Caractérisation de l'activité l-thrénine désaminasique du foie de cobaye induite par un régime hyperprotidique.

We have elaborated a kinetic method which allows us to evaluate whether a Michaelis-Menten-type enzyme acting on two different substrates has one or two active sites. This method has been used with the rat liver Lthreonine dehydratase, which catalyzes the dehydrative deamination of both serine and threonine. The experimental data can be fitted to the theoretical plot obtained for the case of a single active site.L-Threonine dehydratase is a soluble enzyme from mammalian liver, which catalyzes the dehydrative deamination of both L-serine and L-threonine with the formation of pyruvate + NH3 and 2-oxobutyrate + NH3, respectively.The enzyme has been found in mouse [l, 21, guinea-pig [3], goat [41 and human liver [5], in cell cultures [6, 71 and in tumors [8].Only have given evidence for the existence in guinea-pig liver of a specific L-threonine deaminase and of the usual L-threonine dehydratase having activity toward both threonine and serine. However, the two enzymes have not been separated.The rat liver enzyme is strictly dependent on pyridoxal 5'-phosphate and it has been demonstrated to act on both L-serine and L-threonine, with a ratio of activities which is constant during purification and during various treatments which inactivate the enzyme but at constant assay conditions [9-121. No evidence has been given of the existence of two different enzymes, one acting on L-serine and the other on L-threonine, since the most purified preparations gave a single band when submitted to polyacrylamide gel electrophoresis [13, 141. While most of the literature agrees that in rat liver the dehydrative deamination of both L-serine and of L-threonine is linked to a single enzyme, the behavior with the two substrates changes under different assay conditions, involving variations in the L-threonine/L-serine activity ratio. Dilution or heating of the L-threonine dehydratase lowers its effect on L-threonine more than on L-serine, while an increase in either NH: or K + ion concentration enhances the enzyme's action on L-threonine more than on L-serine [14]. Such results cannot be attributed to two distinct enzymes, since the two activities have never been separated. The current interpretation is that different treatments promote either the dissociation or association of pyridoxal-P from or to the enzyme and the K , values of pyridoxal-P for the two activities are different [15]. However, this interpretation is not unique either since the observed behavior could be due to two different sites on Lthreonine dehydratase, one acting on L-threonine, the other on L-serine. To check this possibility we have elaborated a kinetic method which allows one to differentiate whether a single enzyme acting on two different substrates has one or two active sites.Our results demonstrate that L-threonine dehydratase has a single site acting on both L-threonine and L-serine. MATERIALS AND METHODS MaterialsL-Serine, L-threonine, pyridoxal-P, sodium pyruvate and sodium 2-oxobutyrate were obtained from Merck, dithiothreitol from Sigma. Sephacryl S...

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“…The enzyme has been found in mouse [l, 21, guinea-pig [3], goat [41 and human liver [5], in cell cultures [6, 71 and in tumors [8].…”

mentioning

confidence: 99%

“…Only Vincent-Fiquet et al [3] have given evidence for the existence in guinea-pig liver of a specific L-threonine deaminase and of the usual L-threonine dehydratase having activity toward both threonine and serine. However, the two enzymes have not been separated.…”

mentioning

confidence: 99%

A kinetic method for distinguishing whether an enzyme has one or two active sites for two different substrates

Keleti

Leoncini

Pagani

et al. 1987

European Journal of Biochemistry

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The pAR5 mutation and the allosteric mechanism of Escherichia coli aspartate carbamoyltransferase.

et al. 1987

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Mutation pAR5 replaces residues 145′‐153′ at the C terminus of the regulatory (r) chains of Escherichia coli ATCase by a new sequence of six residues. The mutated enzyme has been shown to lack substrate cooperativity and inhibition by CTP. Solution X‐ray scattering curves demonstrate that, in the absence of ligands, its structure is intermediate between the T form and the R form. In the presence of N‐phosphonacetyl‐L‐aspartate, the mutant is similar to the wild type. An examination of the crystal structure of unligated ATCase reveals that the mutated site is at an interface between r and catalytic (c) chains, which exists only in the T allosteric form. A computer simulation by energy minimization suggests that the pAR5 mutation destabilizes this interface and induces minor changes in the tertiary structure of r chains. The resulting lower stability of the T form explains the loss of substrate cooperativity. The lack of allosteric inhibition may be related to a new electrostatic interaction made in mutant r chains between the C‐terminal carboxylate and a lysine residue of the allosteric domain.

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An improved method for purification of l-threonine deaminase from rat liver

Leoncini

Guerranti

Pagani

et al. 1990

Journal of Biochemical and Biophysical Methods

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Caractérisation de l'activité l-thrénine désaminasique du foie de cobaye induite par un régime hyperprotidique.

Cited by 7 publications

References 11 publications

A kinetic method for distinguishing whether an enzyme has one or two active sites for two different substrates

A kinetic method for distinguishing whether an enzyme has one or two active sites for two different substrates

The pAR5 mutation and the allosteric mechanism of Escherichia coli aspartate carbamoyltransferase.

An improved method for purification of l-threonine deaminase from rat liver

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