucci¶,
Guus
van Zadelhoff
,
Gerrit
Veldink
,
Giampiero
Mei
§,
Nicola
Rosato
§**,
Alessandro
Finazzi-Agrò§, and
Mauro
Maccarrone
§§
From the
INFM, 16152 Genova, Italy, the
§ Department of Experimental Medicine and Biochemical
Sciences, University of Rome, Tor Vergata, 00133 Rome, Italy, the
¶ Department of Pure and Applied Biology, University of L'Aquila,
67100 L'Aquila, Italy,
Bijvoet Center for Biomolecular
Research, Department of Bio-organic Chemistry, Utrecht University, 3584 CH Utrecht, The Netherlands, ** AFaR-CRCCS Divisione di
Neurologia, Ospedale San Giovanni Calibita, Fatebenefratelli,
00100 Rome, Italy, and the 
Department
of Biomedical Sciences, University of Teramo, 64100 Teramo, Italy
Received for publication, November 27, 2002, and in revised form, February 5, 2003
 |
ABSTRACT |
Lipoxygenase-1 (Lox-1) is a member of
the lipoxygenase family, a class of dioxygenases that take part
in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic
digestion of soybean Lox-1 is known to produce a 60 kDa fragment,
termed "mini-Lox," which shows enhanced catalytic efficiency and
higher membrane-binding ability than the native enzyme (Maccarrone,
M., Salucci, M. L., van Zadelhoff, G., Malatesta,
F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò, A. (2001) Biochemistry 40, 6819-6827). In this study, we have investigated the stability of
mini-Lox in guanidinium hydrochloride and under high pressure by
fluorescence and circular dichroism spectroscopy. Only a partial
unfolding could be obtained at high pressure in the range 1-3000 bar
at variance with guanidinium hydrochloride. However, in both cases a
reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0-4.5 M). Both chemical- and
physical-induced denaturation suggest that mini-Lox is more hydrated
than Lox-1, an observation also confirmed by
1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have
also investigated the occurrence of substrate-induced changes in the
protein tertiary structure by dynamic fluorescence techniques. In
particular, eicosatetraynoic acid, an irreversible inhibitor of
lipoxygenase, has been used to mimic the effect of substrate binding.
We demonstrated that mini-Lox is indeed characterized by much
larger conformational changes than those occurring in the native Lox-1
upon binding of eicosatetraynoic acid. Finally, by both activity and
fluorescence measurements we have found that
1-anilino-8-naphthalenesulfonate has access to the active site of
mini-Lox but not to that of intact Lox-1. These findings
strongly support the hypothesis that the larger hydration of mini-Lox
renders this molecule more flexible, and therefore less stable.
 |
INTRODUCTION |
Lipoxygenases (Loxs)1
form a homologous family of non-heme, non-sulfur iron containing
lipid-peroxidizing enzymes, which catalyze the dioxygenation of
polyunsatured fatty acids to the corresponding hydroperoxy derivatives.
Mammalian Loxs have been implicated in the pathogenesis of several
inflammatory conditions such as arthritis, psoriasis, and bronchial
asthma (1). They are also thought to have a role in atherosclerosis,
brain aging, human immunodeficiency virus infection, kidney diseases,
and terminal differentiation of keratinocytes, because they are key
enzymes in the arachidonate cascade, together with cyclooxygenases (2).
In plants, lipoxygenases are active in germination, in the synthesis of
traumatin and jasmonic acid, and in the response to abiotic stress (3).
Recently, Lox activity has been shown to be instrumental in inducing
irreversible damages to organelle membranes (4-5), a process that
might be the basis for the critical role of Loxs in programmed cell
death induced by various pro-apoptotic stimuli (6). The biological activities of Loxs have attracted a growing interest on both their functional and structural properties. Plant and mammalian Loxs are made
by a single polypeptide chain folded in a two-domain structure (7). The
N-terminal domain is a
-barrel of ~110-115 (mammals) and 150 (plants) residues, whereas the larger C-terminal domain is mainly
helical and contains the catalytic site.
