Yeast Phenylalanyl‐tRNA Synthetase

Abstract

The stoichiometries of phenylalanyl‐tRNA‐synthetase · tRNA Phe 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 tRNA Phe . The two former techniques allow the isolation of a complex composed of active enzyme (which is only a fraction of total enzyme used) and tRNA; the two last methods take into account the input protein. All the observed stoichiometries are consistent with the existence of two binding sites for tRNA Phe . Under the experimental conditions used (pH 7.0 and 12°C) a dissociation constant of 3 × 10 −8 M was calculated from the fluoresence quenching of tRNA Phe by the enzyme. tRNA Phe protects the enzyme against heat denaturation. A protection constant of 9 × 10 −7 M was found at 55°C, pH 7.0. The presence of phenylalanine and ATP did modify the strength of interaction of the enzyme‐tRNA system. They induce, however, a synergistic effect of the protection afforded by tRNA Phe . The binding of phenylalanine was examined by equilibrium dialysis in presence of tRNA Phe . The stoichiometry obtained indicates two binding sites for phenylalanine. The two sites however do not possess the same affinity: the corresponding K d 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‐tRNA formation. With regard to the site having the highest affinity, a 20‐fold higher K d value was found, in the absence of tRNA Phe , when binding of the amino acid was followed by the phase partition technique.