Chemical studies on methionyl-tRNA synthetase from Escherichia coli

Abstract

A technique is described which enables the amino acid sequence of small peptides to be determined when only nanomole quantities are available. Both the dimeric and tetrameric forms of methionyl-tRNA synthetase have Ala-Gly-Gly-Thr- as the only amino-terminal sequence, suggesting that the subunits are identical and that conversion of tetramer to dimer does not involve proteolysis. The dimeric form of the enzyme can bind two molecules of l-methionine, of ATP or of both the tRNA Met species, and there is no interaction between the binding sites. In all cases the binding constants are comparable to the kinetically determined Michaelis constants. Four of the five thiol groups in each subunit of the dimer react rapidly with 2,2′-dithiobis-(5-nitrobenzoic acid) or iodoacetic acid, but one reacts only as the subunits dissociate. The fully disulphide-exchanged monomers can be reactivated. In the presence of methionyl-adenylate, only one thiol group reacts and the product is still active and dimeric. By treating methionyl-tRNA with p-nitrophenyl chloroformate, a derivative was prepared which specifically inhibited methionyl-tRNA synthetase by reacting with a lysine residue in the unique sequence Phe-Thr-Tyr-Gln-Lys-Leu-His-Asn.