Soybean lipoxygenase-1 (Lox-1) is widely used as a prototype for
studying the structural and functional properties of lipoxygenases from
tissues of different species (7). Lox-1 has a 30-kDa N-terminal domain
and a 60-kDa C-terminal domain containing the catalytically active iron
and the substrate-binding pocket. The function of the N-terminal
-barrel domain has been elusive for several years, for Lox-1 as well
as for other Loxs (7). Recently, the N-terminal domain has been shown
to be essential for calcium binding and activation of 5-lipoxygenase
activity (8) and for nuclear membrane translocation of this Lox isoform
(9-10). Equilibrium unfolding experiments on Lox-1 have shown that the
C-terminal domain is less stable than the N-terminal domain, undergoing
chemical denaturation in the early steps of the complex protein
unfolding process (11). The smaller N-terminal domain seems to retain a
large part of its
-barrel structure even at high urea concentration,
suggesting that this portion of the protein structure might be
important for the overall protein stability. Limited proteolytic
cleavage of Lox-1 into the N- and C-terminal domains, and their
subsequent isolation, have added new important structural and
functional information (12). In particular, the electrophoretic,
chromatographic, and spectroscopic analyses of the purified 60-kDa
C-terminal domain of Lox-1 (termed "mini-Lox") have shown that the
trimmed enzyme is still folded. Surprisingly, mini-Lox was shown to
have a greater catalytic activity than intact Lox-1, thus suggesting a
built-in inhibitory role for the N-terminal domain (12). In addition, mini-Lox displayed a higher binding affinity than Lox-1 for artificial membranes, attributable to the enhanced surface hydrophobicity exposed
after removal of the 30-kDa N-terminal fragment. Interestingly, similar
results have been recently reported about the effect of the N-terminal
domain removal on the activity and membrane-binding ability of the
reticulocyte-type 15-lipoxygenase (13).
A three-dimensional representation of mini-Lox can be generated from
the Lox-1 crystallographic data (Fig. 1).
The high number of trypthophans (12) renders this enzyme
spectroscopically very complex, as demonstrated by the great
heterogeneity of the mini-Lox fluorescence spectrum (12). A somewhat
greater solvent accessibility of aromatic chromophores of mini-Lox with
respect to Lox-1 is apparent probably associated to a conformational
change after proteolytic cleavage (12).

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Fig. 1.
Three-dimensional structure of mini-Lox.
Ribbon model of mini-Lox obtained from the Lox-1 crystallographic file
(Protein Data Bank code 2sbl) removing the first 277 aminoacids.
The 12 trypthophan residues are evidenced.
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In our study we have investigated the relationship between the activity
of mini-Lox (e.g. enhanced enzymatic activity) and its
structural features checked by circular dichroism,
1-anilino-8-naphthalenesulfonate (ANS) binding, steady-state, and
dynamic fluorescence. Because the balance between the loss of
hydrophobic interactions and the enhanced solvation upon the N-terminal
removal is crucial to understand the mini-Lox properties, we have also
studied the stability of mini-Lox using complementary techniques, such
as chemical equilibrium unfolding measurements and denaturation by
hydrostatic pressure. The results demonstrate that the removal of the
N-terminal domain makes the mini-Lox more sensitive to
denaturation by both guanidinium hydrochloride (GdHCl) and pressure.
Furthermore, dynamic fluorescence measurements and ANS binding
experiments provide new evidence that the active site is more
accessible in the mini-Lox than in the Lox-1 and that quite different
conformational changes follow the substrate binding in the two enzymes.
All together these results provide a new structural rationale that
might explain the peculiar mini-Lox features.
 |
EXPERIMENTAL PROCEDURES |
Materials and Enzymes--
Linoleic (9,12-octadecadienoic) acid
and 5,8,11,14-eicosatetraynoic acid (ETYA) were purchased from
Sigma. Ultrapure guanidinium hydrochloride and ANS were
purchased from US Biochemical Corp. and Molecular Probes Inc.,
respectively. Lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC
1.13.11.12; Lox-1) was purified from soybean (Glycine max
[L.] Merrill, Williams) seeds as reported (14), and mini-Lox
was prepared as previously described (12). Briefly, Lox-1 was digested
with trypsin at a Lox-1/trypsin 10:1 (w/w) ratio, which allowed
completion of the reaction within 30 min. Chromatographic separation of
the tryptic fragments was performed by high performance liquid
chromatography gel-filtration on a Biosep-SEC-S3000 column (600 × 7.8 mm, Phenomenex, Torrance, CA). Fractions corresponding to the
60-kDa fragment eluted after 10 min as a single peak, and were pooled,
dialyzed against water, and concentrated on Centricon 30 ultrafiltration units (Amicon, Beverly, MA) (12). This 60-kDa fragment,
referred to as mini-Lox, was found to be electrophoretically
pure on 12% SDS-polyacrylamide gels, and its N-terminal amino acid
analysis showed the sequence STPIEFHSFQ, which corresponds to a unique
trypsin cleavage site between lysine 277 and serine 278 (12). Such a
cleavage should indeed remove a 30-kDa N-terminal domain of Lox-1,
leading to a fragment of the expected molecular mass of 63,695 Da (12). Protein concentration was determined according to Bradford (15), using
bovine serum albumin as a standard. Dioxygenase activity of mini-Lox in
100 mM sodium borate buffer (pH 9.0) was assayed spectrophotometrically at 25 °C by recording the formation of conjugated hydroperoxides from linoleic acid at 234 nm (16). Except for
lifetime measurements, all the experiments were performed dissolving
Lox-1 and mini-Lox in 0.1 M Tris-HCl buffer (pH 7.2) at a
final protein concentration of 1.5 µM. For lifetime
measurements, Lox-1 and mini-Lox were used at 10 µM, to
obtain a good signal to noise ratio.
