9
Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
Vol. 1 (1) 2012
ISSN: 2169-1878 (online)
doi: xxxxx
Available at http://sci-edit.net/journal/index.php/ijibr/index
Biochemical Characterization of a Novel Aspartic Protease
from Plutella xylostella, Diamondback moth (Lepidoptera:
Plutellidae) Midgut: Inactivation by an Aspartic Protease
Inhibitor)
Ajit Kumar*1, Feroz Khan2 and Mala Rao1
1
Division of Biochemical Sciences, National Chemical Laboratory, Dr. Homi Bhabha road, Pune- 411008, India. Tel.:
+91-20-25902228; Fax: +91-20-2590-2648; E-mail: mb.rao@ncl.res.in.
2
Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences, University of
California, 707 Parnassus Avenue, San Francisco, CA 94143
ABSTRACT
An aspartic protease from Plutella xylostella (PxAP) was purified to homogeneity by ammonium sulphate precipitation;
gel filtration and affinity chromatography with 110 fold purification. The enzyme exhibited a Mr. of 46.1 kDa on SDSPAGE with an optimum pH and temperature of 3.5 and 37 °C respectively. The enzyme hydrolyzed hemoglobin with a KM
value of 538 µM. The enzyme properties were consistent with those of aspartic proteases as the exzyme was inhibited by
pepstatin and also by an aspartic protease inhibitor, API. The inhibition of PxAP by API followed a hyperbolic pattern with
increasing concentrations of inhibitor with an IC50 value of 44 μM. The inhibition constants Ki determined by LineweaverBurk and Dixon plot were found to be 59 μM and 55 μM, respectively. The Kinetic studies on the PxAP-API interactions
reveal a slow-tight binding competitive mechanism of inhibition with the overall inhibition constant Ki*=3.70.3 nM. The
inhibition progress curves were time-dependent and consistent with slow-tight binding inhibition: E+I⇌ (k3, k4) EI⇌ (k5, k6)
EI*. The rate constant k5=6.640.710-2 s-1 reveals a fast isomerization of enzyme-inhibitor complex [EI] and a very slow
dissociation of EI* complex, which is proved by rate constant k6=5.670.910-6s-1. The Rate constants from the intrinsic
tryptophanyl fluorescence data were in agreement with those obtained from the kinetic analysis, therefore the induced
conformational changes in the enzyme were correlated to the isomerization of EI to EI*.
Keywords: Plutella xylostella; aspartic protease; inhibitor; inhibition kinetics; slow tight binding inhibition; fluorescence
*Corresponding author: Current address: Discipline of
Microbiology, School of life Sciences, University of KwaZulu Natal (Westville Campus), Durban-4000, South
Africa. Tel.: +27-31-2607405.
E-mail: ajitkanwal@yahoo.com.
Received: 17 July, 2012; Accepted 2 August, 2012
Abbreviations used: PxAP, Plutella xylostella aspartic
protease; API, aspartic protease inhibitor, TLCK, Na-ptosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-Lphenyl alanine chloromethyl ketone, BSA, bovine serum
albumin; SDS–PAGE, sodium dodisyl sulphate polyacrylamide gel electrophoresis; EDTA, ethylene-diaminetetraacetic acid; REA, relative enzyme activity.
INTRODUCTION:
The aspartic proteases such as pepsin [1], plasma
renin [2], HIV-1 protease [3], plasmepsin [4] and
lysosomal cathepsin D [5] are widely distributed in
nature and participate in the control of several
biological processes. It has been suggested that
aspartic proteases are involved in the hydrolysis of
storage and intracellular proteins in insects like
Ceratitis capitata [6], flesh fly Sarcophaga
peregrine [7], Lygus Hesperus [8], Drosophila
melanogaster [9], mosquito [10], Aedes aegypti [11],
Cockroaches
[12],
Colorado
potato
beetle
(Leptinotarsa decemlineata) [13], Callosobrochus
maculatus and Zabrotes subfasciatus [14] where
they play very important roles according to the
requirement of the insect. The efficacies of plant
defensive proteins are dependent upon the fates of
those proteins in the digestive systems of insects
where insects can compensate for the inhibition of
major proteolytic activities by applying different
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Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
ways [15]. A thorough characterization of the types
and activities of digestive proteases is crucial to
understanding the degradation of nutritional
proteins and to determining the effects those
xenobiotic proteins have on an insect’s digestive
system.
