Inactivation of Anopheles gambiae Glutathione Transferase ε2 by Epiphyllocoumarin

Abstract

Glutathione transferases (GSTs) are part of a major family of detoxifying enzymes that can catalyze the reductive dehydrochlorination of dichlorodiphenyltrichloroethane (DDT). The delta and epsilon classes of insect GSTs have been implicated in conferring resistance to this insecticide. In this study, the inactivation of Anopheles gambiae GSTε2 by epiphyllocoumarin (Tral 1) was investigated. Recombinant AgGSTε2 was expressed in Escherichia coli cells containing a pET3a-AGSTε2 plasmid and purified by affinity chromatography. Tral 1 was shown to inactivate GSTε2 both in a time-dependent manner and in a concentration-dependent manner. The half-life of GSTε2 in the presence of 25 μM ethacrynic acid (ETA) was 22 minutes and with Tral 1 was 30 minutes, indicating that Tral 1 was not as efficient as ETA as an inactivator. The inactivation parameters k inact and K I were found to be 0.020 ± 0.001 min(-1) and 7.5 ± 2.1 μM, respectively, after 90 minutes of incubation. Inactivation of GSTε2 by Tral 1 implies that Tral 1 covalently binds to this enzyme in vitro and would be expected to exhibit time-dependent effects on the enzyme in vivo. Tral 1, therefore, would produce irreversible effects when used together with dichlorodiphenyltrichloroethane (DDT) in malaria control programmes where resistance is mediated by GSTs.

Figure 1

The conjugation reaction of 1-chloro-2,4-dinitrobenzene and DDT (a) as catalyzed by GSTs and (b) the compounds used in this study for the determination of inactivation properties of AgGSTε-2.

Figure 2

Electrophoretic analysis of the different fractions of AgGSTε2. Lane 1 contained molecular weight markers. Lane 2 contained the supernatant fraction obtained after lysis, sonication, and centrifugation of E. coli cells. Lane 3 contained the nonbound fraction containing protein that did not bind the affinity chromatographic ligands hexyl GSH/GSH. Lane 4 contained the wash fraction. Lane 5 contains the affinity pool fraction. The concentrate fraction is contained in lane 6 whilst lane 7 contained the dialysate fraction. Lanes 2 and 3 contained numerous bands indicating the presence of unwanted proteins. The single bands shown in lanes 5, 6, and 7 indicate that GSTε2 was purified to homogeneity.

Figure 3

Percentage activity against time for the time-dependent inactivation of AgGSTε2 by Tral 1 and ETA. The activity was determined every 15 min over a 2 h period. The t _1/2 was determined by nonlinear regression.

Figure 4

Percentage activity against time for the concentration-dependent inactivation of GSTε2 by Tral 1. The activity was determined every 15 min over a 2 h period. The t _1/2 was determined by nonlinear regression.

Figure 5

The rate of loss of GSTε2 activity with increase in Tral 1 concentration after 30 min incubation. This graph was obtained by analysing data according to the method of Maurer and Fung [11] so as to determine the inactivation parameters. K _I is the concentration of inhibitor which gives half the maximal rate of inactivation.

Figure 6

Pathways for irreversible inhibition [11]. K _i is a measure of the binding affinity for the inactivator for the free enzyme. K _i′ is a measure of the binding affinity for the inactivator for the enzyme-substrate complex and k _inact describes the rate at which inhibitor-enzyme complex is irreversibly transformed into EI^∗.