Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism

Abstract

Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.

Detoxification of insecticides, allechemicals and heavy metals by glutathione S‐transferase SlGSTE1 in the gut of Spodoptera litura

“…Glutathione S ‐transferases (GSTs), a super family of detoxifying enzymes, play an important role in detoxification by catalyzing conjugation of reduced glutathione (GSH) with electrophilic endogenous and xenobiotic compounds, including chemosynthetic insecticides and natural phytochemicals, converting them to less toxic water‐soluble forms (Grant & Matsumura, ; Singh et al ., ). Two domains of GSTs are responsible for the detoxification function, a GSH‐binding domain, which is well conserved in the N‐terminal thioredoxin‐fold region of different classes of GSTs, and a hydrophobic substrate binding domain, which is structurally diverse in the C‐terminal α‐helical region of GSTs (Mannervik & Danielson, ).…”

Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions

“…[8][9][10] Studies aimed at measuring GST activity have traditionally been performed using the chromogenic substrate 1-chloro-2,4dinitrobenzene (CDNB). 11 Unfortunately, however, the available change in absorption at 340 nm is oen limited by the low sensitivity and selectivity of this substrate, and some GSTs have low activity with CDNB. As a consequence, several groups have developed uorogenic [12][13][14][15][16] and bioluminogenic 17 probes to provide higher levels of sensitivity for the detection of GST activity.…”