We are interested in basic and applied aspects
of an aspartic protease from Plutella xylostella
(Diamond backmoth, Lepidoptera) midgut. Plutella
xylostella is known worldwide as a serious pest of
cruciferous vegetables [16] and is the only insect
pest that has developed resistance to B.
thuringiensis formulations under field conditions
[17]. However, little is known concerning the
enzymes responsible for the proteolytic processing
of the protoxin to the active toxin, whereas, some
serine proteases were found to play a major role in
proteolytic processes in Lepidopteran larval guts
[18]. Inhibitors of aspartic proteases are relatively
uncommon. Few examples include proteins from
the plants such as potato, squash, inhibitor from
sea anemone and yeast [19]. Pepstatin, a low
molecular
weight
inhibitor
isolated
from
Streptomyces is universal inhibitor of aspartic
proteases [20] and has been widely exploited as a
research tool for unraveling the mechanism of this
group of enzymes [21].
Here for the first time, we report an aspartic
protease from Plutella xylostella and its
interactions with an aspartic protease inhibitor
API. API inhibits pepsin and recently reported
from our laboratory [22]. The inhibitor progress
curve analysis demonstrates the time and
concentration dependent inhibition of PxAP and
indicates a slow-tight binding competitive
inhibition mechanism. The fluorescence studies
revealed that the binding of API induced localized
conformational changes in the enzyme, as reflected
during the isomerization of the EI complex to the
EI* complex [23-25]. Considering the physiological
importance of the aspartic proteases and their role
in various proteolytic activities of insects, there is
a lacuna in the studies of the mechanism of
inhibition by slow tight-binding inhibitors. Our
study on PxAP-API interaction would be helpful to
grasp the mechanism of action of enzymes from
insect midgut and their implication in host
pathogen interactions.
MATERIALS AND METHODS
Materials
Hemoglobin, Na-p-tosyl-L-lysinechloromethylketone (TLCK), N-tosyl-L-phenylalanine chloromethylketone (TPCK), Bovine Serum Albumin (BSA)
(electrophoresis-marker
grade),
PepstatinAagarose and Bovine Trypsin were purchased from
Sigma Chemical (St. Louis, MO, USA). The
reagents for SDS-PAGE and other chemicals were
of analytical grade.
Rearing of Plutella xylostella and preparation of
midgut homogenate
The diamondback moth, P. xylostella larvae were
reared on cabbage leaves at 25±1 °C with a
light:dark cycle of 16:8 and 60-70 % relative
humidity. Before larvae emerged as adults, pupae
were separated, collected and placed into plastic
honey jars until moth emergence. The moths were
maintained on a diet of honey and water. Fifth
instar larval (those reared in laboratory conditions)
gut homogenate was prepared by extracting
midgets (dissecting in 0.5 M NaCl) with 50 mM
acetate buffer, pH 3.5 and homogenizing manually
using a close fitting glass pestle maintained in ice.
The homogenate was centrifuged at 15,000 ×g for
20 min at 4 °C and the resulting supernatant was
used for further studies.
Enzymes assays
The proteolytic activity of gut homogenate and
purified enzyme was measured by incubating with
hemoglobin (1 mM) in 0.2 M acetate buffer, pH 3.5
for 30 min [26]. The following inhibitors, pepstatin,
EDTA (Ethylenediaminetetraaceticacid), E-64 (Ltrans-epoxysuccinyl-leucyl-amido
(4-guanidino)butane), TPCK (tosyl-phenylalanyl-chloromethylketon and TLCK (tosyl-lysyl-chloromethyl-keton)
were utilized for inhibition studies. One unit of
enzyme was defined as the amount of enzyme that
produced an increase in absorbance of 0.001 at 280
nm per minute under assay conditions [27].
Purification of Plutella xylostella aspartic protease
(PxAP)
The P. xylostella midgut homogenate extracted
from 500 midguts was precipitated with 40-60%
ammonium sulphate at 4 oC for 2 hours,
centrifuged at 15000 ×g for 20 min and the pellets
were re-suspended in 50 ml volume of same buffer.
1 ml (1 mg/ml) homogenate was then applied to a
Sephadex G-75 column (2.0 × 50.0 cm) equilibrated
and eluted with the same buffer. Fractions (2.0 ml)
were collected and those containing hemoglobin
hydrolyzing activity sensitive to pepstatin were
pooled and applied to a pepstatinA-agarose affinity
column (1.0 × 5.0 cm) equilibrated with 50 mM
acetate buffer, pH 3.5. The enzyme was eluted
with 50 mM Tris/HCl buffer at pH 7.0. Active
fractions showing aspartic protease activity were
pooled, concentrated and the pH was adjusted at
3.5. 12% SDS-PAGE was carried out to check the
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Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
purity of the active fractions; gels were stained by
silver staining [28]. Protein concentration was
determined by dye binding assay of Bradford [29],
using BSA as standard.
