Kinetic properties of the membrane-bound human liver mitochondrial protoporphyrinogen oxidase.
We studied the kinetic properties of the membrane-bound human liver mitochondrial protoporphyrinogen oxidase. The activity was monitored by direct recording of protoporphyrin fluorescence appearance in the incubation medium without extraction or dilution. The human liver enzyme shows some different catalytic properties than the rat enzyme since its optimum pH was found at 7.2. We also measured the optimum pH on partially purified protoporphyrinogen oxidase from solubilized human mitochondrial membranes. Like the rat enzyme, the human enzyme had a molecular weight of congruent to 32,000 as determined by gel filtration, but its optimum pH was the same as that of the membrane-bound enzyme. The apparent Km for protoporphyrinogen IX of the membrane-bound enzyme was a function of the pH; Km = 0.16 microM at pH 7.2, 0.33 microM at pH 8.0, and 0.55 microM at pH 8.5. Moreover, there was inhibition by excess protoporphyrinogen IX (KI = 5 microM at pH 7.2). The human enzyme was able to catalyze the oxidation of mesoporphyrinogen IX to mesoporphyrin IX. In this case, the Lineweaver-Burk plot of the data showed a biphasic curve with two different apparent Km's for mesoporphyrinogen IX of 0.5 microM (Vmax = 2.40 nmol h-1 mg-1) and 4 microM (Vmax = 5.7 nmol h-1 mg-1). Human liver protoporphyrinogen oxidase was sensitive to inhibition by some metalloporphyrins such as Mn- and Co-protoporphyrin, and to a lesser degree by Cd-, Ni- and Fe-protoporphyrin (heme). Cu-, Mg-, Sn-, and Zn-protoporphyrins were not inhibitors of the activity. A detailed study of the inhibition of protoporphyrinogen oxidase by cobalt-protoporphyrin shows a noncompetitive mechanism of inhibition with respect to protoporphyrinogen IX (KIapp = 0.8 microM).
D002850:Chromatography, Gel; D005420:Flavoproteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008665:Metalloporphyrins / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology*; D024101:Mitochondrial Proteins; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011524:Protoporphyrins / Q000378:metabolism
D005420:Flavoproteins; D008665:Metalloporphyrins; D024101:Mitochondrial Proteins; D011524:Protoporphyrins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493978:PPOX protein, human; D050768:Protoporphyrinogen Oxidase