Equilibrium Unfolding Measurements--
Protein denaturation by
GdHCl was obtained after a 12 h incubation at 4 °C in the
presence of different amounts of denaturant. Fluorescence and CD
spectra were recorded at 20 °C after 30 min of incubation. Unfolding
and refolding pathways were independent of protein concentration.
Refolding of fully unfolded mini-Lox samples was achieved by diluting
the denaturant concentration with buffer. The analysis of the unfolding
transition was performed as described elsewhere (17), according to a
two step denaturation pathway according to Scheme 1,
where N, I, and U represent the native, intermediate, and
unfolded protein fractions. They were directly evaluated as a
linear combination from the fluorescence signal.
K1 and K2 are the two equilibrium constants related to the respective free energy values
G1 and
G2, which are supposed to vary
linearly with the denaturant concentration [D] as shown in Equation 1
(where i = 1.2).
|
(Eq. 1)
|
A non-linear least-squares fit was used to evaluate the
parameters reported in Table I, according to the minimum
2 value. A single transition model was instead
sufficient to fit both the CD and activity data.
Circular Dichroism, Steady-State, and Dynamic Fluorescence
Measurements--
CD spectra were recorded on a Jasco-710
spectropolarimeter, at 20 °C, using a 0.1 cm quartz cuvette.
Steady-state fluorescence spectra have been recorded using an ISS-K2
spectrofluorometer (ISS), at 20 °C upon excitation at 280 nm.
No differences were observed in fluorescence spectra when the
excitation wavelength was varied from 280 to 295 nm due to a very
efficient energy transfer from tyrosines to tryptophans. High pressure
measurements were performed with the same instrument, using the high
pressure ISS cell equipped with an external bath circulator. The
analysis of the high pressure unfolding transition was performed
assuming a two-state equilibrium model between native and intermediate species as shown in Equation 2 as follows,
|
(Eq. 2)
|
with
G =
RTlnK1 and
V = (
lnK1)/
P).
The fluorescence emission decay of both Lox-1 and mini-Lox was
extrapolated from the phase-shift and demodulation data, obtained with
the cross-correlation technique (18) upon excitation at 280 nm, using
the frequency-modulated light of an arc-xenon lamp, in the range 5-200
MHz. The emission was observed through a 305 nm cutoff filter to avoid
the contribution of scattered light.
1-Anilino-8-naphthalenesulfonate Binding Measurements--
ANS
binding was measured as follows. Fluorescence spectra in the range
420-550 nm were recorded as a function of the amount of ANS
(
exc = 350 nm), at fixed protein concentration, then
each spectrum was resolved according to the linear combination in
Equation 3
|
(Eq. 3)
|
where Sexp, SF, and SB are
column vectors corresponding to the experimental, ANS-free, and
ANS-bound spectra. CF and CB represent the
extrapolated linear correlation coefficients representing the
percentage of free and bound ANS, respectively. The spectrum of the
totally bound ANS (SB) was extrapolated, in a separate experiment, by varying the protein concentration, in the presence of a
fixed amount of ANS (19). The ANS binding data have been represented as
Scatchard plots and fitted according to models for one or two
independent classes of sites (20) as follows,
|
(Eq. 4)
|
where
represents the moles of ANS bound per moles of
protein, [L] is the concentration of free ANS,
i = 1 or 2, and ni and
ki are the number of binding sites and the
association constant of each class of sites. The fits were performed
using the SPW 1.0 version of the Sigmaplot scientific graphic software
(by Jandel Scientific).
 |
RESULTS AND DISCUSSION |
Mini-Lox Is a Rather Unstable Molecule--
The stability of
mini-Lox has been studied by equilibrium unfolding measurements at
increasing GdHCl concentrations (Fig. 2).
