Prostaglandin H synthase. Kinetics of tyrosyl radical formation and of cyclooxygenase catalysis
Hydroperoxides are known to induce the formation of tyrosyl free radicals in prostaglandin (PG) H synthase. To evaluate the role of these radicals in cyclooxygenase catalysis we have analyzed the temporal correlation between radical formation and substrate conversion during reaction of the synthase...
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Veröffentlicht in: | The Journal of biological chemistry 1992-09, Vol.267 (25), p.17753-17759 |
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Zusammenfassung: | Hydroperoxides are known to induce the formation of tyrosyl free radicals in prostaglandin (PG) H synthase. To evaluate the
role of these radicals in cyclooxygenase catalysis we have analyzed the temporal correlation between radical formation and
substrate conversion during reaction of the synthase with arachidonic acid. PGH synthase reacted with equimolar levels of
arachidonic acid generated sequentially the wide doublet (34 G peak-to-trough) and wide singlet (32 G peak-to-trough) tyrosyl
radical signals previously reported for reaction with hydroperoxide. The kinetics of formation and decay of the doublet signal
corresponded reasonably well with those of cyclooxygenase activity. However, the wide singlet free radical signal accumulated
only after prostaglandin formation had ceased, indicating that the wide singlet is not likely to be an intermediate in cyclooxygenase
catalysis. When PGH synthase was reacted with 25 equivalents of arachidonic acid, the wide doublet and wide singlet radical
signals were not observed. Instead, a narrower singlet (24 G peak-to-trough) tyrosyl radical was generated, similar to that
found upon reaction of indomethacin-treated synthase with hydroperoxide. Only about 11 mol of prostaglandin were formed per
mol of synthase before complete self-inactivation of the cyclooxygenase, far less than the 170 mol/mol synthase produced under
standard assay conditions. Phenol (0.5 mM) increased the extent of cyclooxygenase reaction by only about 50%, in contrast
to the 460% stimulation seen under standard assay conditions. These results indicate that the narrow singlet tyrosyl radical
observed in the reaction with high levels of arachidonate in this study and by Lassmann et al. (Lassmann, G., Odenwaller,
R., Curtis, J.F., DeGray, J.A., Mason, R.P., Marnett, L.J., and Eling, T.E. (1991) J. Biol. Chem. 266, 20045-20055) is associated
with abnormal cyclooxygenase activity and is probably nonphysiological. In titrations of the synthase with arachidonate or
with hydroperoxide, the loss of enzyme activity and destruction of heme were linear functions of the amount of titrant added.
Complete inactivation of cyclooxygenase activity was found at about 10 mol of arachidonate, ethyl hydrogen peroxide, or hydrogen
peroxide per mol of synthase heme; maximal bleaching of the heme Soret absorbance peak was found with 10 mol of ethyl hydroperoxide
or 20 mol of either arachidonate or hydrogen peroxide per mol of synthase heme. The peak concentration of the |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(19)37108-X |