The formation of stable fatty acid substrate complexes in prostaglandin H-2 synthase-1

We have developed a protocol to purify apoovine (o) prostaglandin endoperox ide H-2 synthase-1 (PGHS-1) to homogeneity from ram seminal vesicles. The r esulting apo enzyme can then be reconstituted with Co3+-protoporphyrin IX i nstead of Fe3+-protoporphyrin IX to produce a native-like, but functionally inert, enzyme suitable for the production of enzyme:fatty acid substrate c omplexes for biophysical characterization. Co3+-protoporphyrin IX reconstit uted oPGHS-1 (Co3+-oPGHS-1) displays a Soret band at 426 nm that shifts to 406 nm upon reduction. This behavior is similar to th at of cobalt-reconsti tuted horseradish peroxidase and myoglobin and suggests, along with resonan ce Raman spectroscopy, that the Co3+-protoporphyrin IX group is one in a si x-coordinate, cobalt(III) state. However, Co3+-oPGHS-1 does not display cyc looxygenase or peroxidase activity, nor does the enzyme produce prostagland in products when incubated with [1-C-14]arachidonic acid, The cocrystalliza tion of Co3+-oPGHS-1 and the substrate arachidonic acid (AA) has been achie ved using sodium citrate as the precipitant in the presence of the nonionic detergent N-octyl-beta-D-glucopyranoside, Crystals are hexagonal, belongin g to the space group P6(5)22, with cell dimensions of a b = 181.69 Angstrom and c = 103.74 Angstrom, and a monomer in the asymmetric unit, GC-MS analy sis of dissolved crystals indicates that unoxidized AA is bound within the crystals. (C)2000AcademicPress.