Mammalian ferrochelatase. Expression and characterization of normal and two human protoporphyric ferrochelatases.
Ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Herein we report the expression, purification, and characterization of the mature processed form of human and mouse ferrochelatase in Escherichia coli JM109. Metal analysis of the recombinant normal human ferrochelatase reveals that there are approximately 2 iron atoms/molecule of enzyme. This, along with the presence of spectral absorbance near 320 nm, is strongly suggestive that recombinant mammalian ferrochelatase as expressed in E. coli may contain an iron sulfur cluster. Two human protoporphyric ferrochelatases, F417S and M267I, were also expressed and characterized. The M267I mutant possesses the same Km and Vmax as the normal enzyme but exhibits increased thermolability when compared with normal human ferrochelatase. The F417S mutant has less than 2% of the normal activity. Since the Phe-->Ser substitution in this mutation is both chemically and structurally significant, three single amino acid substitutions (Lys, Tyr, and Trp) were engineered and characterized. None of these resulted in a protein with wild type activity. Additionally the carboxyl-terminal 10-amino acid segment, which contains Phe-417, from the yeast sequence was substituted, but this construct had no activity. Elimination of the carboxyl-terminal 30 amino acid residues (which include Phe-417) results in a protein the same length as the bacterial ferrochelatases, but it is an inactive enzyme.
Dailey
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HA
Department of Microbiology, University of Georgia, Athens 30602-2605.
Sellers
V M
VM
Dailey
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TA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005294:Ferrochelatase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011166:Porphyrins / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011166:Porphyrins; D011994:Recombinant Proteins; D005294:Ferrochelatase