PROSTAGLANDIN-H SYNTHASE - CHEMICAL MODIFICATION OF HISTIDINE-RESIDUES IN DEFERENT FORMS OF THE ENZYME WITH DIETHYL PYROCARBONATE

Prostaglandin H synthase (PGHS) as the apo- and holo-enzymes and the h oloenzyme inactivated during conversion of arachidonic acid into prost aglandin H-2 were modified with diethylpyrocarbonate (DEPC). DEPC reac ted rapidly with all three forms of the enzyme, but with quantitative differences. Exhaustive reaction with DEPC resulted in the modificatio n of 7 histidine residues in apo-PGHS, 4 residues in holo-PGHS, and 18 residues in the enzyme inactivated during catalysis. Modification of apo-PGHS was accompanied by corresponding loss of total (cyclooxygenas e plus peroxidase) and peroxidase activities, which were practically n ot detectable after modification of 2-3 histidine residues. The veloci ties of tryptic cleavage of the three forms of the enzyme into two fra gments (33 and 38 kD) were rather different but independent of modific ation. The histidine residues protected by heme were shown to be part of the C-teminal fragment of PGHS. Two of these are probably His-309 a nd His-388. From the accessibility of all 18 histidine residues for mo dification with DEPC in the enzyme after its interaction with arachido nic acid and from the abnormally high rate of tryptic cleavage of this enzymatic form, we suggest that fast and dramatic changes in the prot ein structure occur during the catalysis.