Section 45
Chapter 44,995

Yeast phenylalanyl-tRNA synthetase. Molecular weight and interaction with tRNA Phe and phenylalanine

Fasiolo, F.; Remy, P.; Pouyet, J.; Ebel, J.P.

European Journal of Biochemistry 50(1): 227-236


ISSN/ISBN: 0014-2956
PMID: 4615901
DOI: 10.1111/j.1432-1033.1974.tb03891.x
Accession: 044994063

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The stoichiometries of phenylalanyl-t RNA-synthetase · t RNAPhe complexes were found close to 2, as determined by the following methods: zone centrifugation in sucrose gradient, Sephadex filtration, sedimentation boundary technique and fluorescence quenching of Y base of t RNAPhe. The two former techniques allow the isolation of a complex composed of active enzyme (which is only a fraction of total enzyme used) and t RNA; the two last methods take into account the input protein. All the observed stoichiometries are consistent with the existence of two binding sites for t RNAPhe. Under the experimental conditions used (p H 7.0 and 12°C) a dissociation constant of 3 × 10−8 M was calculated from the fluoresence quenching of t RNAPhe by the enzyme. t RNAPhe protects the enzyme against heat denaturation. A protection constant of 9 × 10−7 M was found at 55°C, p H 7.0. The presence of phenylalanine and Atp did modify the strength of interaction of the enzyme-t Rna system. They induce, however, a synergistic effect of the protection afforded by t RNAPhe. The binding of phenylalanine was examined by equilibrium dialysis in presence of t RNAPhe. The stoichiometry obtained indicates two binding sites for phenylalanine. The two sites however do not possess the same affinity: the corresponding Kd values are 6 and 30μM. The presence of more than one active site is also manifested by the curvature of the double-reciprocal plots of v versus [S] for phenylalanine, when measuring the initial rates of phenylalanyl-t Rna formation. With regard to the site having the highest affinity, a 20-fold higher Kd value was found, in the absence of t RNAPhe, when binding of the amino acid was followed by the phase partition technique.

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