Reconstitution of the two terminal enzymes of the heme biosynthetic pathway into phospholipid vesicles.
Purified mouse protoporphyrinogen oxidase (EC 1.3.3.4) and ferrochelatase (EC 4.99.1.1), the two terminal enzymes of the heme biosynthetic pathway, have been reconstituted into phospholipid vesicles, and the kinetics of the enzymes in the reconstituted systems were compared with the values obtained with the free enzymes. The apparent Km for free protoporphyrinogen oxidase in detergent solution is 5.61 +/- 0.62 microM for free protoporphyrinogen. The Km was lower when the enzyme was inserted into phospholipid vesicles (0.78 +/- 0.28 microM) and when both enzyme and substrate were incorporated into phospholipid vesicles (0.61 +/- 0.14 microM). In the presence of cardiolipin, a phospholipid present mainly in the inner mitochondrial membrane, the value of the Km for the substrate decreased 3-fold (0.20 +/- 0.02 microM). For reconstituted ferrochelatase similar kinetic analyses were carried out and it was found that the apparent Km values were only weakly affected by the lipid environment. Studies on the orientation of ferrochelatase demonstrated that approximately 50% of the enzyme in the reconstituted system had the active site located in the inner face of the phospholipid vesicle. This is in contrast to intact mitochondria where the active site is located on the matrix side of the inner mitochondrial membrane. The activation energies for both enzymes were determined for free and reconstituted enzymes. It was found that for both enzymes the activation energies were lower for the reconstituted systems than for the free enzymes.
D000818:Animals; D001665:Binding Sites; D002308:Cardiolipins / Q000494:pharmacology; D002850:Chromatography, Gel; D005294:Ferrochelatase / Q000378:metabolism*; D005420:Flavoproteins; D005614:Freeze Fracturing; D006418:Heme / Q000096:biosynthesis*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008190:Lyases / Q000378:metabolism*; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010713:Phosphatidylcholines / Q000502:physiology; D010743:Phospholipids / Q000502:physiology*; D050768:Protoporphyrinogen Oxidase; D013025:Glycine max / Q000032:analysis; D013696:Temperature; D013816:Thermodynamics
D002308:Cardiolipins; D005420:Flavoproteins; D008081:Liposomes; D024101:Mitochondrial Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; D006418:Heme; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493979:Ppox protein, mouse; D050768:Protoporphyrinogen Oxidase; D008190:Lyases; D005294:Ferrochelatase