Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants.
Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn(2+) results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.
Franco
R
R
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
Pereira
A S
AS
Tavares
P
P
Mangravita
A
A
Barber
M J
MJ
Moura
I
I
Ferreira
G C
GC
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D005294:Ferrochelatase / Q000235:genetics*; D014644:Genetic Variation; D018698:Glutamic Acid; D005973:Glutamine; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011166:Porphyrins / Q000378:metabolism*; D011524:Protoporphyrins / Q000378:metabolism
D011166:Porphyrins; D011524:Protoporphyrins; D005973:Glutamine; D018698:Glutamic Acid; C028025:protoporphyrin IX; D005294:Ferrochelatase; D000409:Alanine