Determination of molecular weight
The molecular weight of PxAP was determined by
SDS-PAGE [28]. The relative electrophoretic
mobility (Rf) i.e. the ratio of the distance of protein
migration to the distance of dye migration were
plotted against the log of molecular weight of
standard proteins and the molecular weight of the
protein was determined by regression analysis.
The molecular weight of PxAP was also
determined by gel filtration on Sephadex G-75
column. The void volume of the column (Vo) was
determined with Blue Dextran 2000 (2×106Da).
Marker proteins and PxAP (2 mg/ml) were loaded
separately on the column and eluted with the same
buffer. Molecular weights of the markers used
were bovine serum albumin (66 kDa), ovalbumine
(45 kDa), carbonic anhydrase (30 kDa), trypsin
inhibitor (20.1 kDa) and α-lactalbumine (14.4 kDa).
Fractions of 2.0 ml each were collected at a flow
rate of 10 ml/h. The elution volume (Ve) of each
protein was determined by measuring the
absorbance at 280 nm. The molecular weight of the
protein was determined by plotting the graph of
Ve/Vo vs log Mr.
Optimum pH and temperature of the purified
enzyme
The effect of pH on the proteolytic activity was
determined by assaying the enzyme at
concentration as described in enzyme assays
section in the range of pH 2.0 to 10.0 by adjusting
with variety of 100 mM buffers (pH 3.0 to 5.0),
phosphate (pH 5.0 to 8.0), and Tris-HCL (pH 8.0
to 10.0). The effect of temperature on the
proteolytic activity was determined at pH 3.5 in
the range of 25 oC to 80 oC.
Purification and biochemical characterization of
aspartic protease inhibitor (API)
The inhibitor was purified and characterized from
extra cellular culture filtrate of Bacillus
licheniformis as described previously [22]. Briefly,
the API was obtained after culturing Bacillus
licheniformis on a minimal medium of pH 7 and
50 °C containing 2 % Soyameal. The extracellular
culture filtrate was centrifuged at 10000 ×g, at
4 °C for 20 min. Further the API was purified by
ultrafiltration (UM-10 and UM-3), gel filtration
(sephadex G-25) and rp-HPLC by using prepacked
UltroPac column (fluka RP-C 8) pre-equilibrated
with 90 % acetonitrile (CH3CN) and 0.05 %
trifluoroacetate (TFA). The fractions were eluted
on a linear gradient of 0-90 % acetonitrile with
H2O containing 0.05 % TFA at a flow rate of 0.5
ml/min and monitored at a wavelength of 210 nm.
The eluted sample was lyophilized and dissolved in
de-ionized water to check the anti-PxAP activity.
The amino acid sequence of the purified peptide
was analyzed with a protein sequencer (ABI
Procise Clc Protein Sequencer), and the sequence
homology was done manually after retrieving the
peptide sequences from the data bank. The
isoelectric point of the inhibitor was determined as
described previously [22].
Assay for PxAP inhibition by API
100 l of enzyme (5 M) and 100 l (100 M) API
solution were incubated at 37 °C for 30 min in 0.2
M acetate buffer, pH 3.5. Then 0.5 ml of
hemoglobin (1 mM) dissolved in the same buffer
was added and incubated at 37 °C for 30 min. The
reaction was stopped by the addition of 5 %
trichloric acid (TCA) acidified with 2.25 % HCl and
the product was measured at 280 nm. The enzyme
was
incubated
with
different
inhibitor
concentrations (10-100 M) to determine the IC50
value.
Initial kinetic analysis for the determination of KM
and Ki
The kinetic parameters for the substrate
hydrolysis were determined by measuring the
initial rate of enzymatic activity. For the
Lineweaver-Burk analysis, enzyme (10 M) was
incubated with API at 0, 50 M and 100 M and
assayed
with
increased
concentration
of
hemoglobin (0.1-1.0 mM) at 37 °C for 30 min. The
reciprocals of substrate hydrolysis (1/v) for each
API concentration were plotted against the
reciprocals of the substrate concentrations to
determine the Km and Ki values by fitting the
resulting data in a computer software. In Dixon’s
method [30], hydrolytic activity of enzyme (10 μM)
was measured in the presence of 0.5 mM and 1.0
mM hemoglobin, at concentrations of API ranging
from 10-100 μM at 37 °C for 30 min. The
reciprocals of substrate hydrolysis (1/v) were
plotted against the API concentration and the Ki
was determined by fitting the data in ORIGIN 6.1.
