EXAMINATION OF FERROCHELATASE MUTATIONS THAT CAUSE ERYTHROPOIETIC PROTOPORPHYRIA

Ferrochelatase (E.C. 4.99.1.1), the enzyme that catalyzes the terminal step in the heme biosynthetic pathway, is the site of defect in the h uman inherited disease erythropoietic protoporphyria (EPP). Previously it has been demonstrated that patients with EPP may have missense mut ations leading to amino acid substitutions, early chain termination, o r exon deletions. While it has been clearly demonstrated that two miss ense mutations result in lowered enzyme activity, it has never been sh own what effect specific exon deletions may have. In the current work, recombinant human ferrochelatase has been engineered to have individu al exon deletions corresponding to exons 3 through 11. When expressed in Escherichia coli, none of these possesses significant enzyme activi ty and all lack the [2Fe-2S] cluster, One of the human missense mutati ons, F417S, and a series of amino acid replacements at this site (ie, F417W, F417Y, and F417L) were examined. With the exception of F417L, a ll lacked enzyme activity and did not contain the [2Fe-2S] cluster in vivo or as isolated in vitro. (C) 1998 by The American Society of Hema tology.