Analysis of the Amino Acid Binding to the Proline Transfer Ribonucleic Acid Synthetase of Escherichia coli

The prolyl-tRNA synthetase of Escherichia coli has been purified. The enzyme is reversibly inhibited by p-mercuribenzoate when two to four groups are bound per molecule of enzyme. Many analogues of proline were tested as substrates and inhibitors. The requirements for activation and transfer appear to be the same: a secondary l-amino acid with a side chain not much bulkier than the ring of proline. Substitutions of hydroxyl or methyl groups on carbon 3 or carbon 4 cause steric inhibition that minimize or prevent binding, but allo-hydroxyproline and trans-3-methyl proline are substrates at high concentrations. Double bonds in the ring destroy substrate activity but pyrrole is bound as a competitive inhibitor. The carboxyl group is not essential for binding and probably does not form an ionic bond since the amide is bound almost as well. d-Proline is not bound to the enzyme. Several open chain compounds are activated and transferred to tRNApro. The broad specificity of the enzyme suggests that mechanisms used to confer great specificity on other activating enzymes (induced fit) do not apply to the proline enzyme. It is also suggested that open chain analogues might be incorporated into proteins in place of proline.