Inhibition progress curve analysis
The inhibition progress curves for the interaction
between API and PxAP were analyzed using
Equation 1.
p= p0 + Vst + (Vo - Vs)[1 - exp (-kappt)]/kapp
(1)
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Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
Each individual progress curve was fitted
separately. The local fitting parameters were the
initial velocity V0, the steady-state velocity Vs, the
apparent first order rate constant kapp, and the
instrumental offset p0. These fitting parameters
were analyzed to extract approximate inhibition
constants [31].
Table 1. P. xylostella larval mid-gut aspartic protease purification
Step
Homogenate
Ammonium
sulphate
SG-75
PepstatinAagarose
Proteolytic
activity (Units/ml)
protein
(mg/ml)
Specific activity
(units/mg)
Purification
73
57
12.4
0.56
5.88
101.78
1
17.31
41
33
0.15
0.051
273.33
647
48.48
110
90
A
Enzyme activity (Units)
80
70
60
50
40
30
20
10
0
2
3
4
5
6
7
8
9
10
pH
Fluorescence spectroscopic analysis
B
100
90
80
Enzyme activity(U)
Figure 1. Partial purification and SDS-PAGE of P.
xylostella mid-gut enzyme. 60% ammonium
sulphate precipitated mid-gut homogenate dialyzed
and loaded on a Sephadex G-75 gel-filtration
chromatography
column.
The
column
was
equilibrated and run in 50 mM acetate buffer, pH 3.5.
Fractions were each of 2.0 ml. Proteolytic activities
against hemoglobin was measured in the absence (●)
and presence of pepstatin (). Absorbance at 280 nm
(■).
70
60
50
40
30
20
10
0
0
Fluorescence measurements were performed
according to Kumar and Rao 2006 [25]. Titration of
PxAP with API was performed by the addition of
different concentrations of API each time to a new
PxAP solution. The magnitude of the rapid
fluorescence decrease (F0−F) occurring at each
inhibitor concentration was fitted to the equation
(F0−F) =ΔFmax /{1+ (Ki/[I])} to determine value of Ki
and ∆Fmax. The first order rate constants for the
slow loss of fluorescence, kobs, at each API
concentration [I] were calculated by fitting of the
remaining data, starting 1 sec after inhibitor
addition, to a first order equation y=a+b*exp(-kobs*t)
using ORIGIN 6.1 software.
10
20
30
40
50
60
70
80
o
Temperature ( C)
Figure 2. Optimum pH and temperature of the
enzyme. (A) Effect of pH on the proteolytic activity of
the purified enzyme. (B) Effect of temperature on the
proteolytic activity of PxAP.
These values of kobs were then applied to the
equation kobs= k5 [I]/ (Ki + [I]) for the determination
of k5 under the assumption that, for a tight-binding
inhibitor, k6 can be considered negligible at the
onset of the slow loss of fluorescence [32, 33].
Background buffer spectra were subtracted to
remove the contribution from Raman scattering.
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Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
Statistics
RESULTS
Data are presented as mean ±SEM (standard error
of the mean). All the kinetic parameters were
determined by fitting the data using software
ORIGIN 6.1 (Origin Lab). For the kinetic analysis
and rate constant determinations, the assays were
carried out in triplicates, and the average value
was considered throughout this work.
Detection of aspartic protease activity in gut
homogenate
A
The gut homogenate of P. xylostella showed
hemoglobin hydrolyzing activity at pH 3.5
suggesting the presence of acidic protease type
activity. The 35 % inhibition of mid-gut
homogenate proteolytic activity by a universal
inhibitor of aspartic proteases, pepstatin confirms
the aspartic protease type activity.
80
A
70
Percent Inhibition
60
50
40
30
20
10
1.0
10
0
0.8
B
20.1 kd
0.7
0.5
10
20
30
40
50
60
70
80
90
100
API(M)
7
6
20.1 kd
0
8
30.0 kd
14.4 kd
0.6
Rf Value
9
14.4 kd
44.0 kd PxAP
70
65
5
30.0 kd
66.0 kd
0.4
Ve/Vo
0.9
60
4
50
44.0 kd
97 kd
66.0 kd
3
45
40
2
0.1
1
0.0
0
1/V
0.3
0.2
B
55
35
30
25
4.0
4.2
4.4
4.6
4.8
20
5.0
15
Log(Mol.Wt.)
10
5
Figure 3. Determination of homogeneity and
molecular weight of PxAP. (A) The fractions from
PepstatinA-Agarose affinity chromatography were
concentrated and applied to 12% SDS-PAGE. Lane1
shows 2μg enzyme, Lane2 shows 5μg enzyme and
lane 3 shows the standard molecular weight markers.
