Linked Life Data

10079084

Source:http://linkedlifedata.com/resource/pubmed/id/10079084

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
1999-4-15
pubmed:abstractText
Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Triphosphatases, http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Triphosphate, http://linkedlifedata.com/resource/pubmed/chemical/Arginine, http://linkedlifedata.com/resource/pubmed/chemical/Asparagine, http://linkedlifedata.com/resource/pubmed/chemical/Biotin, http://linkedlifedata.com/resource/pubmed/chemical/Carbon-Nitrogen Ligases, http://linkedlifedata.com/resource/pubmed/chemical/Glutamic Acid, http://linkedlifedata.com/resource/pubmed/chemical/Histidine, http://linkedlifedata.com/resource/pubmed/chemical/Magnesium, http://linkedlifedata.com/resource/pubmed/chemical/N1'-carboxybiotin, http://linkedlifedata.com/resource/pubmed/chemical/Peptides, http://linkedlifedata.com/resource/pubmed/chemical/biotin carboxylase, http://linkedlifedata.com/resource/pubmed/chemical/polyhistidine
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
16
pubmed:volume
38
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3393-400
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin.
pubmed:affiliation
Division of Biochemistry and Molecular Biology, Louisiana State University, Baton Rouge 70803-1806, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't