The non-coincidence of the fluorescence and CD measurements demonstrates that the unfolding transition was not cooperative and that
the presence of stable intermediate species had to be taken into
account. The simplest denaturation model, which successfully fitted the
experimental data, was a three-state process, N
I
U (see
"Experimental Procedures"), whose parameters are reported in Table
I. As shown by the very early and steep
increase of the fluorescence signal (Fig. 2), a small free
energy of unfolding (Table I) characterizes the first transition
(0-1.5 M GdHCl). No relevant change occurred in the CD
signal at low GdHCl concentration, and the data could be fitted
according to a two-state model (Table I). The overall stabilization
energy obtained from the spectroscopic measurements
(
G
4.0 ± 0.3 kcal/mol) demonstrated that mini-Lox is rather unstable, specially if compared with intact Lox-1 (
G
26 kcal/mol (11)). Combined refolding and proteolysis experiments have
suggested that the C-terminal domain of Lox-1 is the less stable part
of the intact enzyme. This domain, which roughly corresponds to the whole mini-Lox molecule, is probably fully unfolded in the intermediate state found in the Lox-1 denaturation pathway (11). The transition from
the native structure to this intermediate species is characterized by a
quite large free energy change (
G
14 kcal/mol), as compared with the total stabilization energy of
mini-Lox (Table I). It can be therefore concluded that the low
stability of mini-Lox is due to the removal of the 30-kDa N-terminal
domain. As a matter of fact this hypothesis is indirectly supported by
the mi values reported in Table I. The
m parameter, which describes the cooperativity of the
unfolding process, is strictly correlated to the change in
protein-accessible surface area upon denaturation (21). In particular,
greater hydration has been found to correspond to larger m
values and vice versa (21). It is also well known that
m values obtained with GdHCl are about 2.2× larger than
those obtained with urea (21). Thus, even though different unfolding
agents have been used in the equilibrium unfolding measurements of
Lox-1 and mini-Lox (urea and GdHCl, respectively), it is possible to
compare the different results obtained for the two proteins. In
particular, from the results reported by Sudharshan and Appu Rao (11),
it can be expected that the GdHCl-induced denaturation of Lox-1 would
yield m1
4.4 kcal/mol M and
m2
3.5 kcal/mol, for the first and second step of its unfolding pathway. On the other hand, the total
m value for mini-Lox (Table I) is
3 kcal/mol,
i.e. 30% less than that obtained in the first transition of
Lox-1. This lower value stands for a smaller change in the
solvent-exposed surface area, indicating that the native mini-Lox is
more hydrated than the C-terminal domain of Lox-1. Thus, the N-terminal
Lox-1 domain plays a fundamental structural role, shielding the other
domain from the solvent and therefore enhancing its stability.

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Fig. 2.
Denaturation of mini-Lox by GdHCl. The
denaturation at different GdHCl concentrations was monitored by
steady-state fluorescence ( ex = 280 nm, filled
circles; ex = 295 nm, open circles) and
CD at 220 nm (squares). Solid lines represent the
best fit obtained using a three-state (fluorescence) or two-state (CD)
denaturation model (Table I). In the inset the mini-Lox
enzymatic activity is reported as a function of GdHCl (filled
triangles). The activity recovered upon refolding is instead
indicated by empty symbols.
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Table I
Physical Parameters Characterizing the Unfolding of Mini-Lox
The parameters have been obtained by fitting CD, enzymatic activity,
and fluorescence data (Figs. 2 and 3) according to a two- or
three-state denaturation process (see "Experimental Procedures").
The errors on the calculated parameters were directly obtained by the
fitting routine.
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Characterization of Mini-Lox Unfolding Intermediate under
Hydrostatic Pressure--
Despite its low stabilization energy, the
mini-Lox unfolding pathway was complex (Fig. 2), suggesting the
presence of stable (or partially stable) intermediate species.
Measurements of enzymatic activity demonstrated that the protein
biological activity was progressively lost between 0 and 1.5 M GdHCl (Fig. 2, inset). The corresponding free
energy value (
G
1.0 ± 0.2 kcal/mol), very close to
that calculated for the first fluorescence phase (Table I), reflects
the high instability of the enzyme, especially around the active site
region. In fact, in the same range a significant loosening of the
protein tertiary structure was taking place, as revealed by the change
(50%) of the protein intrinsic fluorescence signal (Fig. 2). These
findings suggest that the intermediate state is partially unfolded, but
it retains a native-like secondary structure. Such features resemble
those of the so-called molten globule state, an unfolding intermediate
species described in the denaturation pathway of several globular
proteins (22-23). The most relevant property of this structure is
indeed a greater exposure of the protein hydrophobic moieties to the
solvent. In the case of mini-Lox, the great heterogeneity of the
fluorescence spectrum (12), due also to a high number of tryptophan
residues (Fig. 1), makes it impossible to dissect the contribution of
each domain to the fluorescence of the intermediate state. In the last years, several studies have demonstrated that molten globule
intermediates may be also produced by hydrostatic pressure, which may
force solvent into the protein core (24).
