Structure-function studies of yeast ferrochelatase. Identification and functional analysis of amino acid substitutions that increase Vmax and the KM for both substrates.
The molecular basis of the ferrochelatase defects was investigated in two "protoporphyric" and partially heme-deficient yeast mutants. Ferrochelatase, a mitochondrial inner membrane-bound enzyme, catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. The mutant cells made normal amounts of normal-sized ferrochelatase, as detected by immunoblotting. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant strains genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. Substitution of the conserved Ser-169 by Phe caused a 10-fold increase in Vmax and a 45- and 35-fold increase in the KM for protoporphyrin and metal, respectively. Replacement of Ser-174 by Pro produced the same effects, but to a lesser degree. There was a good correlation between the ferrochelatase defects measured in vitro and the heme synthesis deficiencies estimated in vivo. The decreased in vivo heme synthesis is probably due to the lower affinity of the mutant enzymes for iron. We propose that the region identified by the two close mutations contributes to the binding domains of metal and protoporphyrin.
Abbas
A
A
Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, Université Paris 7, France.
Labbe-Bois
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D004271:DNA, Fungal; D005294:Ferrochelatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011524:Protoporphyrins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
D004271:DNA, Fungal; D011524:Protoporphyrins; D007501:Iron; D005294:Ferrochelatase