3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.
The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded 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 less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate 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 the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects.
Pettigrew
N E
NE
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Brush
E J
EJ
Colman
R F
RF
D019297:2,4-Dinitrophenol / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D017738:Alkanesulfonic Acids / Q000378:metabolism; D000818:Animals; D002021:Buffers; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004976:Ethacrynic Acid / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006531:HEPES / Q000378:metabolism; D007210:Indoleacetic Acids / Q000378:metabolism; D007211:Indoles / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D009025:Morpholines / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000078183:Oxindoles; D013448:Sulfobromophthalein / Q000378:metabolism; D013552:Swine; D014364:Tryptophan / Q000378:metabolism*
D000345:Affinity Labels; D017738:Alkanesulfonic Acids; D002021:Buffers; D004791:Enzyme Inhibitors; D007210:Indoleacetic Acids; D007211:Indoles; D007527:Isoenzymes; D008024:Ligands; D009025:Morpholines; D009842:Oligopeptides; D000078183:Oxindoles; D013448:Sulfobromophthalein; C008440:3-methyleneoxindole; C004550:2-(N-morpholino)ethanesulfonic acid; D014364:Tryptophan; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D005978:Glutathione; D004976:Ethacrynic Acid; C033550:hexylglutathione; C017514:S-methyl glutathione; D019297:2,4-Dinitrophenol; D006531:HEPES