The formation of stable fatty acid substrate complexes in prostaglandin H(2) synthase-1.
We have developed a protocol to purify apo-ovine (o) prostaglandin endoperoxide H(2) synthase-1 (PGHS-1) to homogeneity from ram seminal vesicles. The resulting apo enzyme can then be reconstituted with Co(3+)-protoporphyrin IX instead of Fe(3+)-protoporphyrin IX to produce a native-like, but functionally inert, enzyme suitable for the production of enzyme:fatty acid substrate complexes for biophysical characterization. Co(3+)-protoporphyrin IX reconstituted oPGHS-1 (Co(3+)-oPGHS-1) displays a Soret band at 426 nm that shifts to 406 nm upon reduction. This behavior is similar to that of cobalt-reconstituted horseradish peroxidase and myoglobin and suggests, along with resonance Raman spectroscopy, that the Co(3+)-protoporphyrin IX group is one in a six-coordinate, cobalt(III) state. However, Co(3+)-oPGHS-1 does not display cyclooxygenase or peroxidase activity, nor does the enzyme produce prostaglandin products when incubated with [1-(14)C]arachidonic acid. The cocrystallization of Co(3+)-oPGHS-1 and the substrate arachidonic acid (AA) has been achieved using sodium citrate as the precipitant in the presence of the nonionic detergent N-octyl-beta-d-glucopyranoside. Crystals are hexagonal, belonging to the space group P6(5)22, with cell dimensions of a = b = 181.69 A and c = 103.74 A, and a monomer in the asymmetric unit. GC-MS analysis of dissolved crystals indicates that unoxidized AA is bound within the crystals.
Malkowski
M G
MG
Department of Biochemistry, Michigan State University, Room 522, Biochemistry Building, East Lansing, Michigan 48824-1319, USA.
Theisen
M J
MJ
Scharmen
A
A
Garavito
R M
RM
D000818:Animals; D001051:Apoenzymes; D003035:Cobalt / Q000378:metabolism; D003460:Crystallization; D051545:Cyclooxygenase 1; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D007501:Iron / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009195:Peroxidase / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D011524:Protoporphyrins / Q000378:metabolism; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013059:Spectrum Analysis, Raman
D001051:Apoenzymes; D005227:Fatty Acids; D007527:Isoenzymes; D011524:Protoporphyrins; D003035:Cobalt; C028025:protoporphyrin IX; D007501:Iron; D009195:Peroxidase; D051545:Cyclooxygenase 1; D011451:Prostaglandin-Endoperoxide Synthases