Use of mutant enzymes to demonstrate the presence of two active sites on phenylalanyl‐tRNA synthetase from Escherichia coli

Abstract

Phenylalanyl-tRNA synthetase (PRS) from Escherichia coli is a tetramer with a molecular weight of 267 000 and the apparent subunit composition a& where (Y and 0 have molecular weights of 39 000 and 94 000, respectively [ 1,2] . Consistent with the quarternary symmetry of this enzyme is the recent finding, that there are two binding sites for the substrates phenylalanine, ATP and tRNAPhe [3,4]. Using physico-chemical methods Baytmann et al. [3] have provided evidence that the two :nolecules of substrates bound to PRS can be converted by the enzyme into two molecules of reaction products which indicates that two active sites are functioning on the enzyme. In this paper we demonstrate that biochemical and genetic experiments with the help of mutant enzymes are leading to the same conclusion. The basis for this study was provided by the findings (i) that the (Y subunits in the PRS seem to carry the phenylalanine binding sites [5] and (ii) that two different types of PRS mutants are available which have an altered cw subunit [5,6] clearly distinguished by their unique substrate specificities. The principle of our experimental approach was to construct a hybrid mutant PRS which should contain these two distinct (Y subunits in one molecule. The question of whether or not two active sites are present can then be answered in the following manner: if the. unique substrate specificity of only one of the two mutant (II subunits is measurable, the presence of only one active site seems to be indicated; if, however, the two distinct enzymatic activities of both OL subunits in the hybrid enzyme are expressed, the presence