Inhibitory kinetics of quercetin on phenoloxidase from loopworm

Insect Science 12, 435J441

Inhibitiory kinetics of quercetin on phenoloxidase 435

Introduction

Cuticle sclerotization is a vital process that occurs duringeach stage of insect development to harden and stabilizethe newly secreted exoskeleton. The initial step in thesclerotization process involves the formation of highlyreactive tanning agents by the oxidation of polyphenolsor diphenols to their corresponding electrophlilic o-benzoquinones, which is catalyzed by phenoloxidase(PO; monophenol, dihydroxyphenolalanine: oxygen oxi-doreductase; EC. 1.14.18.1 and also known as tyrosinase)(Kramer et al., 2001). Oxidization of phenols and diphenolswith the enzyme depends on the presence of copper atomsat the active site (Olivaries et al., 2002). Since PO is oneof the key enzymes in the insect moulting process (Kim

et al., 2002; Kubo et al., 2003; Dowd, 1999), someresearchers focused on PO in agricultural pests, such asPlutella xylostella, Spodoptera exigua and Pieris rapae(Wang et al., 2005; Luo et al., 2005; Gao et al., 2004; Xueet al., 2004). Fenoll et al. (2003) and Chen and Kubo(2002) found that quercetin could inhibit the activities oftyrosinase from mushroom. However, no reports werefound to test the role of quercetin on PO from forestinsects.

Loopworm (Semiothsa cinerearia Bremer et Grey)(Homoptera: Geometridae) is a kind of dangerous herbivo-rous insect in forests. In the present study, we investigatedthe effect of quercetin on PO from loopworm to ultimatelyprovide clues to the control of this forest pest (Kubo et al.,2003; Dowd, 1999). To discover whether or not quercetincan act on the PO from insects, the effects of quercetin onPO from loopworm were investigated and the kinetictheory of the substrate reaction during modification ofenzyme activity as previously described by Tsou (1988)was applied to study the kinetics of the course of inactiva-tion by quercetin.

Inhibitory kinetics of quercetin on phenoloxidase fromloopworm

X I A O - Y U N WA N G 1 , C H U N - Y I N G L I U 1 , J I E - D A O Z H A N G 1 a n d WA N -C H U N L U O 2

1College of Life Science and 2 College of Plant Protection, Shandong Agricultural University, Tai�an, China

Abstract Phenoloxidase (PO; monophenol or dihydroxyphenolalanine: oxygenoxidoreductase; EC. 1.14.18.1 and also known as tyrosinase) is one of the key enzymes inthe insect moulting process, so screening based on discovery of inhibitors of the enzymemight ultimately provide clues for enhancing the control of insect pests. In the presentinvestigation, the inactivation of phenoloxidase from loopworm Semiothisa cinereariaBremer et Grea (Homoptera: Geometridae) by quercetin were studied. The results showedthat low concentration to 180 µmol/L quercetin could inhibit PO activity, and the IC50

(inhibition concentration showing 50% of the maximum inhibition) was estimated to be111.5 µmol/L. In addition, quercetin was proven to be a competitive inhibitor, and theequilibrium constant for the inhibitor binding was determined. The results also showed thatinactivation of the enzyme by quercetin was a slow, reversible reaction with fractionalremaining activity, and the microscopic reaction rate constants of the inhibitor with theenzyme were determined.

Key words inactivation, loopworm, phenol oxidase, quercetinDOI 10.1111/j.1744-7917.2005.00055.x

www.blackwellpublishing.com/ins 435

Insect Science (2005) 12, 435J441

Correspondence: Wan-Chun Luo, College of Plant Protection,Shandong Agricultural University, Shandong, Tai�an 271018,China. Tel: +86 538 8242983; fax: +86 538 8242983; e-mail:[email protected]


436 X. Y. Wang et al.

Materials and methods

Chemicals

Quercetin and L-dihydroxyphenylalanine (L-DOPA)were purchased from Sigma. All other reagents were localproducts of analytical grade.