(B) Molecular weight determination of purified PxAP
by 12% SDS-PAGE (■) and gel-filtration sephadex G75 (●).
Table 2. Best fit values of adjustable parameters,
obtained in fitting the progress curves shown in
figure 5A (100 μM PxAp, 1mM hemoglobin, pH 3.5,
37 ˚C) to eq. 1.
[I]o μM
0
20
40
60
100
VS (REA)
79.24
34.62
17.68
12.09
09.24
V0 (REA)
79.24
1.73
0.88
0.60
0.46
-1
kapp (s )
0.0
0.22
0.31
0.36
0.39
-0.04
-0.02
0
0.00
0.02
0.04
0.06
0.08
0.10
1/[S] [mg/ml]
C
Figure 4. Binding of API to PxAP and inhibition
kinetics analysis. (A) The hyperbola curve indicates
the best fit for the percentage inhibition data obtained,
and the IC50 value was calculated from the graph. (B)
PxAP (10μM) was incubated, without (■) or with API
at 50 μM (●) and 100 μM (▲) concentrations and
assayed at increasing concentrations of the substrate.
(C) Enzymatic activity of PxAP (10μM) was estimated
using hemoglobin= 0.5mM (●) and 1.0 mM (■) at
different concentrations of API (10-100 μM).
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Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
Purification of aspartic protease from the gut
homogenate
A
Relative Enzyme Activity X 10
-2
100
80
60
40
20
0
0
5
10
15
20
25
30
Time (min)
B
200
150
60
50
100
40
F0-F
Fluorescence Intensity (a.u)
The enzyme was purified from P. xylostella mid-gut
homogenate extract by 40-60 % ammonium
sulphate precipitation, Sephadex G-75 column and
pepstatinA-agarose affinity column. Gel filtration
in sephadex G-75 coupled with protease assay
employing the substrate hemoglobin (at pH 3.5) in
the presence and absence of inhibitor pepstatin
showed the presence of an aspartic protease
(Figure 1). The fractions from 30-45 showed
proteolytic activity at pH 3.5 also suggest the
presence of aspartic protease which was confirmed
by the inhibition of the proteolytic activity by
pepstatin. The active fractions were concentrated
and applied to pepstatinA-agarose affinity column.
250
30
20
50
10
0
0
10
20
30
40
50
60
70
80
90
100
[I]  M
0
300
320
340
360
380
400
Wavelenght (n.m)
Figure 6. Steady-state fluorescence emission
spectra of PxAP as a function of inhibitor. (A)
Titration of PxAP (100μM) with APT at 0.0 (■), 10 (●),
20 (▲), 30 (▼), 50 (), 60 (◄), 70 (►), 80 (o), 100
( ★ ) μM concentrations. The curve in the inset
represents the fluorescence changes (F0-F) plotted
against the API concentrations. The resulting
hyperbola curve was fitted to the equation (F0 - F) = Δ
Fmax / (1 + (Ki/ [I]) to give the calculated value of Ki.
The gut homogenate showed 73U of acidic protease
with a specific activity of 5.88 U/mg. The
proteolytic activity remains 33 U with a specific
activity of 647 U/mg after affinity column and the
enzyme was purified to 110 fold (Table 1). The
purified enzyme showed an optimum pH 3.5
(Figure 2A) and optimum temperature 37 oC
(Figure 2B). Pepstatin shows 82 % inhibition of the
purified enzyme while other inhibitors had no
significant effect (data not shown).
Molecular weight of PxAP
80
0.4
70
60
0.3
40
0.2
Kappa
velocity
50
30
20
0.1
10
0.0
0
0
20
40
60
80
100
[I]M
Figure 5. Least-squares fit of progress curves
from the inhibition of PxAP (100 μM) by API to equ.
1. (A) the initial substrate concentration was 1mM
and the API concentrations were 0 (■), 20 (●), 40 (▲),
60 (▼) and 100 () μM. (B) Least-squares fit of the
initial velocities V0 (■) from Table 2 to eq. 2a, the
steady-state velocity VS (●) to eq. 2b, and the
apparent first-order rate constant kapp (▲) to eq. 2c.
SDS-PAGE showed a single band of Mr 46.1 kDa
(Figure 3A) and also confirms the homogeneity of
the enzyme. The average molecular weight of the
purified PxAP as determined by gel filtration on
Sephadex G-75 (45.3 kDa) revealed the
homogeneity of the enzyme and its monomeric
nature. The molecular weight determined by gel
filtration and SDS-PAGE were found comparable
suggesting the absence of subunits in the protein
(Figure 3B).
