Purification and properties of ferrochelatase from the yeast Saccharomyces cerevisiae. Evidence for a precursor form of the protein.
Ferrochelatase was purified to homogeneity from yeast mitochondrial membranes and found to be a 40-kDa polypeptide with a pI at 6.3. Fatty acids were absolutely necessary to measure the activity in vitro. The Michaelis constants for protoporphyrin IX (9 x 10(-8) M), ferrous iron (1.6 x 10(-7) M), and zinc (9 x 10(-6) M) were determined on purified enzyme preparations in the presence of dithiothreitol. However, the Km for zinc was lower when measured in the absence of dithiothreitol (K-m(Zn2+) = 2.5 x 10(-7) M, Km(protoporphyrin) unchanged). The maximum velocities of the enzyme were 35,000 nmol of heme/h/mg of protein and 27,000 nmol of zinc-protoporphyrin/h/mg of protein. Antibodies against yeast ferrochelatase were raised in rabbits and used in studies on the biogenesis of the enzyme. Ferrochelatase is synthesized as a higher molecular weight precursor (Mr = 44,000) that is very rapidly matured in vivo to the Mr = 40,000 membrane-bound form. This precursor form of ferrochelatase was immunoprecipitated from in vitro translation (in a rabbit reticulocyte lysate system) of total yeast RNAs. The antibodies were used to characterize two yeast mutant strains deficient in ferrochelatase activity as being devoid of immunodetectable protein in vivo and ferrochelatase mRNA in vitro translation product. The N-terminal amino acid sequence of the purified protein has been established and was found to be frayed.
D000595:Amino Acid Sequence; D004229:Dithiothreitol / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D005294:Ferrochelatase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007526:Isoelectric Point; D008190:Lyases / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011524:Protoporphyrins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D015032:Zinc / Q000378:metabolism
D004792:Enzyme Precursors; D005296:Ferrous Compounds; D011524:Protoporphyrins; D012333:RNA, Messenger; C028025:protoporphyrin IX; D008190:Lyases; D005294:Ferrochelatase; D015032:Zinc; D004229:Dithiothreitol