Fig. 3 reports the fluorescence spectra
of mini-Lox at 1 and 2400 bar. A shift of the emission signal toward
longer wavelengths at higher pressure values is observed (Fig. 3,
inset), supporting the hypothesis of a progressive hydration
of the internal tryptophylic residues. It almost perfectly overlaps the
steady-state spectrum in the presence of 1.5 M GdHCl (Fig.
3), demonstrating that physically induced unfolding may neatly
reproduce chemical denaturation. Actually, the two-state fit reported
in the inset of Fig. 3 yields the same free energy of
unfolding (
1.5 kcal/mol) characterizing the first denaturation
transition in GdHCl (Table I). The corresponding volume change has also
been evaluated (see "Experimental Procedures") and resulted to be
quite small (
69 ± 9 ml/mol), despite the large protein size
(
63,000 Da) as usually found by compression of globular proteins
(24-25). Recent studies on the pressure-induced molten globule state
of cytochrome c (26),
-lactalbumin (27), apomyoglobin
(28), and the Ras domain of RalGDS (29) reported
V values
from 15 to 70 ml/mol, quite similar to that found here for mini-Lox,
which, however, is a much larger molecule. In this line, the results
obtained with point mutants of staphylococcal nuclease (30) have
suggested that the collapse of internal cavities under pressure might
be the main source of the volume changes. Because the number and size
of cavities in a protein have been found to be related to its molecular
weight (31-33), the low
V value obtained for mini-Lox could be
explained by assuming that most protein cavities are already solvated
at ambient pressure. Not only this hypothesis is consistent with the
chemical denaturation experiments, but it could also explain the easier
resiliency of mini-Lox with respect to Lox-1. In fact, at
variance with the native enzyme (11, 34-35), both pressure and
GdHCl unfoldings of mini-Lox were fully reversible, being activity, CD,
and fluorescence spectra fully recovered after restoring the initial
conditions.

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Fig. 3.
High pressure effect on mini-Lox.
Steady-state fluorescence spectra of mini-Lox, native (dashed
line), partially unfolded in 1.5 M GdHCl (dotted
line), or at 2400 bar (solid line). In the
inset the center of mass (com) of mini-Lox
fluorescence spectrum is reported as a function of pressure. The
solid line represents the best fit obtained with a two-state
unfolding model.
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Mini-Lox Conformational Changes upon ETYA Binding--
Because
mini-Lox is more active but less stable than Lox-1, we have
investigated which conformational changes were involved in the
biological activity. To this aim, both steady-state and dynamic
fluorescence measurements were performed in the presence of ETYA, an
irreversible inhibitor of lipoxygenase that rapidly binds at the active
site (36).
Normalized steady-state spectra of mini-Lox and Lox-1 in the presence
and absence of ETYA are reported in Figs.
4a and
5a, respectively. The decrease
in the emission intensity indicated that ETYA strongly quenches the
intrinsic fluorescence of both proteins, yet the overall effect was
remarkably different in the two cases. First of all, as revealed by the
peak position (
325 nm) and the full width at half-maximum, the
spectral shape of Lox-1 spectrum did not change after incubation for 30 min in the presence of the inhibitor. The difference spectrum was
symmetrical and centered at the same wavelength (
325 nm), indicating
that quenching is affecting in the same way both exposed and buried tryptophans. Instead, the fluorescence spectrum of mini-Lox appeared to
be red-shifted and narrowed in the presence of ETYA (Fig.
4a). The difference spectrum, peaking around 315 nm, was
diagnostic of a preferential quenching of the less hydrated
tryptophylic residues in the protein. It is worth mentioning that the
drop in fluorescence intensity following ETYA addition was not sudden, but required almost 30 min to reach equilibrium for both proteins (see
insets of Figs. 4a and 5a). This
finding strongly suggests that the quenching mechanism must be
indirect, probably due to an induced protein conformational change upon
ETYA binding. As a matter of fact, spectroscopic (37-38), structural
(39), and chemical denaturation studies (40) have shown that some
(1-3) tryptophylic residues are essential for the enzymatic activity of Lox-1, located in the hydrophobic substrate-binding site. Thus, a
simple diffusion of ETYA into the active site would have led to a much
faster quenching process than observed for both proteins (insets of Figs. 4a and 5a).
