Interconversion of Two Forms of Leucyl-tRNA Synthetase

Abstract

Two forms of leucyl-tRNA synthetase were isolated from E. Coli. Both forms, named E_I and E_II, catalyse the formation of leucyladenylate and the ATP-pyrophosphate exchange reaction with identical Km for leucine and ATP. Each form binds specifically tRNAs acceptor of leucine but only E_II is able to transfer the activated aminoacid onto tRNA. Moreover if E_II is highly specific for leucine, E_I catalyses pyrophosphate exchange also in the presence of valine, isoleucine and methionine. The titration of sulfhydryl groups shows that two groups are masked in E_I. One SH group in E_II is rapidly reacting with pMB and is essential to the transfer of leucine to tRNA. E_II with one SH blocked resembles E_I by its catalytic properties but remains specific for leucine and cannot be separated from native E_II. The two forms E_I and E_II are interconvertible in the presence of cell extracts. It was found that two factors are involved in this process; F₁ which is a protease and releases a peptide of 3 000 MW from E_II, thereby converting E_II into E_I. This peptide which is the factor F₂ can reassociate with E_I leading to an enzyme with catalytic and chromatographic properties of E_II. The results indicate that E_II is constituted from two subunit-like fragments which are in E_I associated by low-energy bonds and in the E_II also by two peptide bonds at the ends of peptide F₂. The association of F₂ with the two “subunits” is necessary for full E_II activity. Low-energy bonds are sufficient to keep this association (E_I-F_I) and reconstituted enzyme from E_I and F_l maintains a conformation and properties close to those of native E_II.