Seibold Steve A, Ball Terry, Hsi Linda C, Mills Denise A, Abeysinghe Rajeewa D, Micielli Renee, Rieke Caroline Jill, Cukier Robert I, Smith William L
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
J Biol Chem. 2003 Nov 14;278(46):46163-70. doi: 10.1074/jbc.M306319200. Epub 2003 Sep 2.
Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG2 to PGH2. The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr385 to a tyrosyl radical within the cyclooxygenase site. His386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr385, which protrudes into the cyclooxygenase site, and His388, the proximal ligand of the peroxidase heme. When His386 was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10-30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS-1. The low peroxidase activity and decreased affinity for heme of H386A PGHS-1 imply that His386 helps optimize heme binding. Molecular dynamic simulations suggest that this is accomplished in part by a hydrogen bond between the heme D-ring propionate and the N-delta of Asn382 of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N-delta of His386 and the backbone carbonyl oxygens of Asn382 and Gln383. We speculate that the binding of cyclooxygenase inhibitors or AA to the cyclooxygenase site of ovine H386A PGHS-1 reopens the constriction in the cyclooxygenase site between the extended helix and a helix containing Gly526 and Ser530 and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.
前列腺素内过氧化物H合酶（PGHSs）具有一种环氧化酶，可从花生四烯酸（AA）加氧气生成前列腺素（PG）G2，还有一种过氧化物酶，可将PGG2还原为PGH2。过氧化物酶激活环氧化酶。这涉及过氧化物酶血红素基团首先被氢过氧化物氧化，随后环氧化酶位点内的Tyr385被氧化为酪氨酰自由基。PGHS-1的His386并非正式属于任何一个活性位点，但位于延伸螺旋中，该螺旋介于伸入环氧化酶位点的Tyr385和过氧化物酶血红素的近端配体His388之间。当PGHS-1中的His386被丙氨酸取代时，突变体保留的天然过氧化物酶活性不到2.5%，但保留了超过20%的天然环氧化酶活性。然而，通过用环氧化酶抑制剂或AA处理H386A PGHS-1可恢复过氧化物酶活性（10 - 30%），但用亚油酸处理则不能；相反，仅仅占据天然PGHS-1的环氧化酶位点对过氧化物酶活性没有影响。血红素滴定表明，H386A PGHS-1与血红素的结合比天然PGHS-1更松散。H386A PGHS-1的低过氧化物酶活性和对血红素亲和力的降低意味着His386有助于优化血红素结合。分子动力学模拟表明，这部分是通过血红素D环丙酸酯与延伸螺旋中Asn382的N-δ之间的氢键实现的。延伸螺旋的结构又反过来由His386的N-δ与Asn382和Gln383的主链羰基氧之间稳定的氢键强烈支持。我们推测，环氧化酶抑制剂或AA与绵羊H386A PGHS-1的环氧化酶位点结合会重新打开延伸螺旋与包含Gly526和Ser530的螺旋之间环氧化酶位点的收缩，并使延伸螺旋恢复类似天然的结构。由于亚油酸的体积比AA小，显然无法重新打开这种收缩。
