Abstract
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.
Highlights
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
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
We found that the loss of the amide group of Gln-189 slightly increased the rate of Compound I formation without structural perturbations around the heme environment, implying that PGHS-2 does not need the interaction of Gln-189 with the iron bound peroxide, which is a unique molecular mechanism for the peroxidase reaction
Summary
Materials—General molecular biology supplies were obtained from Invitrogen, Qiagen (Valencia, CA), and Amersham Biosciences. The concentration of the purified enzyme lovirus stock solution was added to the medium [31, 32]. Cells were tration was calculated exactly by using an extinction coefficient harvested by centrifugation at 3500 rpm for 5 min and washed of 58.4 mMϪ1 cmϪ1 at 386 nm [35]. Enzyme concentration was calculated by using an pended in homogenization buffer PGHS-2 was added to a solution containing 1 mM ABTS and peroxide (15-hydroperoxyeicosatetraenoic acid (HPETE) or H2O2) in 500 l of the reaction buffer (50 mM Tris-HCl, pH 7.5, 0.1% CHAPS) until the final concentration of PGHS-2 was 20 nM. The rate constant of Compound I formation (k1) and kcat was obtained from Equations 5 and 6, respectively [38]
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