Hsi L C, Tsai A L, Kulmacz R J, English D G, Siefker A O, Otto J C, Smith W L
Department of Biochemistry, Michigan State University, East Lansing 48824.
J Lipid Mediat. 1993 Mar-Apr;6(1-3):131-8.
The active site sequence 385-YHWH-388 of ovine prostaglandin endoperoxide synthase-1 (PGHS-1) has residues critical for cyclooxygenase and peroxidase catalysis. Tyr385 is essential for cyclooxygenase activity, His386, for peroxidase activity, and His388, for both activities. To determine the importance of Trp387, we used site-directed mutagenesis to replace Trp387 of PGHS-1 with arginine, phenylalanine, and serine. W387R and W387S lacked significant activity. W387F retained both cyclooxygenase and peroxidase activities. Thus, we conclude that Trp387 is not essential for catalysis by PGHS-1. Purified PGHS-1 is a homodimer. There are two putative leucine zipper regions in ovine PGHS-1 involving residues 345-366 and 487-508. We tested for a role of these leucine zippers as determinants of dimer formation. Helix-breaking proline mutations were introduced at Leu359 or Leu501. Neither of these residues proved to be essential for peroxidase activity; but, mutations at each residue greatly reduced or eliminated cyclooxygenase activity. Both mutant proteins chromatographed as dimers on Sephacryl G-200. Thus, neither of these putative leucine zipper regions alone is responsible for PGHS-1 dimer formation.
绵羊前列腺素内过氧化物合酶-1（PGHS-1）的活性位点序列385-YHWH-388含有对环氧化酶和过氧化物酶催化至关重要的残基。Tyr385对环氧化酶活性至关重要，His386对过氧化物酶活性至关重要，His388对两种活性都至关重要。为了确定Trp387的重要性，我们使用定点诱变将PGHS-1的Trp387分别替换为精氨酸、苯丙氨酸和丝氨酸。W387R和W387S缺乏显著活性。W387F保留了环氧化酶和过氧化物酶活性。因此，我们得出结论，Trp387对PGHS-1的催化不是必需的。纯化的PGHS-1是一种同源二聚体。绵羊PGHS-1中有两个假定的亮氨酸拉链区域，涉及残基345-366和487-508。我们测试了这些亮氨酸拉链作为二聚体形成决定因素的作用。在Leu359或Leu501处引入了破坏螺旋的脯氨酸突变。这些残基对过氧化物酶活性都不是必需的；但是，每个残基处的突变都大大降低或消除了环氧化酶活性。两种突变蛋白在Sephacryl G-200上均以二聚体形式进行色谱分离。因此，单独的这两个假定的亮氨酸拉链区域都不负责PGHS-1二聚体的形成。
