Multiple interactions of threonine with an aspartokinase-homoserine dehydrogenase complex

Cunningham, Glenn N.; Maul, Stephen B.; Shive, William

doi:10.1016/0006-291x(68)90464-6

Cited by 23 publications

(10 citation statements)

References 7 publications

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“…Cunningham et al [20] actually conclude that the molecular weight of the active protein is in the 80,000-120,000 range, with a sedimentation coefficient of 6.3 S in absence of threonine. However, these experiments were performed in the cold and in a buffer containing no KCl, and Patte et al [14] have noted that polymerization also occurs upon addition of KC1, K+ being an essential cofactor of both catalytic activities [14,10].…”

Section: Discussioementioning

confidence: 99%

“…Observing a change in the elution pattern and sedimentation in sucrose gradients of the aspartokinase [lo] and homoserine dehydrogenase [20,21] activities, several groups have proposed that a major effect of threonine is a polymerization of the enzyme. Cunningham et al [20] actually conclude that the molecular weight of the active protein is in the 80,000-120,000 range, with a sedimentation coefficient of 6.3 S in absence of threonine.…”

Section: Discussioementioning

confidence: 99%

See 1 more Smart Citation

The Threonine-Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12. Binding of Threonine and of Pyridine Nucleotides: Stoichiometry and Optical Effects

Janin

Truffa‐Bachi

et al. 1969

Eur J Biochem

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The aspartokinase I-homoserine dehydrogenase I of Escherichk coli K 12, composed of six subunits of molecular weight 60,000, binds cooperatively six molecules of L-threonine. A maximum of three molecules of NADP+ or NADPH, measured by a number of techniques, is bound in the presence or in the absence of threonine.Characteristic effects of L-threonine on the protein include a perturbation of its absorption and fluorescence spectra and a quenching of the fluorescence of the protein-NADPH complex. These findings are discussed in view of the structure of the protein.Physical and chemical studies of the protein carrying the aspartokinase I and homoserine dehydrogenase I of E . coli K12 [i, 21 suggest that its six constitutive subunits are identical. The two enzymic functions imply binding sites for their respective substrates, for activating cations (K+ and Mg++) and for the allosteric inhibitor, L-threonine. Kinetic data gave an approximation of the affinities for the different ligands ; threonine and the pyridine nucleotides were found to be most amenable to study. The stoichiometry of their binding is presented here and discussed in relation with the subunit structure. Effects of threonine upon the ultraviolet spectrum of the protein and the fluorescence of its complex with NADPH are also demonstrated. MATERIAL AND METHODS Preparation of the ProteinThe pure, electrophoretically homogeneous protein was obtained from extracts of E . wli K12 as described by Truffa-Bachi et al. [l]. The specific activity of homoserine dehydrogenase was a t least 47 units per mg in fresh preparations, and the experiments described here were performed on freshly purified protein unless specified. Enzymic activities were measured and expressed as described in [i]. BuffersBuffer P contained 20 mM potassium phosphate pH 7.2, 2 mM magnesium titriplex and 1 mM dithiothreitol. Buffer A has been described earlier [ l ] and is actually buffer P plus 0.15 M KC1 and 4mM m-threonke. Buffer B is buffer P plus 0.15 M KC1.The protein resuspended from 5001, ammonium sulfate precipitate was equilibrated with buffer by passing it on a small column of Sephadex G 25; exchanging buffers was done the same way. Chemicals and RadiochemicalsThe salts and chemicals used were the same as in [i]. L-Threonine (allo-free) was obtained from Fluka, NADP+ and NADPH from Sigma.~-[~4C]Threonine (uniformly labelled, 81.6 mC/ m o l e ) was obtained from the Commissariat 21 1'Energie Atomique, [carbonyl-14C]nicotinamide dinucleotide phosphate 18.7 mC/mmole, from the Radiochemical Centre (Amersham, England). The purity of the labelled nucleotides was checked by thin layer chromatography. Protein DeterminationProtein was determined by biuret related to dry weight by the following method: about 5 mg of purified protein was dialyzed for 2days against distilled water, and lyophilized. Two fractions were then weighed on a microbalance and subjected to the biuret reaction. The remainder was analyzed for C, H, N, content on a Technicon CHN Analyzer. The measured content in carbon...

