Linked Life Data

19349157

Source:http://linkedlifedata.com/resource/pubmed/id/19349157

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
9
pubmed:dateCreated
2009-4-20
pubmed:abstractText
This study describes the development of a universal phosphorylated peptide-binding protein designed to simultaneously detect serine, threonine and tyrosine kinases. The Escherichia coli alkaline phosphatase (EAP) is a well-defined nonspecific phosphated monoesterase and Ser-, Thr- or Tyr-phosphorylated peptides served as substrates for EAP in preliminary experiments. Based on the known catalytic mechanism of EAP, the recombinant site-directed mutant EAP-S102L was generated, whose catalytic activity was blocked, but its binding ability was preserved. For EAP-S102L the catalytic rate constant, k(cat), was reduced by a factor of 1000, while the Michaelis-Menten constant, K(m), remained almost unchanged. Crystallographic analysis of the EAP-S102L/phophorylated peptide complex revealed that EAP-S102L could bind the phosphate group of the phosphorylated peptide but lacked nucleophilic attack potential which was essential for the catalytic ability of EAP. Finally, by combining the fluorescence-labeled EAP-S102L with non-phophorylated peptide chips, kinases could be detected from tumor cell samples. The recombinant EAP-S102L construct is perhaps the first functional binding protein derived from a native enzyme, illustrating how one single mutation tremendously alters protein function.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
1873-4235
pubmed:author
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
24
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2871-7
pubmed:meshHeading
pubmed:year
2009
pubmed:articleTitle
Development of a universal phosphorylated peptide-binding protein for simultaneous assay of kinases.
pubmed:affiliation
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Evaluation Studies