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Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenylalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The α and β subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl-tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant a subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation. [ABSTRACT FROM AUTHOR]

This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of non-covalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 μM. The enzyme binds FAD by a simple second-order process with Kd 0.67 μM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme. [ABSTRACT FROM AUTHOR]

We have studied the ribonucleosidediphosphate reductase of a deoxyuridine-requiring mutant LD195 of Escherichia coli K. This enzyme was purified 50 fold from the mutant and a control strain. In all steps of the purification the mutant strain yielded normal activity of the Bi subunit of the enzyme but approximately 10 % of the wild-type activity of the B2 subunit The activity is similarly reduced for all three substrates tested CDP, UDP, and GDP. The mutation appears to affect the physical structure of the B2 subunit, since addition of high concentrations of sodium acetate can restore enzymatic activity to the partially purified B2 subunit from the mutant strain. In contrast, the same concentrations strongly inhibit the wild- type B2 subunit. Although the mutant has a requirement of deoxyuridine at 42 °C but not at 30 °C (as shown in an accompanying paper), mutant protein B2 is not more thermo-labile than wild-type protein B2 This requirement therefore probably reflects an increased demand of deoxyribonucleotides at higher temperatures. Hydroxyurea inhibits mutant and wild-type ribonucleosidediphosphate reductase to the same extent in vitro. In spite of this, hydroxyurea inhibits growth of the mutant strain at a 20 fold lower concentration than that required for comparable inhibition of growth of the parent. This is explained by the greatly decreased protein B2 activity of the mutant. [ABSTRACT FROM AUTHOR]