Biochemical properties of API and determination of
KM and Ki
API, a peptidic inhibitor of aspartic proteases,
produced extracellularly by thermo tolerant
Bacillus licheniformis has an amino acid sequence
of Asp-Leu-Tyr-Asp-Gly-Trp-Lys-His-Glu-Ala-GluCys-Ile with a pI value of 4.3. The predominance of
the charged amino acid residues in the inhibitor
sequence indicates its hydrophilic nature. The
15
Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
Simplified inhibition progress curve analysis for
API
The results of a preliminary analysis, based on the
assumption of rapid equilibrium, are shown in
Figure 5A. The progress curves obtained at 0 µM,
20 μM, 40 μM, 60 μM and 100 μM API
concentrations were fitted individually to Eq. 1.
The best-fit values of adjustable parameters, for
each concentration of the API, are listed in Table 2.
For a one-step inhibition mechanism, without an
intermediate enzyme-inhibitor complex, the initial
velocity V0 should be constant, and the apparent
rate constant kapp should increase linearly with the
inhibition concentration [I]0 [34]. The properties of
the two-step mechanism are expressed in eq. 2a-c,
The initial velocity should decrease with the
inhibitor concentration, by following a typical
binding curve, and the apparent rate constant
should depend on [I]0 as a hyperbola.
V0=Vmax [S]0/ ([S]0+KM (1+[I]0/Ki))
VS=Vmax [S]0/ ([S]0+KM (1 + [I]0/Ki*))
kapp=k6 + k5 ([I]0/Ki)/ (1+[S]0/KM+[I]0/Ki)
(2a)
(2b)
(2c)
conformational changes induced in PxAP due to the
binding of API. The tryptophanyl fluorescence of
PxAP exhibited an emission maxima (λmax) at 342
nm, as a result of the “radiative” decay of the π-π*
transition from the Trp residues. The binding of
inhibitor resulted in a concentration-dependent
quenching of the fluorescence with saturation
reaching at or above 100 μM inhibitor
concentrations (Figure 6). The absence of blue or
red shift in λmax negated any drastic gross
conformational changes in the three-dimension
structure of the enzyme due to API binding.
Binding of API resulted in an exponential decay of
the fluorescence intensity as indicated by a sharp
decrease in the quantum yield of fluorescence
followed by a slower decline to a stable value.
Furthermore, titration of inhibitor against PxAP
revealed that the magnitude of the initial rapid
fluorescence loss (F0-F) increased in a saturationtype manner which corroborated the two-step slowtight binding inhibition of PxAP by inhibitor. The
value of Ki determined by fitting the data for the
magnitude of the rapid fluorescence decrease ( F0-F)
was 67.0 μM (Figure 6 inset).
102
A
100
98
Fluorescence Intensity [a.u.]
inhibition of PxAP followed a hyperbolic pattern
with increasing concentrations of the inhibitor API
and showed an IC50 value of 44 μM (Figure 4A).
The Lineweaver-Burk reciprocal plot showed that
the KM value of PxAP for hemoglobin was 538 μM.
The inhibition constant Ki determined by
Lineweaver-Burk (Figure 4B) and Dixon plot
(Figure 4C) was found to be 59 μM and 55 μM
respectively.
a
96
94
92
90
b
88
86
84
82
0
Effect of API binding on the Fluorescence of PxAP
The kinetic analysis revealed a two-step inhibition
mechanism, where the EI complex isomerizes to a
tightly bound, slow dissociating EI* complex. This
isomerization
is
a
consequence
of
the
100
200
300
400
500
Time [sec]
B
78
76
74
72
kobs
The parameters listed in Table 2 favor the twostep inhibition mechanism, because the initial
velocity does decrease with the concentration of the
inhibitor, as predicted by eq. 2a. Also, the increase
of the apparent rate constant with [I] 0 is hyperbolic,
instead of linear. The nonlinear least squares fit of
V0, VS, and kapp to eq. 2a-c are shown in Figure 5B.
From fitting of V0 to eq. 2a, the dissociation
constant Ki of the initial complex was 48.3 5.1 μM.
From fitting of VS to eq. 2b, the overall dissociation
constant Ki* was 3.70.3 nM. From fitting of kapp to
eq. 2c, the initial inhibition constant Ki was 57.9 8
μM. The isomerization rate constants were k5=6.64
0.710-2s-1 and k6 =5.67 0.910-6s-1, from which
the total dissociation constant Ki*=Kik6/k5 was 4.9
nM.
