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Identification of glutamic acid 479 as the gluzincin coordinator of zinc in FtsH (HflB).
- Source :
-
Biochemistry [Biochemistry] 2002 Feb 12; Vol. 41 (6), pp. 1861-8. - Publication Year :
- 2002
-
Abstract
- Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent metalloprotease. Its cytoplasmic domain contains a zinc-binding motif, H(417)EXXH, whose histidine residues have been shown to be functionally important. Although they are believed to be involved directly in zinc coordination, nothing is known about the third zinc ligand of this protease. Sequence alignment indicates that glutamic acid residues are conserved among the FtsH homologues at positions corresponding to Glu(479) and Glu(585) of E. coli FtsH. We replaced each of them by Gln, Asp, Lys, or Val. Mutations at position 479 compromised the proteolytic functions of FtsH in vivo. In vitro proteolytic activities of the E479Q, E479V, and E479D mutant enzymes were much lower than that of the wild-type protein and were significantly stimulated by a high concentration of zinc ion. These mutant proteins retained the wild-type levels of ATPase activities, and their trypsin susceptibilities as well as CD spectra were essentially indistinguishable from those of the wild-type protein, indicating that the mutations did not cause gross conformational changes in FtsH. They exhibited reduced zinc contents upon purification. From these results, we conclude that Glu(479) is a zinc-coordinating residue.
- Subjects :
- ATP-Dependent Proteases
Amino Acid Sequence
Amino Acid Substitution
Bacterial Proteins genetics
Bacterial Proteins metabolism
Base Sequence
Circular Dichroism
DNA, Bacterial genetics
Escherichia coli enzymology
Escherichia coli genetics
Escherichia coli Proteins
Glutamic Acid chemistry
Kinetics
Membrane Proteins genetics
Membrane Proteins metabolism
Metalloendopeptidases genetics
Metalloendopeptidases metabolism
Metals analysis
Mutagenesis, Site-Directed
Sequence Homology, Amino Acid
Zinc analysis
Zinc chemistry
Bacterial Proteins chemistry
Membrane Proteins chemistry
Metalloendopeptidases chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 41
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 11827531
- Full Text :
- https://doi.org/10.1021/bi015748o