The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5'-p-fluorosulfonylbenzoyladenosine.
Int J Biochem Cell Biol
; 33(3): 249-59, 2001 Mar.
Article
em En
| MEDLINE
| ID: mdl-11311856
ABSTRACT
The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hundred micromolar FSBA inactivated PI4K230 specifically with apparently first-order kinetics and resulted in 50% loss of enzyme activity in 36--130 min. The specificity of the reaction with FSBA was demonstrated by the lack of inactivation with 5'-p-fluorosulfonylbenzoyl chloride and by protection with ATP and ATP analogues against inactivation. Most ATP analogues competed with FSBA inactivation in order of their increasing hydrophobicity, parallel to their inhibitory potency in activity measurements. The specific binding of FSBA to PI4K230 was demonstrated also by Western-blot experiments. These results suggest that FSBA-reactive group(s) involved in the enzyme activity are located near to the ATP-binding site in a hydrophobic region of native PI4K230. Experiments with site-directed mutagenesis indicate that the conserved Lys-1792 plays essential role in the enzyme activity and serves as one target of affinity labelling by FSBA. Prevention of both Lys-1792-directed and Lys-1792-independent binding of FSBA by Cibacron Blue 3GA suggest that these sites are located spatially close to each other.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Encéfalo
/
Adenosina
/
Trifosfato de Adenosina
/
1-Fosfatidilinositol 4-Quinase
Limite:
Animals
Idioma:
En
Revista:
Int J Biochem Cell Biol
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2001
Tipo de documento:
Article
País de afiliação:
Hungria