70
68
66
64
20
40
60
80
100
120
140
160
[I]M
Figure 7. Time-dependent effect of API on the
fluorescence quenching of PxAP. (A) API, 0 μM (a),
100 μM (b) was added to PxAP (100 μM) at the
specified time (indicated by the arrow), and the
fluorescence emission was monitored for 500 s, at a
data acquisition time of 0.1 s. (B) The slow decay of
PxAP fluorescence at each concentration was fitted
to a first order rate equation to obtain kobs. The kobs
values were then computer fitted to the equation
kobs=k5 [I]/(Ki+[I]) to give calculated values of Ki and k5.
16
Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
k1
E+S
ES
k2
k7
E+P
k3
(a) E + I
(b) E I
k4
EI
k5
k6
E I*
Scheme 1. Schematic representation of slow tight
binding inhibition mechanism. E stands for the free
enzyme, I is the free inhibitor, EI is a rapidly forming
pre-equilibrium complex, and EI* is the final enzymeinhibitor complex. Binding between the enzyme and
inhibitor may either involve a single step, having slow
association and dissociation rates (Scheme 1a), or
have an initial fast binding step, followed by a slow
reversible transformation of EI to another entity, EI*
(Scheme 1b).
The subtle conformational changes induced during
the isomerization of EI to EI* was monitored by
analyzing the tryptophanyl fluorescence of the
complexes as a function of time (Figure 7A). The k5
and Ki values were determined from the data
derived from the slow decrease in fluorescence
were 4.620.210-2 s-1 and 705 μM respectively
(Figure 7B). These rate constants are in good
agreement with that obtained from the kinetic
analysis. Therefore, the initial rapid fluorescence
decrease can be correlated to the formation of the
reversible complex EI, whereas the slow, timedependent decrease reflected the accumulation of
the tight bound slow dissociating complex EI*.
DISCUSSION
The presence of serine protease type activity in
Plutella xylostella larvae mid-gut was previously
reported by Mohan and Gujar, 2003 [35]. The
present work demonstrates that the hemoglobin
hydrolyzing activity of gut homogenate is partly
due to aspartic protease also, as it was found to be
sensitive to pepstatin. The purified enzyme was
inhibited by pepstatin (which is a known inhibitor
of aspartic proteases) while the inhibitors TPCK
and TLCK (serine), E-64 (Cystein) and EDTA
(Metalloprotease) had shown no effect on its
hemoglobin hydrolyzing activity. The optimum pH
of isolated fraction (purified PxAP) for hemoglobin
hydrolysis was 3.5 while the optimum pH of midgut homogenate on the same substrate was 6-7.
This was also observed by Houseman et al., 1984
[36] and Kitch and Murdock, 1986 [37] when
working with a Cathapsin D-like proteinase from R.
prolixus and C. maculates respectively. The
utilization of aspartic and cysteine proteases for
extracellular digestion was first described in
Hemiptera feeding in blood or seeds by Houseman
et al., 1984 [36]. Later cathapsin B like proteases
were described in the bruchids C. maculates [37]
and A. obtectus [38]. After detection and
characterization of aspartic protease (this work)
and serine [35] in the larval mid-gut of P. xylostella,
it seems that both kinds of proteases contribute in
digestion of food. The present reported protease
suggest that larvae of P. xylostella have a great
potential for protein digestion which could be
related to the capacity of P. xylostella larvae to
survive on cabbage plants. To gain further insight
into the details of the hydrolytic mechanism of
aspartic proteases from insect source, specific
inhibitors like pepstatin are necessary, which can
act as mechanistic and structural probes.
In this study, we report a novel aspartic
protease from Plutella xylostella mid-gut and its
inhibition by API. The kinetic interactions lead to a
better understanding of the relationship of the
enzyme-inhibitor interactions. Moreover, in the
absence of structure knowledge and crystallization
data of the enzyme, the inhibition study will be
significant in deciphering the catalytic mechanism
of the enzyme. During the initial kinetic analysis,
PxAP was found to be inhibited by the Inhibitor
with an IC50 and Ki value of 44 μM and 48 μM-70
μM respectively. The Lineweaver-Burk and Dixon
plots (Fig. 4B and 4C) show that inhibitor was a
competitive inhibitor of PxAP.