Interestingly, the time course of this process was not the same in the
two proteins: the fluorescence intensity of mini-Lox decreased
exponentially, whereas much slower kinetics were observed in the case
of Lox-1 (insets of Figs. 4a and 5a).
One possible explanation for this different behavior might be the
larger degrees of freedom experienced by the mini-Lox molecule. Thus,
larger conformational changes might be expected for mini-Lox upon ETYA
binding.

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Fig. 4.
ETYA binding to mini-Lox. a,
steady-state emission spectra of mini-Lox (solid line) and
of mini-Lox incubated for 45 min in the presence of 20 molar excess of
ETYA (dotted line). The dashed line is the
difference spectrum. The inset shows the total fluorescence
intensity of mini-Lox as a function of time after addition of ETYA.
b, fluorescence lifetime distributions of mini-Lox
(solid line) and of mini-Lox in the presence of 20 molar
excess of ETYA after 45 min incubation (dotted line).
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Fig. 5.
ETYA binding to Lox-1. a,
steady-state emission spectra of Lox-1 (solid line) and of
Lox-1 incubated for 45 min in the presence of 20 molar excess of ETYA
(dotted line). The dashed line is the difference
spectrum. The inset shows the total fluorescence intensity
of Lox-1 as a function of time after addition of ETYA. b,
fluorescence lifetime distributions of Lox-1 (solid line)
and of Lox-1 in the presence of 20 molar excess of ETYA after 45 min
incubation (dotted line).
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This hypothesis has been checked by dynamic fluorescence measurements,
which are in fact extremely sensitive to changes in the protein
tertiary structure (41). The fluorescence decay of large,
multi-tryptophan-containing proteins is heterogeneous and generally
best described by continuous distributions of lifetimes, rather than by
few discrete components (42-44). Previous measurements have shown that
the dynamic fluorescence of Lox-1 may be resolved into a pair of
lorentzian lifetimes distributions, probably associated to two distinct
classes of tryptophylic residues, depending on their relative exposure
to the solvent molecules (35). In the present study the phase and
demodulation technique (18) has been used to characterize the
fluorescence decay of mini-Lox and Lox-1 upon excitation at 280 nm. The
results, shown in Figs. 4b and 5b, demonstrate
that in both cases a double distribution is required to fit the
experimental data. Despite the peak positions being quite similar
(i.e. c1
1.25 ns and c2
3.5 ns), the relative fractional intensities were inverted for mini-Lox
(i.e. F1 was greater than F2). This
result rules out the possibility that the short-lived component was
essentially due to tryptophans located in the N-terminal domain of
Lox-1, as previously suggested by urea unfolding measurements (35). The
average lifetimes, evaluated from the distribution analysis of Lox-1
and mini-Lox, were
Lox-1
1.18 ns and
mini-Lox
1.00 ns, respectively, indicating a 20%
more efficient dynamic quenching in the case of mini-Lox. This result
could be indeed expected for a more solvated structure, as suggested
for mini-Lox (see above). The analysis of the dynamic fluorescence
measurements performed in the presence of ETYA yielded very similar
distribution profiles (Figs. 4b and 5b),
characterized by an overall shift toward shorter lifetimes
and a marked decrease in the fractional intensity of the first
component, F1. The last effect was more evident for
mini-Lox and paralleled the asymmetrical change of the fluorescence
spectrum upon ligand binding (Fig. 4a). In conclusion,
kinetics, steady-state and dynamic fluorescence measurements point to
the occurrence of larger conformational changes in the tertiary
structure of mini-Lox than of Lox-1.
ANS Binding Experiments--
ANS is a well known fluorescent
probe, whose quantum yield considerably increases upon binding to
hydrophobic pockets of proteins. Its emission spectrum also depends on
the environment, being blue-shifted the deeper and tighter its
interaction with the protein matrix (45). Hence ANS is a very suitable
probe to investigate the formation of protein-fatty acids complexes
(46-47). Previous measurements on Lox-1 have demonstrated that the
native enzyme has one binding site for ANS and that the enzymatic
activity is not inhibited by the fluorescent probe (48). It was
therefore concluded that ANS does not interact with the fatty
acid-binding site, but rather interacts with another hydrophobic patch
present in the protein structure. The above reported unfolding
measurements and spectroscopic assays of mini-Lox have pointed out that
the main structural differences between the two enzymes are associated
with the solvent accessibility, at the level of their tertiary
structure. We have therefore analyzed the binding of ANS to the
protein. Fig. 6a reports the
Scatchard plot for ANS binding to mini-Lox, showing a clear biphasic
behavior indicative of the presence of multiple binding sites. Because no evidence for cooperativity was obtained by other procedures (e.g. Hill plots), the data were fitted according to models
taking into account one or two different classes of independent binding sites (see "Experimental Procedure"). This last fit gave the best results (Fig. 6a), with one and four binding sites for the
two classes, respectively, hence confirming that the mini-Lox surface is characterized by a larger number of hydrophobic patches than Lox-1.
