Prostaglandin endoperoxide synthase substituted with manganese protoporphyrin IX. Formation of a higher oxidation state and its relation to cyclooxygenase reaction.
The heme in prostaglandin endoperoxide synthase (PGH synthase) was substituted with Mn(III)-protoporphyrin IX. The resulting enzyme, Mn-PGH synthase, showed full cyclooxygenase activity but only 0.9% of the peroxidase activity of the native iron enzyme. During the reaction with exogenous or endogenously produced hydroperoxides, a spectral intermediate of Mn-PGH synthase was observed. The electronic absorption bands of the resting enzyme at 376, 472, and 561 nm decreased, and the intermediate's bands at 417, around 513, and 625 nm appeared. The rate constant of the formation of the intermediate was about 10(4) M-1.s-1 at 22 degrees C, three orders of magnitude lower than with the iron enzyme. Spectral properties, conditions of formation, and the suppressed formation in the presence of electron donors provide evidence for a higher oxidation state of Mn-PGH synthase, tentatively a Mn(IV) species. This species was assigned to an intermediate in the peroxidase reaction of Mn-PGH synthase, the low activity of which was explained by the rate-limiting slow reaction of Mn-PGH synthase with hydroperoxides. The findings and interpretation are consistent with the published properties of other manganese-substituted peroxidases. Although the cyclooxygenase activity was similar to that of Fe-PGH synthase, the cyclooxygenase reaction of Mn-PGH synthase showed distinct differences in comparison with Fe-PGH synthase. A longer activation phase was observed which resembled the time course of the formation of the higher oxidation state. Glutathione peroxidase with glutathione, a hydroperoxide-scavenging system, inhibited the cyclooxygenase of Mn-PGH synthase at concentrations where the activity of Fe-PGH synthase was not affected. It is demonstrated that Mn-PGH synthase requires higher concentrations of hydroperoxides for the activation of the cyclooxygenase. These findings suggest that the substitution of iron with manganese in PGH synthase does not change the mechanism of the enzyme. The main difference is the much lower rate of the reaction with hydroperoxides which affects both the peroxidase activity and the hydroperoxide-dependent activation of the cyclooxygenase. A reaction scheme for Mn-PGH synthase is proposed analogous to that suggested for Fe-PGH synthase (Karthein, R., Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 313-320).
Strieder
S
S
Fraunhofer-Institut für Biomedizinische Technik and Fachrichtung Medizintechnik, Universität des Saarlandes, St. Ingbert, Federal Republic of Germany.
Schaible
K
K
Scherer
H J
HJ
Dietz
R
R
Ruf
H H
HH
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000494:pharmacology; D007501:Iron / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D010084:Oxidation-Reduction; D011451:Prostaglandin-Endoperoxide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D011524:Protoporphyrins / Q000378:metabolism / Q000494:pharmacology*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013053:Spectrophotometry
D011524:Protoporphyrins; C038654:manganese protoporphyrin; D016718:Arachidonic Acid; D007501:Iron; D005979:Glutathione Peroxidase; D011451:Prostaglandin-Endoperoxide Synthases; D005978:Glutathione