Preparation of the PO

Four-day loopworms were homogenized using a blenderin ice-cold extraction buffer (0.02 mol/L phosphate buffersolution [PBS], pH 7.0). The homogenate was centrifugedat 1 000 g for 10 min. The supernatant was the crudeenzyme extract.

Purification of the PO

The crude enzyme extract was used for the furtherpurification including ammonium sulfate precipitation,extensive dialysis against deionized water, Sephadex G-100 gel filtration (Liu et al., 2004). All operations werecarried out at 0J4�, unless otherwise stated. The purifiedenzyme solution was used in the present investigation. Thespecific activity of the enzyme was 366.1 U/mg protein (1unit = 1 µmol/L of substrate transformed per minute, µmol/L�min-1).

Protein estimation

Total protein concentration was determined byCoomassie blue G-250 dye-binding using bovine serumalbumin as standard (Bradford, 1976).

PO assay

Enzyme activity was assayed at 25� by following theincreasing absorbance at 475 nm accompanying the hy-drolysis of L-DOPA (Chen & Kubo, 2002). The progressof the substrate reaction method previously described byTsou (1988) was used to study the inhibition kinetics of POby quercetin. In this method, 50 µL enzyme solution wasadded to the assay substrate solution with 100 µL ofdimethysulfoxide (DMSO) containing different concen-trations of quercetin. The final reaction condition was that1 mL system contained different concentrations of L-DOPA (0.8J2 mmol/L) in 0.02 mol/L PBS (pH 7.0)containing different concentrations of quercetin and 10%DMSO. The absorption changes were detected by anUltrospec 4300 pro UV/visible spectrophotometer(Pharmacia Co.).

Results

Determination of kinetic parameters of PO

Under the conditions employed in the present study, theoxidization of L-DOPA of loopworm phenol oxidase fol-lowed Michaelis-Menten kinetics. The kinetic parametersfor the enzyme were determined using a Lineweaver-Burkplot, with the results shown in Fig.1. The results show thatKm and Vmax are 1.022 mmol/L and 30.87 µmol/L�min-1.

The effect of quercetin on the activity of phenol oxidase

As other researchers did, DMSO was used as a solventwhich had no effects on the activity of PO at lowconcentrations, and the same amount of DMSO was addedto the control (Wang et al, 2005; Chen & Kubo, 2002). Theeffect of quercetin on the oxidation of L-DOPA by phenoloxidase was studied. The inhibition of the enzyme byquercetin was concentration-dependent (Fig. 2). When theconcentration of the quercetin reached approximately 111.5µmol/L, the enzyme activity decreased by 50%. The pres-ence of quercetin resulted in the inhibition of the enzymeat low concentration to 180 µmol/L, which indicates thatthe inhibition of quercetin on the enzyme is a reversiblereaction with remaining enzymatic activity.

Fig. 1 Lineweaver-Burk plot for the determination of Km andVmax of loopworm phenoloxidase for the catalysis of L-DOPA at25�. Assay conditions: 1 mL system containing 0.02 mol/L PBS(pH 7.0) containing different concentrations of L-DOPA. Thefinal concentration of the enzyme was 0.095 mg protein/mL.



Determination of the inhibition type of quercetin on PO

In order to determine the inhibition type of quercetin, aLineweaver-Burk plot was made (Fig. 3a). The resultsshow that quercetin is a competitive inhibitor, since in-creasing the quercetin concentration results in a family oflines with the same intercept on the 1/v axis but withdifferent slopes. The equilibrium constant for the inhibitorbinding, KI, was obtained from the plot of the apparentMichaelis-Menten constant (Km

) versus the concentrationof quercetin (Fig. 3b). The obtained constant is summa-rized in Table 1.

