EXPRESSION OF 2 DIFFERENT FORMS OF CDNA FOR THROMBOXANE SYNTHASE IN INSECT CELLS AND SITE-DIRECTED MUTAGENESIS OF A CRITICAL CYSTEINE RESIDUE

cDNA coding for human placental thromboxane synthase (EC 5.3.99.5) was amplified by PCR from a human placental cDNA library and sequenced. T his cDNA and a shorter cDNA isolated from a human lung cDNA library wi th a deletion of 163 bp near the 3' end were expressed in Spodoptera f rugiperda (Sf9) insect cells using a baculovirus expression system. Th e cDNA from human placenta was expressed as an active enzyme (60 kDa) with a specific activity higher than those reported from other cell ty pes, whereas the shorter cDNA was expressed in an inactive form (52 kD a). The active recombinant enzyme appeared to be unglycosylated as the molecular mass and the enzyme activity were not altered in the presen ce of tunicamycin. Site-directed mutagenesis was performed to convert a cysteine at position 480 in thromboxane synthase to a serine. This c ysteine is found to be highly conserved in related cytochrome P-450 en zymes. The mutant enzyme was found to be inactive, although Western bl ot, immunoprecipitation and SDS/PAGE analysis indicated that the mutan t enzyme was expressed at a level comparable with the wild-type enzyme . These results suggest that Cys-480 is essential for the enzyme catal ytic activity and that the short-form cDNA may be a non-functional tra nscript.