The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli

Patte, Jean‐Claude; Truffa‐Bachi, Paolo; Cohen, Georges N.

doi:10.1016/0926-6593(66)90003-8

Cited by 122 publications

(61 citation statements)

References 23 publications

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“…The precipitate was dissolved in Buffer B supplemented with 0.5 M KCl and 0.5 M ammonium sulfate (final pH = 7.2), and applied to the column. The enzyme assay was performed as described by Patte et al [7]. The buffer used was 0.01 M Tris-acetate, pH 7.1, containing 0.1 M NaCl, 0.01 M MgClz and 0.01 M &mercaptoethanol (Buffer C).…”

Section: Aspartokinase I-homoserine Dehydrogenase Imentioning

confidence: 99%

Purification of E. coli enzymes by chromatography on amphiphilic gels

1975

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Section: Aspartokinase I-homoserine Dehydrogenase Imentioning

confidence: 99%

Purification of E. coli enzymes by chromatography on amphiphilic gels

1975

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“…Activity was measured by the hydroxamate ferric chloride method [l J modified as in [13]. A modification of this last procedure was also used, with 20 mM potassium phosphate pH 7.2 instead of 200 mM Tris-HC1 pH 8.1.…”

Section: Measurements Of Enzyme Activitymentioning

confidence: 99%

The Role of Lysine and Leucine Binding on the Catalytical and Structural Properties of Aspartokinase III of Escherichia coli K 12

Richaud

Mazat

Felenbok

et al. 1974

European Journal of Biochemistry

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The lysine-sensitive aspartokinase of Escherichia coli K12, a 100 000-molecular weight dimer, possesses four binding sites for L-lysine, its allosteric inhibitor. The lysine binding is cooperative.In the presence of leucine this cooperative effect disappears, that may explain the synergistic inhibition phenomenon observed between these two amino acids. The four sites are non-equivalent two by two, with a K of, respectively, 8 pM and 100 pM. The tetramerization of the protein observed in the presence of lysine appears not to be related to the inhibition of enzyme activity. Lysine or leucine binding leads to transconformational changes evidenced by difference spectra and modification in -SH titration rates. All the results are consistent with the hypothesis that the lysine-sensitive aspartokinase belongs to the class V of allosteric enzymes.

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“…Activity was measured by the hydroxamate ferric chloride method as modified in [13]. Units of enzyme activity are defined as micromoles of aspartohydroxamate formed per minute.…”

Section: Strains Used and Enzyme Purificationmentioning

confidence: 99%

Subunit Structure of Aspartokinase III of Escherichia coli K12

Richaud

Patte

Mazat

et al. 1973

European Journal of Biochemistry

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The lysine-sensitive aspartokinase I11 of Escherichia coli K12 is composed of subunits of 50000 molecular weight. By determination of terminal residues and inspection of tryptic hydrolysis products, the subunits appear identical. The native enzyme is a dimer of 105000 molecular weight in the presence of 0.15 M KC1 in the absence of lysine. Two types of equilibrium can be evidenced : an association at high ionic strength in the presence of lysine leading to a tetrameric form and a dissociation towards monomer at low ionic strength in the absence of lysine. The two equilibria are dependent on protein concentration.The lysine-sensitive aspartokinase of Escherichia coli K12 is one of the three enzymes catalyzing the phosphorylation of the /?-carboxyl of aspartate in these bacteria [1,2]. Lysine is the feed-back inhibitor of this activity [l] The protein from E. coli K12 has been purified to homogeneity [6 -81 ; molecular weights of 100 000 [6], 177000 [7] and 130000 [S] have been proposed for the native enzyme. von Dippe et al.[9] reported a subunit molecular weight of 39000 for the E . coli B enzyme, the native enzyme undergoing a dimertetramer transition in the presence of lysine and Mg2+.As this paper was to be submitted for publication a paper by Niles and Westhead appeared [lo] where they proposed for aspartokinase I11 from E. coli K12 a tetrameric structure with 48000-molecular-weight subunits ; the tetramer was obtained in 0.1 M KC1 and 1 mM lysine at high protein concentration. A variable quaternary structure of the enzyme is described and proposed to be related to the growth phase and to an adenylation of the enzyme [lo, 111 : one form is isolated in the tetrameric state, while the second is a stable dimer showing no aggregation process. We present results for the subunit structure of E . coli K12 aspartokinase I11 in agreement with the one proposed by Niles and Westhead [lo]. I n addition, we show that the enzyme subunits are identical, that a dimeric form is observed in the presence of KC1 without lysine and that this dimer either aggregates in KC1 plus lysine or dissociates a t low ionic strength in the absence of the ligand. MATERIALS AND METHODS Strains Used and Enzyme PurificationAspartokinase I11 was purified from Escherichia coli K12. I n addition to the strains HfrH grown as previously described [S] two other strains were used which produce larger amounts of enzyme: DO6 is a partially constitutive mutant for aspartokinase I11 isolated in our laboratory (J. C. Patte, unpublished), in which the specific activity of this enzyme is 3 to 5-fold increased when the st.rain is grown in minimal medium. G6 MD3 is a mutant carrying a deletion for the gene coding for aspartic semialdehyde dehydrogenase (asd) [12], kindly provided by M. Schwartz ; when this strain is grown in the presence of 20 pM diaminopimelic acid (plus 0.2 mM m-methionine and 2 mM DL-threonine) the aspartokinase I11 synthesis is 10 to 15-fold derepressed (probably due to lysine limitation).Strain DO6 was grown in fermentor of 200 1 a t ...

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The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli

Cited by 122 publications

References 23 publications

Purification of E. coli enzymes by chromatography on amphiphilic gels

Purification of E. coli enzymes by chromatography on amphiphilic gels

The Role of Lysine and Leucine Binding on the Catalytical and Structural Properties of Aspartokinase III of Escherichia coli K 12

Subunit Structure of Aspartokinase III of Escherichia coli K12

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