Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae.
Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. It is encoded by the HEM15 gene in the yeast Saccharomyces cerevisiae. Five hem15 mutants causing defective heme synthesis and protoporphyrin accumulation were investigated. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. The substitution L62F caused a five-fold increase in Vmax and 32-fold and four-fold increases in the KM's for protoporphyrin and metal. Replacements of the conserved G47 by S and S102 by F increased the KM for protoporphyrin 10-fold without affecting the affinity for metal or enzyme activity. Two amino acid changes, L205P and P221L, produced a thermosensitive phenotype. In vivo heme synthesis, the amount of immunodetected protein, and ferrochelatase activity measured in vitro were more affected in cells grown at 37 degrees C than at 30 degrees C. The effects of these mutations on the enzyme function are discussed with respects to ferrochelatase structure and mechanism of action.
Góra
M
M
Polish Academy of Sciences, Institute of Biochemistry and Biophysics, Warsaw, Poland.
Chaciñska
A
A
Rytka
J
J
Labbe-Bois
R
R
D000483:Alleles; D000595:Amino Acid Sequence; D004271:DNA, Fungal / Q000737:chemistry; D005294:Ferrochelatase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005838:Genotype; D006418:Heme / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013054:Spectrophotometry, Atomic
D004271:DNA, Fungal; D006418:Heme; D005294:Ferrochelatase