Detecting S-adenosyl-L-methionine-induced conformational change of a histone methyltransferase using a homogeneous time-resolved fluorescence-based binding assay.
Anal Biochem
; 423(1): 171-7, 2012 Apr 01.
Article
em En
| MEDLINE
| ID: mdl-22342622
ABSTRACT
A homogeneous time-resolved fluorescence (HTRF)-based binding assay has been established to measure the binding of the histone methyltransferase (HMT) G9a to its inhibitor CJP702 (a biotin analog of the known peptide-pocket inhibitor, BIX-01294). This assay was used to characterize G9a inhibitors. As expected, the peptide-pocket inhibitors decreased the G9a-CJP702 binding signal in a concentration-dependent manner. In contrast, the S-adenosyl-L-methionine (SAM)-pocket compounds, SAM and sinefungin, significantly increased the G9a-CJP702 binding signal, whereas S-adenosyl-L-homocysteine (SAH) showed minimal effect. Enzyme kinetic studies showed that CJP702 is an uncompetitive inhibitor (vs. SAM) that has a strong preference for the ESAM form of the enzyme. Other data presented suggest that the SAM/sinefungin-induced increase in the HTRF signal is secondary to an increased ESAM or Esinefungin concentration. Thus, the G9a-CJP702 binding assay not only can be used to characterize the peptide-pocket inhibitors but also can detect the subtle conformational differences induced by the binding of different SAM-pocket compounds. To our knowledge, this is the first demonstration of using an uncompetitive inhibitor as a probe to monitor the conformational change induced by compound binding with an HTRF assay.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
S-Adenosil-Homocisteína
/
Cromatografia Líquida de Alta Pressão
/
Histona-Lisina N-Metiltransferase
/
Espectrometria de Massas em Tandem
/
Corantes Fluorescentes
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article