Purification and Properties of an N‐Formylmethionyl‐tRNA Hydrolase

The isolation and properties of a novel N‐formylmethionyl‐tRNA hydrolase (hydrolase II) from Escherichia coli are described. This enzyme is difficult to detect in crude extracts; purification, however, unmasks the activity. Sedimentation and gel filtration parameters of this enzyme differ from those of the previously described peptidyl‐tRNA hydrolase (hydrolase I), and preparations can be obtained where the two activities are free of each other. A mutant of hydrolase I has wild‐type levels of hydrolase II. These data indicate that hydrolase II is a different enzyme, or an altogether different form of hydrolase 1. The bulk of the enzymic activity occurs in the ribosome‐free cytoplasm; the remainder is found on intact or dissociated 70‐S ribosomes. Purified preparations of hydrolase II analyzed by two‐dimensional gel electrophoresis contain 2 protein bands. These 2 proteins do not coincide in electrophoretic mobility with any known ribosomal proteins. Analysis after mixing experiments verifies this conclusion. The purified enzyme (hydrolase II) is inhibited by ribosomes bearing bound N‐formylmethionyl‐tRNA. The inhibition is potentiated by sparsomycin and other antibiotics that block specifically peptide‐bond synthesis. The relationship of this enzyme to other hydrolytic activities, including a newly described ribosome‐dependent hydrolase, are discussed.