ROLE OF DIMERIZATION IN YEAST ASPARTYL-TRANSFER RNA-SYNTHETASE AND IMPORTANCE OF THE CLASS-II INVARIANT PROLINE

Cytoplasmic aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) from yeast i s, as are most class II synthetases, an alpha2 dimer. The only invaria nt amino acid in signature motif 1 of this class is Pro-273; this resi due is located at the dimer interface. To understand the role of Pro-2 73 in the conserved dimeric configuration, we tested the effect of a P ro-273 --> Gly (P273G) substitution on the catalytic properties of hom o- and heterodimeric AspRS. Heterodimers of AspRS were produced in viv o by overexpression of their respective subunit variants from plasmid- encoded genes and purified to homogeneity in one HPLC step. The homodi mer containing the P273G shows an 80% inactivation of the enzyme and a n affinity decrease for its cognate tRNA(Asp) of one order of magnitud e. The P273G-mutated subunit recovered wild-type enzymatic properties when associated with a native subunit or a monomer otherwise inactivat ed having an intact dimeric interface domain. These results, which can be explained by the crystal structure of the native enzyme complexed with its substrates, confirm the structural importance of Pro-273 for dimerization and clearly establish the functional interdependence of t he AspRS subunits. More generally, the dimeric conformation may be a s tructural prerequisite for the activity of mononucleotide binding site s constructed from antiparallel beta strands.