Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.
Subject | Predicate | Object | Context |
---|---|---|---|
http://purl.uniprot.org/cit... | rdf:type | uniprot:Journal_Citation | lld:uniprot |
http://purl.uniprot.org/cit... | rdfs:comment | Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed. | lld:uniprot |
http://purl.uniprot.org/cit... | skos:exactMatch | http://purl.uniprot.org/pub... | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:name | Biochemistry | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Shimizu T. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Minami M. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Kurokawa H. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Sagami I. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Yamauchi S. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Igarashi J. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Miksanova M. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:date | 2006 | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:pages | 9894-9905 | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:title | Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:volume | 45 | lld:uniprot |
http://purl.uniprot.org/cit... | dc-term:identifier | doi:10.1021/bi060556k | lld:uniprot |
uniprot-protein:Q9Z2R9 | uniprot:citation | http://purl.uniprot.org/cit... | lld:uniprot |
http://linkedlifedata.com/r... | uniprot:source | http://purl.uniprot.org/cit... | lld:uniprot |
http://linkedlifedata.com/r... | rdf:object | http://purl.uniprot.org/cit... | lld:uniprot |