The Asparagine Synthetase of Escherichia coli

Abstract

Abstract Synthesis of l-asparagine was detected in extracts of Escherichia coli K-12 only under conditions where either the amount of asparaginase II was greatly lowered or else its activity was inhibited by 5-diazo-4-oxo-l-norvaline (DONV). Asparagine synthetase, the enzyme responsible for the formation of the amino acid, was lacking in extracts of an auxotrophic mutant which required asparagine. Asparagine repressed the formation of the synthetase, but at a rather high concentration, possibly because asparagine is not readily concentrated by the bacterial cell. A genetic locus for the production of asparagine synthetase (for which we propose the name asn) was found to be between the 73rd and 74th min of the E. coli chromosome by conjugation and transduction experiments. Asparagine synthetase was purified 370-fold from a mutant of E. coli impaired in its ability to form asparaginase II. In order to diminish the amount of residual asparaginase further, extracts were made of this mutant after it was osmotically shocked. The synthetase had a molecular weight of about 80,000; it was stabilized by 2-mercaptoethanol and by 10% glycerol. The synthesis of asparagine required aspartate, ATP-Mg2+, and ammonia, and resulted in the stoichiometric production of pyrophosphate and AMP. Glutamine did not serve as a source of amide nitrogen. The pH optimum was 8.4. The enzyme preparation, shown to be free of aspartyl transfer RNA synthetase, in the absence of added ammonia also catalyzed an aspartate-dependent pyrophosphate exchange with ATP, which was inhibited by asparagine. β-Aspartyl hydroxamate was formed in incubations with hydroxylamine.