Purification and properties of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli

Abstract

Methionyl-t-RNA synthetase (where t-RNA denotes ''soluble'' or transfer RNA) was purified to apparent homogeneity from a ribonuclease I-free strain of E. coli. Polyacrylamide-gel electrophoresis of the final product revealed a single band. The purified enzyme catalyzes the exchange of 450 n moles of pyrophosphate into ATP/mg in 15 min. at 37[degree]. Methionyl-t-RNA synthetase is specific for the L-isomer of methionine but appears to catalyze the methionylation of 2 distinct species of t-RNA, both of which are specific for methionine, but only 1 of which may be subsequently formylated. The Michaelis constant for L-methionine is 2 x 10-4 M in the ATP-PPi exchange assay and 2 x 10-4 M for the acylation of t-RNA. Gel filtration of both crude and highly purified preparations of methionyl-t-RNA synthetase on Sephadex G-200 indicates that the active species of enzyme has a molecular weight of about 190,000. The amino-acid composition of the enzyme is similar to those reported for the iso-leucine and tyrosine enzymes from E. coli.