Lysyl-tRNA synthetase from Bacillus stearothermophilus. Purification, and fluorometric and kinetic analysis of the binding of substrates, L-lysine and ATP.
J Biochem
; 119(4): 680-9, 1996 Apr.
Article
en En
| MEDLINE
| ID: mdl-8743569
ABSTRACT
Lysyl-tRNA synthetase [L-lysinetRNA(Lys)ligase (AMP forming); EC 6.1.1.6] was purified from Bacillus stearothermophilus NCA1503 approximately 1,100-fold to homogeneity in PAGE. The enzyme is a homodimer of M(r) 57,700 x 2. The molar absorption coefficient, epsilon, at 280 nm is 71,600 M-1.cm-1 at pH8.0. Enzyme activity in the tRNA aminoacylation reaction and the ATP-PPi exchange reaction increases up to 50 degrees C at pH 8.0, but is lost completely at 70 degrees C. The pH-optima of the two reactions are 8.3 at 37 degrees C. In the tRNA aminoacylation reaction, the Km values for L-lysine and ATP are 16.4 and 23.2 muM, respectively, and in the ATP-PPi exchange reaction, the Km values for L-lysine and ATP are 23.6 and 65.1 muM, respectively at 37 degrees C, pH 8.0. Interaction of either L-lysine or ATP with the enzyme has been investigated by using as a probe the ligand-induced quenching of protein fluorescence and by equilibrium dialysis. These static analyses, as well as the kinetic analysis of the L-lysine dependent ATP-PPi exchange reaction indicate that the binding mode of L-lysine and ATP to the enzyme is sequential ordered (L-lysine first). The interaction of lysine analogues with the enzyme has also been investigated.
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Banco de datos:
MEDLINE
Asunto principal:
Geobacillus stearothermophilus
/
Adenosina Trifosfato
/
Lisina
/
Lisina-ARNt Ligasa
Idioma:
En
Revista:
J Biochem
Año:
1996
Tipo del documento:
Article