Bovine ferrochelatase. Kinetic analysis of inhibition by N-methylprotoporphyrin, manganese, and heme.
The terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase EC 4.99.1.1), has been purified to apparent homogeneity from bovine liver mitochondria using a scheme similar to that reported by Taketani and Tokunaga (Taketani, S. and Tokunaga, R. (1981) J. Biol. Chem. 256, 12748-12753) for purification of the enzyme from rat liver. The final yield was 49% with a 2000-fold purification. Ferrochelatase has an apparent molecular weight of approximately 40,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and column chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. The purified enzyme was only slightly stimulated by added lipid and was inhibited by Mn2+, Pb2+, and Hg2+. Bovine ferrochelatase utilized proto-, meso-, and deuteroporphyrin, but not disubstituted porphyrins (2,4-disulfonic and 2,4-bisglycol deuteroporphyrin). N-Methylprotoporphyrin, a toxic by-product of the metabolism of some drugs, was found to inhibit ferrochelatase in a competitive fashion with respect to porphyrin with a Ki of 7 nM and uncompetitive with respect to iron. Manganese inhibits ferrochelatase competitively with respect to iron (Ki = 15 microM) and noncompetitively with respect to the porphyrin substrate. Heme, one of the products, is a noncompetitive inhibitor with respect to iron. These findings lead to a sequential Bi Bi kinetic model for ferrochelatase with iron binding occurring prior to porphyrin binding and heme being released prior to the release of two protons.
D000818:Animals; D002417:Cattle; D005294:Ferrochelatase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D006418:Heme / Q000494:pharmacology*; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors*; D008345:Manganese / Q000494:pharmacology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011166:Porphyrins / Q000494:pharmacology*; D011524:Protoporphyrins / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D011166:Porphyrins; D011524:Protoporphyrins; D006418:Heme; D008345:Manganese; C028842:N-methylprotoporphyrin IX; D008190:Lyases; D005294:Ferrochelatase