Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function.
Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5'-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.
Andersson
C
C
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Morgenstern
R
R
D000818:Animals; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010658:Phenylglyoxal / Q000494:pharmacology; D010713:Phosphatidylcholines; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry
D008081:Liposomes; D010713:Phosphatidylcholines; D011732:Pyridoxal Phosphate; D005982:Glutathione Transferase; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; D005033:Ethylmaleimide