Publications

• Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases.

  Yeh H.C., Tsai A.L., Wang L.H.

  Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H(2) (PGH(2)) to produce prostacyclin (PGI(2)) and thromboxane A(2) (TXA(2)). PGI(2) and TXA(2) have opposing actions on platelet agg ... >> More

  Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H(2) (PGH(2)) to produce prostacyclin (PGI(2)) and thromboxane A(2) (TXA(2)). PGI(2) and TXA(2) have opposing actions on platelet aggregation and blood vessel tone. In this report, we use a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to explore the active site characteristics of PGIS and TXAS. The two enzymes transformed 15-HPETE not only into 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid (13-OH-14,15-EET), like many microsomal P450s, but also to 15-ketoeicosatetraenoic acid (15-KETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). 13-OH-14,15-EET and 15-KETE result from homolytic cleavage of the O-O bond, whereas 15-HETE results from heterolytic cleavage, a common peroxidase pathway. About 80% of 15-HPETE was homolytically cleaved by PGIS and 60% was homolytically cleaved by TXAS. The V(max) of homolytic cleavage is 3.5-fold faster than heterolytic cleavage for PGIS-catalyzed reactions (1100 min(-1)vs. 320 min(-1)) and 1.4-fold faster for TXAS (170 min(-1)vs. 120 min(-1)). Similar K(M) values for homolytic and heterolytic cleavages were found for PGIS ( approximately 60 microM 15-HPETE) and TXAS ( approximately 80 microM 15-HPETE), making PGIS a more efficient catalyst for the 15-HPETE reaction. << Less

  Arch. Biochem. Biophys. 461:159-168(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mutational analysis of the role of the distal histidine and glutamine residues of prostaglandin-endoperoxide synthase-2 in peroxidase catalysis, hydroperoxide reduction, and cyclooxygenase activation.

  Landino L.M., Crews B.C., Gierse J.K., Hauser S.D., Marnett L.J.

  Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed ... >> More

  Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wtPGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to approximately 60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS. << Less

  J. Biol. Chem. 272:21565-21574(1997) [PubMed] [EuropePMC]

• Unique peroxidase reaction mechanism in prostaglandin endoperoxide H synthase-2: compound I in prostaglandin endoperoxide H synthase-2 can be formed without assistance by distal glutamine residue.

  Ichimura S., Uchida T., Taniguchi S., Hira S., Tosha T., Morishima I., Kitagawa T., Ishimori K.

  Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H2, but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bond ... >> More

  Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H2, but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine (Gln-189) mutants, Arg (Gln-->Arg), Asn (Gln-->Asn), and Val (Gln-->Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln residue might serve as a functionally equivalent residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a six-coordinate to a five-coordinate structure. Introduction of the bulky amino acid residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases. << Less

  J Biol Chem 282:16681-16690(2007) [PubMed] [EuropePMC]