Arginyl-tRNA synthetase from Escherichia coli K12. Purification, properties, and sequence of substrate addition

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Abstract
Arginyl-tRNA synthetase from E. coli K12 was purified more than 1000-fold with a recovery of 17%. The enzyme consists of a single polypeptide chain of about 60,000 MW and has only 1 cysteine residue which is essential for enzymatic activity. tRNA completely protects the enzyme against inactivation by p-hydroxymercuribenzoate. The enzyme catalyzes the esterification of 5000 nmol of arginine to tRNA in 1 min/mg of protein at 37.degree. C and pH 7.4. One mol of ATP is consumed for each mole of arginyl-tRNA formed. The sequence of substrate binding was investigated by using initial velocity experiments and dead-end and product inhibition studies. The kinetic patterns are consistent with a random addition of substrates with all steps in rapid equilibrium except for the interconversion of the central quaternary complexes. The Kd of the different enzyme-substrate complexes and of the complexes with the dead-end inhibitors homoarginine and 8-azido-ATP were calculated on this basis. Binding of ATP to the enzyme is influenced by tRNA and vice versa.