Trp387 and the putative leucine zippers of PGH synthases-1 and -2.
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.
Hsi
L C
LC
Department of Biochemistry, Michigan State University, East Lansing 48824.
Tsai
A L
AL
Kulmacz
R J
RJ
English
D G
DG
Siefker
A O
AO
Otto
J C
JC
Smith
W L
WL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002645:Chickens; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics*; D016350:Leucine Zippers / Q000235:genetics*; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013045:Species Specificity; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D007527:Isoenzymes; D014364:Tryptophan; D011451:Prostaglandin-Endoperoxide Synthases