3-methyleneoxindole: An affinity label of glutathione S-transferase pi which targets tryptophan 38

The compound 3-methyleneoxindole (MOI), a photooxidation product of the pla nt auxin indole-3-acetic acid, functions as an affinity label of the dimeri c pi class glutathione S-transferase (GST) isolated from pig lung. MOI inac tivates the enzyme to a limit of 14% activity. The k for inactivation by MO I is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglu tathione, suggesting that MOI does not react entirely within the glutathion e site. The striking protection against inactivation provided by S-(hydroxy ethyl)ethacrynic acid indicates that MOI reacts in the active site region i nvolving both the glutathione and the xenobiotic substrate sites. Incorpora tion of [H-3]MOI up to similar to1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits . Fractionation of the proteolytic digest of [3H]MOI-modified GST pi yielde d Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is les s than 4 Angstrom from ethacrynic acid. Similarly, MOI may bind in this sub strate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from th e rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the e fficacy of alkylating cancer drugs while minimizing toxic side effects.