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Section: Discussioementioning

confidence: 99%

Section: Discussioementioning

confidence: 99%

The Threonine-Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12. Binding of Threonine and of Pyridine Nucleotides: Stoichiometry and Optical Effects

Janin

Truffa‐Bachi

et al. 1969

Eur J Biochem

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“…However, on the basis of a number of other observations, this possibility would appear to be very unlikely. For example, no preparation of the E. coli enzyme examined thus far can be completely inhibited at pH 9 by threonine (Patte et al, 1963;Cunningham et al, 1968). Although the particular enzyme preparation used in the present kinetic investigation was not a highly purified preparation, it displayed a single, sharp, well-defined band of homoserine dehydrogenase activity on disc electrophoresis (Figure 10).…”

Section: Discussionmentioning

confidence: 94%

Homoserine dehydrogenase of Escherichia coli. I. Feedback inhibition by L-threonine and activation by potassium ions

Ogilvie

Sightler²,

Clark³

1969

Biochemistry

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“…Deviations of the slope of the Hill plot at low and high threonine concentrations are expected (Cornish-Bowden & Koshland, 1975) with a value of one at each extreme. At low concentration, however, an activation of HSD was consistently observed which could be due to a stabilization of the tetramer of AK-HSD by low concentrations of threonine (Cunningham et al, 1968).…”

Section: Methodsmentioning

confidence: 96%

Threonine inhibition of the aspartokinase-homoserine dehydrogenase I of Escherichia coli. Stopped-flow kinetics and the cooperativity of inhibition of the homoserine dehydrogenase activity

Bearer¹,

Neet²

1978

Biochemistry

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Threonine interactions with the threonine-sensitive aspartokinase-homoserine dehydrogenase I (AK-HSD) from Escherichia coli K12 have been investigated by studying the relaxation times from the uninhibited to the inhibited form of the enzyme and the kinetic inhibition of the steady state activity. Comparison of changes in the relaxation time, changes in the cooperativity of the inhibition, and changes in binding cooperativity have been made under a range of comparable conditions. The progress curve lag in threonine inhibition of the HSD activity was studied by stopped-flow rapid kinetic techniques as a function of pH, temperature, ionic strength, and ATP concentration. The protein concentration independence and the activation energy of the apparent first-order rate constant for the lag, k i, demonstrated its identity to the R to T isomerization previously studied. Observed relaxation times ranged from 10 ms to 1.5 s. Limiting values of the relaxation time, rmi", and the threonine concentration at half the maximal rate of transition, AT, were obtained from analysis of doublereciprocal plots of k i vs. threonine concentration. rmjn was substantially decreased with increasing temperature. Other changes in experimental conditions resulted in smaller decreases in rmin. Kinetic Hill coefficients obtained from standardThe presence of two distinct, separate sites of threonine binding to aspartokinase-homoserine dehydrogenase I (AK-HSD)* 1 of E. coli (Bearer & Neet, 1978) has raised the question of the role of each in the inhibition of homoserine dehydrogenase activity, and the mechanism by which inhibition occurs. Thus, a study of the initial and steady-state kinetics of the HSD inhibition was undertaken. The characteristics of the steady-state inhibition of HSD by threonine vary according to direction and conditions of assay. Under forward assay conditions (aspartic /3-semialdehyde to homoserine) threonine inhibition is noncompetitive vs. aspartic /3-semialdehyde (Patte et al., 1963). When studied at high pH (8.5) and low potassium (5 mM), no cooperativity is observed (Truffa-Bachi et al., 1974). At lower pH and in the presence of 0.

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Multiple interactions of threonine with an aspartokinase-homoserine dehydrogenase complex

Cited by 23 publications

References 7 publications

The Threonine-Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12. Binding of Threonine and of Pyridine Nucleotides: Stoichiometry and Optical Effects

The Threonine-Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12. Binding of Threonine and of Pyridine Nucleotides: Stoichiometry and Optical Effects

Homoserine dehydrogenase of Escherichia coli. I. Feedback inhibition by L-threonine and activation by potassium ions

Threonine inhibition of the aspartokinase-homoserine dehydrogenase I of Escherichia coli. Stopped-flow kinetics and the cooperativity of inhibition of the homoserine dehydrogenase activity

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