Unique Peroxidase Reaction Mechanism in Prostaglandin Endoperoxide H Synthase-2

Abstract

Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H 2 , 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 (Gln3 Arg), Asn (Gln3 Asn), and Val (Gln3 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 sixcoordinate 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.Prostaglandin endoperoxide H synthase (PGHS, 3 also known as cyclooxygenase or COX) is a membrane-bound heme-containing protein catalyzing the first committed step in prostanoid biosynthesis (1-3) including two sequential enzymatic reactions, bis-oxygenation of arachidonic acid to prostaglandin G 2 (PGG 2 ) (cyclooxygenase reaction) and the reduction of PGG 2 to prostaglandin H 2 (PGH 2 ) (peroxidase reaction). PGH 2 is, then, further metabolized to various kinds of prostaglandins, prostacyclins, and thromboxanes by the appropriate synthase (2). Two isoforms of PGHS have been discovered thus far, PGHS-1 and PGHS-2, both of which are composed of ϳ600 amino acid residues, sharing more than 60% sequence identity (4, 5), and their crystal structures are essentially superimposable (6 -8).Although both the PGHS isozymes function as a homodimer of ϳ70-kDa subunits and have similar catalytic properties, they have distinctly different biological functions. PGHS-1 is a "housekeeping" enzyme, expressed constitutively in most tissues, and produces prostaglandins to regulate cellular responses to hormonal stimulation and to regulate vascular homeostasis. PGHS-2, in contrast, is inducibly expressed i...

Prostaglandin H synthase: Resolved and unresolved mechanistic issues

“…5). Recent mutagenic results, however, indicate that removal of the side chain amine group from Gln203 in either PGHS-1 [32] or PGHS-2 [39] has little effect on heterolytic cleavage of peroxide. Thus, His207 may by itself exert sufficient “pull” to achieve peroxide cleavage, so that Gln203 only provide H-bonding to help orient His207.…”

Boosting the Catalase‐Like Activity of SAzymes via Facile Tuning of the Distances between Neighboring Atoms in Single‐Iron Sites

“…[24] As for surprisingly poor activity of Fe 1 -SAzyme at pH 3, it might be attributed to the low affinity of protonated H 2 O 2 to isolated FeN 4 center (int1!int2, Figure 5b). [25] To derive the intrinsic rate-determining step (R.D.S) for both of Fe 2 -and Fe 1 -SAzyme, the corresponding Gibbs freeenergy profiles were further investigated. (Figure 5c) The R.D.S in Fe 2 -SAzyme cycle was determined as the finial regeneration step of Fe 2 -SAzyme with 0.14 eV barrier (int5!int1, Figure 5a), in contrast to a much higher R.D.S barrier for Fe 1 -SAzyme (0.40 eV, int2!int3, Figure 5b).…”