Kinetics of the substrate reaction in the presence of differentconcentrations of quercetin

The progress of the substrate reaction method previouslydescribed by Tsou (1988) was used for the study of theinhibition kinetics of loopworm PO by quercetin. Figure 4ashows the time course of the oxidization of the substrate inthe presence of different quercetin concentrations. It can beseen that at each concentration of quercetin the rate de-creases with increasing time until a straight line isapproached, the slope of which decreases with increasingquercetin concentration, which indicates that there are stillfractional remaining enzyme activities. The results suggestthat the inhibition of PO by quercetin is a slow, reversible

reaction. According to Tsou�s progress of the substratereaction method,

where [P]calc is the product concentration to be expectedfrom the straight-line portion of the curves in Fig. 4a and[P]t is the product concentration actually obtained at timet. Y denotes the inhibitor. Plots of ln ([P]calc-[P]t) versus t

Fig. 3 Lineweaver-Burk plots for inhibition of quercetin onloopworm PO for the catalysis of L-DOPA. Experimentalconditions were as described for Fig. 1, except the assay systemcontained 2.0 mmol/L L-DOPA, 10% DMSO and differentconcentrations of quercetin. (a) Lineweaver-Burk plots forinhibition of quercetin. Concentration of quercetin for curves 1J5 was 0, 30, 50, 70 and 90 µmol/L, respectively. (b) A plot ofapparent Michaelis-Menten constant (Km ) versus concentrationof quercetin.

app

Fig. 2 Effect of quercetin concentration on the activity of PO forthe catalysis of L-DOPA. Experimental conditions were asdescribed for Fig. 1, except the assay system contained 2.0 mmol/L L-DOPA , 10% DMSO and different concentrations of quercetin,25� for 2 min.

Table 1 Kinetics parameters and microscopic reaction rateconstants of loopworm PO by quercetin.

Index Data Km 1.022 mmol/L Vmax 30.87 U/min IC50 111.5 µmol/L KI 40.03 µmol/L k+0 0.05286 (mmol/L�s)-1

k-0 2.1 � 10-3 s-1

app



give a series of straight lines at different concentrations ofY with slopes of J(A[Y]+B), as shown in Fig. 4b. Theapparent forward and reverse constants, A and B, can beobtained through suitable plots.

Figure 5a shows the kinetics course of the reaction atdifferent substrate concentrations in the presence of 30µmol/L quercetin. Similarly, plots of ln ([P]calc �[P]t)

Fig. 5 Course of reaction at different substrate concentrations in the presence of quercetin. Experimental conditions were described forFig. 3, except for L-DOPA concentrations. The final quercetin concentration was 70 µmol/L. (a) Curves 1J4 are progress curves with2.0, 1.5, 1.0, 0.8 mmol/L L-DOPA, respectively. (b) Semilogarithmic plot of ln ([P]calcJ[P]t) versus time for reaction at different substrateconcentrations of quercetin.

versus t give a series of straight lines at different concen-trations of the substrate with slopes ofJ(A[Y]+B), (Fig.5b).

In a similar method, the kinetics course of the reaction atdifferent substrate concentrations in the presence of differ-ent concentrations of quercetin can be obtained. Figure 6gives plots of the slopes of different substrate concentra-

Fig. 4 Course of substrate reaction in the presence of different concentrations of quercetin. Experimental conditions were as describedfor Fig.1, except the assay system contained 2.0 mmol/L L-DOPA, 10% DMSO and different concentrations of quercetin. (a) Substratereaction course. Concentration of quercetin for curves 0J4 was 0, 30, 50, 70 and 90 µmol/L, respectively. (b) Semilogrithmic plot of ln([P]calcJ[P]t) versus time. Data are taken from curse 1J4 in (a).



Fig. 6 Secondary plots of the slopes of the semilogarithmic plotsversus concentrations of quercetin for a series of fixed substrateconcentrations. Experimental conditions were as described forFig. 4, except for the L-DOPA concentrations. The data for curve4 is from Fig. 4b (at 2.0 mmol/L substrate). Substrate concentrationsfor curves 1, 2 and 3 were 0.8, 1.0, and 1.5 mmol/L, respectively.

