Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin.
The penultimate step of haem biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, was examined with purified murine hepatic protoporphyrinogen oxidase (EC 1.3.3.4) in detergent solution. The kinetic parameters for the two-substrate (protoporphyrinogen and oxygen) reaction were determined. The limiting Km for protoporphyrinogen when oxygen is saturating is 6.6 microM, whereas the Km for oxygen with saturating concentrations of protoporphyrinogen is 125 microM. The kcat. for the overall reaction is 447 h-1. The ratio of kcat. to the Km for protoporphyrinogen is approx. 20-fold greater than the kcat./Km,O2 ratio. The ratio of protoporphyrin formed to dioxygen consumed is 1:3. Ubiquinone-6, ubiquinone-10 and dicoumarol stimulate protoporphyrinogen oxidase activity at low concentrations (less than 15 microM), whereas coenzyme Q0 and menadione show no activation at these concentrations. Above 30 microM, all five quinones inhibit the enzyme activity. FAD does not significantly affect the activity of the enzyme. Bilirubin, a product of haem catabolism, is shown to be a competitive inhibitor of the penultimate enzyme of the haem-biosynthetic pathway, protoporphyrinogen oxidase, with a calculated Ki of 25 microM. The terminal enzyme of haem-biosynthetic pathway, namely ferrochelatase, is not inhibited by bilirubin at concentrations over double the Ki value for the oxidase. In contrast with other enzymic systems, the toxicity of bilirubin is not reversed by binding to albumin.
D000818:Animals; D001663:Bilirubin / Q000494:pharmacology*; D001728:Dicumarol / Q000494:pharmacology; D005420:Flavoproteins; D006418:Heme / Q000096:biosynthesis; D007700:Kinetics; D051379:Mice; D024101:Mitochondrial Proteins; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010100:Oxygen / Q000378:metabolism; D050768:Protoporphyrinogen Oxidase; D011524:Protoporphyrins / Q000378:metabolism; D014451:Ubiquinone / Q000494:pharmacology; D014812:Vitamin K / Q000494:pharmacology
D005420:Flavoproteins; D024101:Mitochondrial Proteins; D011524:Protoporphyrins; D014812:Vitamin K; D014451:Ubiquinone; D006418:Heme; C011539:protoporphyrinogen; D001728:Dicumarol; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493979:Ppox protein, mouse; D050768:Protoporphyrinogen Oxidase; D001663:Bilirubin; D010100:Oxygen