Active site stoichiometry of L-phenylalanine: tRNA ligase from Escherichia coli K(-10).
The existence of two active siter per molecule of L-phenylalanine:tRNA ligase from Escherichia coli K(-10) has been demonstrated by isolation of the E-aminoacyl adenylate and tel filtration and the nitrocellulose filter assay at pH 5.0 revealed the same stoichiometry for the E-tRNAPhe comples as protection against degradation by snake venom phosphodiesterase and equilibrium gel filtration at pH 7.5. Using a fluorescence titration technique, it was found that the dissociation constant for ligase-tRNAPhe complex is decreased 20-fold when the hydrogen ion concentration is changed from pH 6.0 to pH 5.0. The existence of two active sites binding the aminoacyl adenylate intermediate was demonstrated by gel filtration and retention on DEAE-cellulose filters. "Burst" experiments indicated that two sites were involved in a rapid ATP consumption at conditions of catalytic amino acid activation. Furthermore, it was observed that the activated amino acid could be transferred from both sites to cognate tRNA.
D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases; D011485:Protein Binding; D012343:RNA, Transfer; D013050:Spectrometry, Fluorescence
D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D010727:Phosphoric Diester Hydrolases; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase