Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation.

The adenylation of phenylalanine catalyzed by phenylalanyl-tRNA synthetase was investigated in the absence of tRNA, by rapid kinetic measurements using 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS) as a nonspecific fluorescent reporter group. It is shown that each protomer of the enzyme is able to catalyze independently the adenylation of phenylalanine by ATP, as well as the reversion by pyrophosphate, at least in the absence of tRNA. The kinetic rate constants of synthesis and pyrophosphorolysis are respectively found equal to 100 +/- 20 s-1 and 150 +/- 50 s-1. The symmetric behavior of the enzyme is consistent with a symmetric binding of 2 mol of phenylalanine to the enzyme as shown by equilibrium dialysis experiments. The affinity of phenylalanyladenylate for the enzyme could be characterized by an equilibrium constant of 0.2 x 10(9) M-1.