Ichimura Shizuo, Uchida Takeshi, Taniguchi Shuhei, Hira Shusuke, Tosha Takehiko, Morishima Isao, Kitagawa Teizo, Ishimori Koichiro
Division of Chemistry, Graduate School of Science, Hokkaido University, Hokkaido 060-0810, Japan.
J Biol Chem. 2007 Jun 1;282(22):16681-90. doi: 10.1074/jbc.M610785200. Epub 2007 Apr 1.
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.
前列腺素内过氧化物H合酶-2（PGHS-2）具有过氧化物酶活性，可促进前列腺素H2的环氧化酶反应，但过氧化物酶中高度保守的氨基酸残基之一（远端精氨酸）通过氢键相互作用稳定过氧化物上正在形成的负电荷，该残基被中性氨基酸残基谷氨酰胺取代。为了表征PGHS-2中的过氧化物酶反应，我们制备了三个远端谷氨酰胺（Gln-189）突变体，即精氨酸（Gln→Arg）、天冬酰胺（Gln→Asn）和缬氨酸（Gln→Val）突变体，并通过吸收光谱和共振拉曼光谱研究了它们的过氧化物酶活性及其结构特征。尽管先前的一项研究（Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574）表明谷氨酰胺残基可能作为与精氨酸功能等效的残基，但我们目前的结果清楚地表明，缬氨酸和天冬酰胺突变体的过氧化物酶活性与野生型酶相当。此外，CO加合物中的Fe-C和C-O伸缩模式几乎不受突变影响，这意味着Gln-189可能不直接与血红素连接的过氧化物相互作用。相反，精氨酸突变体的过氧化物酶活性降低，同时血红素环境从六配位结构变为五配位结构。引入庞大的氨基酸残基精氨酸会干扰水分子与血红素铁的配位，这表明189位的侧链体积而非酰胺基团对PGHS-2的过氧化物酶活性至关重要。因此，我们可以得出结论，PGHS-2中的O-O键断裂是在过氧化物结合位点没有与带电侧链相互作用的情况下促进的，这与典型植物过氧化物酶有显著不同。