Fig. 7 Plots of A/v versus 1/[S]. The value of v and A wereobtained from the substrate reaction in the absence of quercetinand from Fig. 5.

tions versus the concentration of quercetin. The apparentforward and reverse rate constants A and B can be obtainedthrough suitable plots according to equation 1. The resultshowed that all the straight lines cross the ordinate at thesame point, indicating that B has the same values. Thevalue of B directly gives the microscopic rate constant k-0

given in Table 1.

Figure 7 shows that the plot of A/v versus 1/[S] gives astraight line that passes through the origin. According toequation 2, the slope of the straight line gives the value ofkm�k+0/Vmax . The microscopic rate constant k+0 was thenobtained from the slope and is also shown in Table 1.

Discussion

Phenoloxidase, also known as tyrosinase, is a copper-containing enzyme that catalyzes two distinct reactions ofmelanin synthesis, the hydroxylation of a monophenol ordiphenol and the conversion of an o-diphenol to the corre-sponding o-quinone. The PO is responsible for the moltingprocess in insects, the undesirable browning of fruits andvegetables, and the coloring of skin, hair, and eyes inanimals. The regulation of this enzyme is of wide concern,since the browning of some fruits and vegetables due to POcauses a significant decrease in their nutritional and market

values. Regulation of PO and hence melanogenesis is alsovital to animals and insects. So screening out of inhibitorsof PO should have broad applications in pesticides, cos-metic and medicinal products, and the food industry (Kuboet al., 2003). Some attempts have been made in this area(Kubo & Kinst-Hori, 1999; Weemaes et al., 1999; Valero& García-Carmona, 1998). However, in most cases, mush-room tyrosinase has been used as the research target (Xieet al., 2003; Wu et al., 2003; Seo et al., 2003; Masamoto etal., 2003). Relatively little has been discovered about theinhibitory activity in insects.

Quercetin, as a flavonoid compound, has many functionsin anti-inflammatory activity�the inhibition of cancer,free radicals scavenging and the regulation of the red-oxstate (Agullo et al., 1997; Rodgers & Grant, 1998; dePascual-Teresa et al., 2004; Ueda et al., 2004). Quercetinhas been also found to have inhibitory activity to mush-room tyrosinase (Chen & Kubo, 2002). The results in ourstudy showed that low concentration of quercetin to 180µmol/L could inhibit the activity of PO from loopworm. Itsinhibitory effect was potent, with an IC50 value of 111.5µmol/L. By analyzing inhibitory kinetics, it was shownthat quercetin was a competitive inhibitor and the interac-tion between quercetin and the enzyme exhibited a rela-tively high affinity reflected in an equilibrium constant forthe inhibitor binding; KI was 40.03 µmol/L.

Since the activity of PO can be measured by the progressof the substrate reaction method, the kinetic theory of the



substrate reaction during the irreversible inhibition ofenzyme activity described previously by Tsou (1998) wasapplied to elucidate the type of inactivation and the micro-scopic constants of quercetin. The results in Fig. 4 a showthat the inactivation of the enzyme by quercetin is a slow,reversible reaction. The microscopic constants of k+0 andk-0 were 0.05286 (mmol/L�s)-1 and 2.1�10-3 (s)-1 respec-tively (Table 1). About the inhibitory mechanism, sincequercetin has the property of a substrate analogue, itcompetes with substrate molecules to bind to the activesite, that is, it can pull some of the free enzyme over into theform of the EI complex, which makes v decrease. When theconcentration of the substrate increased sufficiently, theeffect on the velocity can be overcome. Thus there was nochange in the Vmax of the enzyme, but the apparent affinityof the enzyme for its substrate decreased in the presence ofthe inhibitor, and hence Km increased.

We have shown here that, as a natural ingredient oforiental herbal medicines such as ginkgo, tea and mulberry,quercetin is a potential pesticide that can inhibit PO activityand poses little danger to the environment.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No. 30270887 and 30571237).

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Accepted September 14, 2005

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Inhibitory kinetics of quercetin on phenoloxidase from loopworm