The association constants of the two classes (Fig. 6a) were
largely different (the first one being 50 times greater than the
second), indicating that the single site had enhanced affinity for ANS.
At variance with Lox-1, a significant reduction of the enzymatic
activity was obtained in the presence of ANS (Fig.
7, inset), suggesting that
some kind of interaction between the probe and the protein active site
was possibly taking place. To check this possibility another approach
was used, i.e. measuring the spectrum of ANS after the
mini-Lox active site was occupied by the irreversible inhibitor ETYA.
The results demonstrated that the occupancy of the active site by ETYA
reduced the ANS fluorescence by about 22% (Fig. 7), suggesting that
the two molecules compete for the mini-Lox catalytic site. This
hypothesis has been further tested by performing a titration of ANS
binding to the mini-Lox-ETYA complex. In this case the linearity of the
Scatchard plot (Fig. 6b) is a clear indication of similar
binding sites. Indeed, a fit of the data yielded four identical, low
affinity binding sites (Fig. 6b). This finding allows us to
identify the higher affinity ANS-binding site found in the absence of
ETYA with the enzyme active site. No effect of ETYA on the ANS binding
was observed in control experiments performed with Lox-1 (data not
shown).

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Fig. 6.
ANS binding to mini-Lox. a,
Scatchard plot for ANS binding to mini-Lox. The two fits were obtained
using a single class of equivalent binding sites (dashed
line, with n1 2, k1 3 × 105 M 1) or two different classes
of ANS-binding sites (solid line, with n1 1, k1 9 × 105
M 1 and n2 4, k2 2 × 104 M 1). In the
inset the data have been reported as a function of ANS moles
bound per mole of mini-Lox to highlight the presence of a biphasic
behavior. b, Scatchard plot for ANS binding to mini-Lox-ETYA
complex. The fit was obtained using a single class of equivalent
binding sites (solid line, with n 4, k 1.4 × 105 M 1). In the
inset the data have been reported as a function of ANS moles
bound per mole of mini-Lox to highlight the presence of a linear
behavior.
|
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Fig. 7.
Effects of ETYA on ANS binding to
mini-Lox. Solid line, fluorescence spectrum of ANS
bound to mini-Lox after 30 min incubation (at a ANS to mini-Lox ratio
of 5:1). Dotted line, the measurement was repeated by
previously reacting mini-Lox with ETYA (20-fold excess). In the
inset the dependence on substrate concentration of the
dioxygenation of linoleic acid by mini-Lox (circles) and
mini-Lox in the presence of 2 molar excess of ANS (after 30 min
incubation, squares) is reported.
|
|
Kinetic measurements of ANS binding to both mini-Lox and Lox-1 revealed
another interesting difference between the two enzymes (Fig.
8). As shown in Fig. 8, a and
b, the change of ANS fluorescence signal upon binding to
mini-Lox was rather slow, requiring 15-20 min to reach equilibrium.
This time-dependence was observed only at low ANS/protein ratios,
namely between 0.6:1 (Fig. 8a) and 2:1 (Fig. 8b).
In these cases the ANS fluorescence intensity changes are probably due
to re-arrangements of the protein tertiary structure, and in particular
to fluctuations in the polarity of the ANS-binding site.

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Fig. 8.
Fluorescence kinetics of ANS binding.
Time-dependence of the ANS fluorescence spectrum change upon binding to
mini-Lox. The ANS to protein ratio was 0.6:1 (panel a), 2:1
(panel b), or 4:1 (panel c). Solid,
dashed, dotted lines, and circles
correspond to spectra recorded at 0, 10, 20, and 30 min, respectively.
