Prostaglandin H synthase-1 (PGHS-1) is a bifunctional heme protein catalyzing both a peroxidase reaction, in which peroxides are converted to alcohols, and a cyclooxygenase reaction, in which arachidonic acid is converted into prostaglandin G2. Reaction of PGHS-1 with peroxide forms Intermediate I, which has an oxyferryl heme and a porphyrin radical. An intramolecular electron transfer from Tyr385 to Intermediate I forms Intermediate II, which contains two oxidants: an oxyferryl heme and the Tyr385 radical required for cyclooxygenase catalysis. Self-inactivation of the peroxidase begins with Intermediate II, but it has been unclear which of the two oxidants is involved. The kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of PGHS-1 reconstituted with heme or mangano protoporphyrin IX with a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and ethyl hydrogen peroxide (EtOOH). Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels. However, the oxyferryl heme intensity decayed much more rapidly than the tyrosyl radical intensity at high peroxide levels. The rates of reactions for PGHS-1 reconstituted with MnPPIX were approximately an order of magnitude slower, and the initial species formed displayed a wide singlet (WS) radical, rather than the wide doublet radical observed with PGHS-1 reconstituted with heme. Inactivation of the peroxidase activity during the reaction of PGHS-1 with EtOOH or 15-HPETE correlated with oxyferryl heme decay, but not with changes in tyrosyl radical intensity or EPR line shape, indicating that the oxyferryl heme, and not the tyrosyl radical, is responsible for the self-destructive peroxidase side reactions. Computer modeling to a minimal mechanism was consistent with oxyferryl heme being the source of peroxidase inactivation.
Research. Development. Production.
We are a leading supplier to the global Life Science industry with solutions and services for research, biotechnology development and production, and pharmaceutical drug therapy development and production.