Engineering of the myosin-ibeta nucleotide-binding pocket to create selective sensitivity to N(6)-modified ADP analogs.
J Biol Chem
; 274(44): 31373-81, 1999 Oct 29.
Article
en En
| MEDLINE
| ID: mdl-10531338
ABSTRACT
Distinguishing the cellular functions carried out by enzymes of highly similar structure would be simplified by the availability of isozyme-selective inhibitors. To determine roles played by individual members of the large myosin superfamily, we designed a mutation in myosin's nucleotide-binding pocket that permits binding of adenine nucleotides modified with bulky N(6) substituents. Introduction of this mutation, Y61G in rat myosin-Ibeta, did not alter the enzyme's affinity for ATP or actin and actually increased its ATPase activity and actin-translocation rate. We also synthesized several N(6)-modified ADP analogs that should bind to and inhibit mutant, but not wild-type, myosin molecules. Several of these N(6)-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-Ibeta; in doing so, these analogs locked Y61G myosin-Ibeta tightly to actin. N(6)-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Ibeta. Furthermore, a small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-Ibeta. Introduction of Y61G myosin-Ibeta molecules into a cell should permit selective inhibition by N(6)-modified ADP analogs of cellular processes dependent on myosin-Ibeta.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Adenosina Difosfato
/
Miosinas
/
Adenosina Trifosfatasas
/
Isoenzimas
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
J Biol Chem
Año:
1999
Tipo del documento:
Article
País de afiliación:
Estados Unidos