Herbert Dickerman

Control of one-carbon metabolism in a methionine-B12 auxotroph of Escherichia coli

Abstract The control mechanisms of N 5,10 -methylene-H 4 -folate metabolism and de novo methyl group synthesis were studied in a mutant Escherichia coli K 12 , which is auxotrophic for either l -methionine or vitamin-B 12 . N 5,10 -Methylene-H 4 -folate reductase was found to be repressed at high media concentrations of l -methionine. Growth at high media concentrations of purines led to a partial repression of N 5,10 -methylene-H 4 -folate dehydrogenase. This enzyme was also found to be inhibited in vitro by purine ribonucleotide triphosphates, and the kinetics of the inhibition indicated that they were competitive with triphosphopyridine nucleotide. Tracer studies of the incorporation of 3- 14 C- l -serine into nucleic acid, protein, and phospholipid correlated with the preceding in vivo and in vitro effects.

[150b] N10-formyl tetrahydrofolate:methionyl tRNA(F) transformylase from Esherichia coli B

Publisher Summary Formyl-14C tetrahydrofolate is incubated at 37° with either heterogeneous Escheriehia coli B transfer RNA (tRNA) or a partially purified Escheriehia coli B tRNA (F), as well as buffer, magnesium ions, and enzyme dilution. At the end of the incubation, cold 5% trichloroacetic acid is added, and the precipitate is collected on a cellulose nitrate filter. The filter is washed with the dilute trichloroaeetic acid; after drying, radioactivity on the filter is counted by liquid scintillation. After filtration, the membrane containing the precipitated nucleic acid and protein is transferred to counting vials, and 10 ml of naphthalenedioxane counting fluid is added. The membrane is dissolved in the counting fluid, and the sample is then ready for liquid scintillation counting. The assay is linear with enzyme concentrations used, up to 5 minutes. A unit of transformylase catalyzes the formation of 1 millimicromole of N-formylmethionyl tRNA in 5 minutes. The specific activity of the enzyme is defined as the units of activity per milligram of protein. Purification procedure is described in the chapter. The purified Escheriehia coli B transformylase has only three requirements for the reaction—(1) a formyl donor, (2) a formyl acceptor, and (3) magnesium ions. Nl0-formyl tetrahydrofolate is the actual formyl donor although it is more convenient to store the radioactive donor under acidic conditions as the more air-stable compound, N5,10-methenyl tetrahydrofolate.

[20] Formylation of methionyl-tRNAfMet

Publisher Summary This chapter describes the formylation of methionyl-tRNA fMet . The enzyme that catalyzes the formylation of the amino group of methionine esterified to tRNA fMet is similar to the aminoacyl-tRNA synthetases in that a recognition of a specific tRNA molecule underlies the overall reaction. It differs from that category of enzymes in that it modifies an existent aminoacyl tRNA into a product with different biological properties than other aminoacyl tRNAs. fMet-tRNA fMet is a requirement for the factor-mediated formation of the initiation complex on the 30 S ribosomal subunit and is possibly a part of the steric requirement for the formation of the initial bond in polypeptide synthesis. In E.coli, the only reported acceptor of the formyl group is the amino moiety of methionine esterified to tRNA fMet . N-Formylmethionyl-tRNA is quite stable in the presence of the purified formylase, when a reduced pteroyl compound is omitted. The reverse reaction indicates that the transformylase recognizes fMet-tRNA and Met-tRNA.