Modification of Methionyl‐tRNA Synthetase by Proteolytic Cleavage and Properties of the Trypsin‐Modified Enzyme

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Abstract
Earlier studies have shown that native methionyl‐tRNA synthetase from Escherichia coli K12 is composed of four, probably identical, subunits having a molecular weight of 43 000 each.Incubation of the purified enzyme with trypsin results in irreversible conversion into an enzymatically active modified form which, by a number of criteria, including molecular size, catalytic and antigenic properties, electrophoretic and chromatographic behaviour, appears to be undistinguishable from the altered methionyl‐tRNA synthetase previously observed after incubation at 37°C of a crude extract. Furthermore, a similar modification of methionyl‐tRNA synthetase resulted upon incubation of the purified enzyme with several other proteolytic enzymes, including papain, subtilisin and pronase.The structural and catalytic properties of the homogenous trypsin‐modified methionyl‐tRNA synthetase were examined in detail. It has a molecular weight of 64000 established by equilibrium ultracentrifugation, and dissociates in the presence of 8 M urea to yield two, apparently identical subunits of molecular weight 32000. Furthermore, it is shown that proteolysis of the native enzyme by trypsin is accompanied by release of enzymatically inactive fragments corresponding to approximately 20% of the original protein. The simplest interpretation of these results is that proteolysis selectively removes a portion of each original subunit, corresponding to a quarter of its size, with consequent dissociation of the original tetramer into dimers, each now composed of modified subunits.No significant differences in the Michaelis constants for any of the substrates or inhibitors tested were found between native and modified enzyme. Nor was conversion accompanied by any detectable change in the specificity towards the amino acid. The molecular activity (expressed as moles of substrate converted/mole subunit) of the homogenous trypsin‐modified enzyme was found to be 20% lower than that of native methionyl‐tRNA synthetase, for both reactions catalyzed by the enzyme. An interesting difference between the two forms of the enzyme appeared in their magnesium ion requirement for the aminoacylation of tRN As. Thus, under otherwise identical assay conditions, the trypsin‐modified enzyme required significantly less magnesium ions for the aminoacylation of both tRNAMetf and tRNAMetm at optimal rates. Another remarkable feature of the trypsin‐modified enzyme is its enhanced stability against heat inactivation, compared to the native enzyme.A structural model is proposed for methionyl‐tRNA synthetase which attempts to account for its great susceptibility to limited proteolysis, and for the maintenance of its catalytic properties despite the resulting radical changes in its molecular structure.