Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
32
|
| pubmed:dateCreated |
1991-12-23
|
| pubmed:abstractText |
The 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form His-(X)4-His-(X)4-His-(X)17-His-(X)8-His which represents a potential binding site for non heme iron to the protein. The importance of selected amino acids within this His cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems. After single mutation of each of the 5 His residues at positions 363, 368, 373, 391, and 400 by Ser, Cys, or Lys, measurable levels of 5-lipoxygenase activity could be recovered in Escherichia coli only for the Ser363 and Cys363 mutants, with most amino acid substitutions causing a decrease in the levels of expression of the soluble protein. In contrast, 25-80% of soluble 5-lipoxygenase activity was recovered after the replacement of several of the hydrophobic amino acids in this region: Tyr384 by Ser or Phe; Phe394 by Trp and Val375 by Ala. Met436 could be replaced by Leu with little effect on 5-lipoxygenase activity or turnover inactivation half-time. High levels of mutant 5-lipoxygenases containing a Ser residue instead of His at each of the five positions were also expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus. The specific activity (58-75% of control) and the reaction time course of the Ser363, Ser391, and Ser400 mutants were comparable with that of native 5-lipoxygenase whereas inactive proteins were obtained for the Ser368 and Ser373 mutants. These results show that His368 and His373 residues are important for 5-lipoxygenase activity and that the other conserved His363, His391, His400, and Met436 residues are not crucial for the catalytic cycle or for the mechanism of self-inactivation of 5-lipoxygenase.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Arachidonate 5-Lipoxygenase,
http://linkedlifedata.com/resource/pubmed/chemical/Histidine,
http://linkedlifedata.com/resource/pubmed/chemical/Lipoxygenase,
http://linkedlifedata.com/resource/pubmed/chemical/Methionine,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Nov
|
| pubmed:issn |
0021-9258
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
15
|
| pubmed:volume |
266
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
22057-62
|
| pubmed:dateRevised |
2010-11-18
|
| pubmed:meshHeading |
pubmed-meshheading:1939225-Amino Acid Sequence,
pubmed-meshheading:1939225-Animals,
pubmed-meshheading:1939225-Arachidonate 5-Lipoxygenase,
pubmed-meshheading:1939225-Baculoviridae,
pubmed-meshheading:1939225-Biological Evolution,
pubmed-meshheading:1939225-Cell Line,
pubmed-meshheading:1939225-Cloning, Molecular,
pubmed-meshheading:1939225-Genetic Vectors,
pubmed-meshheading:1939225-Histidine,
pubmed-meshheading:1939225-Humans,
pubmed-meshheading:1939225-Immunoblotting,
pubmed-meshheading:1939225-Kinetics,
pubmed-meshheading:1939225-Lipoxygenase,
pubmed-meshheading:1939225-Methionine,
pubmed-meshheading:1939225-Molecular Sequence Data,
pubmed-meshheading:1939225-Moths,
pubmed-meshheading:1939225-Mutagenesis, Site-Directed,
pubmed-meshheading:1939225-Recombinant Proteins,
pubmed-meshheading:1939225-Sequence Homology, Nucleic Acid,
pubmed-meshheading:1939225-Transfection
|
| pubmed:year |
1991
|
| pubmed:articleTitle |
Evaluation of the role of conserved His and Met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase.
|
| pubmed:affiliation |
Department of Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada.
|
| pubmed:publicationType |
Journal Article,
Comparative Study
|