In the insets the time-dependence of the ANS fluorescence
intensity upon binding to Lox-1 (filled bars) and mini-Lox
(empty bars) is reported.
|
|
No time dependence was detected in the case of Lox-1, at any ANS
concentration (Fig. 8, a, b, and c,
insets). Interestingly, the time course characterizing ANS
fluorescence changes was the same found for ETYA binding, indicating
that similar conformational changes might be induced in the protein
tertiary structure to allow their access into the active site.
 |
CONCLUSIONS |
The fact that mini-Lox, the 60-kDa fragment of Lox-1, is more
efficient than the whole native protein in both dioxygenase activity
and membrane-binding ability (12) is rather intriguing. In this paper
we attempted to figure out which possible correlation holds between
biological and structural features of mini-Lox. We have shown that the
removal of the 30-kDa fragment from the 90-kDa molecule generates a
more hydrated protein, which displays an increased affinity for
hydrophobic probes like ANS. Mini-Lox appears to undergo larger
conformational changes than Lox-1 upon ETYA and ANS binding, displaying
greater sensitivity but also better reversibility after pressure- and
GdHCl-induced unfolding. It could be argued that a larger structural
flexibility due to the partial loosening of the tertiary structure is
the main reason for the enhanced activity. In particular, the
possibility of binding ANS in the active site is a direct evidence that
mini-Lox gives the substrates an easier access to the hydrophobic
cavity, where the catalysis takes place. In this context, the
hypothesis that the N-terminal domain of Lox-1 could act as a built-in
inhibitor of the enzymatic activity (12) takes ground. It seems
noteworthy that trypsin-trimmed Lox-1, as well as "reconstituted"
Lox-1 obtained by adding the tryptic fragments of molecular mass <60
kDa to mini-Lox, showed the same activity as isolated mini-Lox,
suggesting that these <60 kDa fragments had no effect on enzyme
activity (12). This result is at variance with a previous report (49).
On the other hand, an inhibition of mini-Lox by these fragments could be expected, in view of the enhanced activity of the trimmed enzyme compared with the native form. It can be proposed that the lack of
effect (either stimulatory or inhibitory) of the <60 kDa fragments on
mini-Lox activity simply reflects a different interaction between the
N-terminal and C-terminal fragments after proteolysis. In addition, the
N-terminal fragment could also make the native enzyme less able to bind
hydrophilic molecules, and more selective toward hydrophobic molecules
like its natural lipid substrates. By the way soybean Lox-1 is a
15-lipoxygenase (7) and interestingly its mammalian counterpart,
i.e. the rabbit reticulocyte 15-lipoxygenase, has a similar
site for trypsin cleavage (50). The C-terminal domain generated by a
cleavage at this site would have almost the same size (535 versus 562 amino acid residues) and an overall structure
like mini-Lox (Fig. 9). Furthermore, the
C-terminal domain of rabbit 15-lipoxygenase is similar to that of human
5-lipoxygenase (51). Therefore, it is tempting to speculate that what
was found with mini-Lox might hold true also for mammalian
lipoxygenases, and that the trimmed enzymes might play a role in
physio(patho)logical conditions, where enhanced lipoxygenase activity
and membrane lipoperoxidation have been observed, such as kidney
disease in humans (52) and programmed cell death in animals (6) and plants (53). Noteworthy, in the latter organisms, a trypsin-like protease, termed SNP1, has been shown to be expressed during infection by pathogens (54), a typical situation where lipoxygenase is known to
be activated (55).

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Fig. 9.
Three-dimensional structure of soybean Lox-1
and of rabbit 15-lipoxygenase. a, ribbon model of Lox-1
(Protein Data Bank code 2sbl) shown in red the N-terminal
domain removed by trypsin digestion and in blue in the
remaining C-terminal fragment (mini-Lox). The trypsin cleavage
site (lysine 277) is shown in yellow. b, ribbon
model of 15-lipoxygenase (Protein Data Bank code 1lox) shown in
red the N-terminal domain and in blue in the
remaining C-terminal fragment. The site of a possible trypsin cleavage
(lysine 127) is shown in yellow.
|
|
 |
FOOTNOTES |
*
This study was supported in part by grants from Istituto
Superiore di Sanità (IV AIDS project), Agenzia Spaziale Italiana (contract I/R/098/00), Ministero dell'Instruzione,
dell'Università e della Ricerca (COFIN 2001), and the Consiglio
Nazionale delle Ricerche target projects MADESS II and Ministero
Politiche Agricole e Forestali (progetto FORMINNOVA 2002), Rome.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.
§§
To whom correspondence should be addressed. Tel.: 39-0861-266875;
Fax: 39-0861-412583; E-mail: Maccarrone@vet.unite.it.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M212122200
 |
ABBREVIATIONS |
The abbreviations used are:
Lox, lipoxygenase;
GdHCl, guanidinium hydrochloride;
CD, circular dichroism;
ETYA, eicosatetraynoic acid;
ANS, 1-anilino-8-naphthalenesulfonate.
 |
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