Some enzymatic reactions display a slow onset
of inhibition in the presence of competitive
inhibitors, such type of inhibition is called slowbinding inhibition, and the inhibitors are called
slow-binding inhibitors [23-25]. The inhibitors that
inhibit the enzyme-catalyzed reactions at the
concentrations comparable to that of the enzyme,
are referred as tight-binding inhibitors. Kinetically
the slow tight binding inhibition mechanism can be
illustrated by Scheme I. When an inhibitor has a
low Ki value and the concentration of I varies in
the region of Ki, both k3I and k4 values would be
low. Thus, a simple second order interaction
between enzyme and inhibitor, and low rates of
association and dissociation would lead to slow
binding inhibition (Scheme Ia). Alternatively, a
two-step model depicts the rapid formation of an
initial collisional complex EI, which slowly
isomerizes to form a tightly bound slow
dissociating complex EI* (Scheme Ib). Scheme I
also describes two alternative models for the time
dependent inhibition. The mechanism in Scheme Ia,
where the binding of the inhibitor to the enzyme is
slow and tight, but occurs in a single step is,
eliminated based on the data of Table 2, because
the inhibitor has a measurable effect on the initial
rates before the onset of slow tight binding
inhibition of PxAP. As in most ground state
17
Int. J. Innov. Biol. Res. 1:1 (2012) 9-19
inhibitors, formation of the EI complex between
PxAP and API was too rapid to be measured at
steady-state kinetics and was likely to be near
diffusion control. However, the isomerization of EI
to the second tightly bound enzyme inhibitor
complex, EI*, was too slow and relatively
independent of the stability of the EI. The k6 values
revealed very slow dissociation of the EI* complex
indicating a highly stable, non-dissociative nature
of the second complex. Therefore for slow tight
binding inhibition the major variable is k6, the
first-order rate constant associated with the
conversion of EI* to EI, and the apparent inhibitor
constant Ki* depends on the ability of the inhibitor
to stabilize the EI*. The half-life as derived from
the k6 value indicated a longer half-life of the EI*
(34 h), which is an essential parameter for an
inhibitor to have biotechnological applications. The
nature of these changes has been discussed [39].
Our results for the inactivation of PxAP therefore
are consistent with the slow tight binding
mechanism as described in Scheme Ib and the
same was also reflected in the quenching pattern of
the fluorescence. On the basis of our fluorescence
studies, we propose that the rapid fluorescence loss
was due to the formation of the reversible PxAPAPI complex, whereas the subsequent slower
decrease was a result of the accumulation of the
tightly bound PxAP-API * complex. The kinetically
observable isomerization of PxAP-API to PxAP-API
* does not involve a major alteration in the threedimensional structure of the enzymes as reflected
in the absence of any shift in the tryptophanyl
fluorescence. Further, agreement of the rate
constant values determined from kinetic and
fluorescence analysis prompted us to correlate the
localized
conformational
changes
to
the
isomerization of PxAP-API to PxAP-API*. Any
changes in the environment of individual
tryptophan residues may result in an alternation of
fluorescence characteristics such as emission
wavelength, quantum yield, and susceptibility to
quenching [40]. Fluorescence quenching can also
result from the energy transfer to an acceptor
molecule having an overlapping absorption
spectrum [33]. As the API has no absorption in the
region of 290-450 nm, we ruled out the quenching
of fluorescence due to the energy transfer between
the inhibitor and the tryptophan residues. The
possibility that can be considered for the above
inhibitor induced fluorescence decrease is due to
the presence of multiple sites; binding at one
induced rapid fluorescence change and at a second
caused the slow fluorescence decrease. To verify
this possibility, a fixed concentration of the
enzymes
was
titrated
with
increasing
concentrations of API. The proteolytic activity of
PxAP
decreased
linearly
with
increasing
concentrations of API, yielding a stoichiometry
close to 1:1 expected for the slow-tight binding
inhibition. Further, the amino acid sequence of API
(Asp-Leu-Tyr-Asp-Gly-Trp-Lys-His-Glu-Ala-GluCys-Ile) revealed the presence of amino acid
residues such as Asp, Lys, and Glu with charged
side chains which prove the possibility of binding of
API to PxAP and interference in the electronic
microenvironment and disruption of the hydrogenbonding network between the catalytic and other
residues involved in catalysis. The results of our
investigation demonstrated the inactivation of
PxAP by inhibitor followed the slow tight binding
inhibition.
The choice of more appropriate inhibitors is a
primary determinant in the success or failure of
any pest control strategy based on extra-cellular
protease inhibition. The development of suitable
protease inhibitors should be based on a detailed
understanding of the biological system considered,
and on the availability of candidate inhibitors with
high affinity for digestive proteases of the target
insects. The use of new, broad spectrum inhibitors
of natural origin are currently considered for the
improvement of protease inhibitor based pest
control strategies, therefore the API and its
derivatives may be suitable lead molecules.
ACKNOWLEDGEMENTS
The senior research fellowship to AK by the
Council of Scientific and Industrial Research, India
is gratefully acknowledged. We profoundly thank to
Dr. Mahesh S. Kulye for the help in collection and
laboratory rearing of Plutella xylostella larvae
throughout the study.
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