ARGINYL-TRANSFER RNA-SYNTHETASE FROM ESCHERICHIA-COLI-K12 - PURIFICATION, PROPERTIES, AND SEQUENCE OF SUBSTRATE ADDITION

Arginyl-tRNA synthetase from Escherichia coli K12 has been purified more than 1000-fold with a recovery of 17%. The enzyme consists of a single polypeptide chain of about 60000 molecular weight and has only one cysteine residue which is essential for enzymatic activity. Transfer ribonucleic acid completely protects the enzyme against inactivation by p-hydroxymercuribenzoate. The enzyme catalyzes the esterification of 5000 nmol of arginine to transfer ribonucleic acid in 1 min/mg of protein at 37 °C and pH 7.4. One mole of ATP is consumed for each mole of arginyl-tRNA formed. The sequence of substrate binding has been 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 dissociation constants of the different enzyme-substrate complexes and of the complexes with the dead-end inhibitors homoarginine and 8-azido-ATP have been calculated on this basis. Binding of ATP to the enzyme is influenced by tRNA and vice versa. © 1979, American Chemical Society. All rights reserved.