Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16.
J Protein Chem
; 9(4): 445-51, 1990 Aug.
Article
en En
| MEDLINE
| ID: mdl-2177336
ABSTRACT
The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme, S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparent Kd of 3-4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [14C]reagent per mole of enzyme subunit. Amino acid analysis of the [14C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.
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Banco de datos:
MEDLINE
Asunto principal:
Fosfotransferasas
/
Plantas
/
Marcadores de Afinidad
/
Fosfotransferasas (Aceptor de Grupo Alcohol)
/
Cisteína
/
Etanolaminas
Idioma:
En
Revista:
J Protein Chem
Año:
1990
Tipo del documento:
Article