Reversible dissociation of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12. The active species is the tetramer. (9/18)

Dimers of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12 have been isolated under very mild conditions. The dimers which cannot be distinguished from the tetramers by their kinetic properties, reassociate in the presence of potassium ions or L-aspartate. The selective sensitivity of aspartokinase I/homoserine dehydrogenase I to mild proteolytic digestion of dimers has been used to probe the reassociation reaction under the conditions of aspartokinase assay. We demonstrate that rapid reassociation occurs and that the protein species present in the assay when dimers are used to test the activity is tetrameric. These results confirm the previously proposed model for the subunit association of aspartokinase I/homoserine dehydrogenase I.  (+info)

Fluorescence studies of threonine-promoted conformational transitions in aspartokinase I using the substrate analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. (10/18)

The trinitrophenyl derivative of ATP, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, has been used as a spectroscopic probe to investigate threonine-promoted conformational changes in the aspartokinase region of aspartokinase-homoserine dehydrogenase I in an attempt to relate the structural effects of threonine binding to inhibition of enzymatic activity. Binding of this analogue substrate to the enzyme is characterized by a 9-fold enhancement in probe fluorescence. Saturating levels of the feedback inhibitor, threonine, produce a 77% increase in fluorescence enhancement, indicating an increase in the rigidity or hydrophobicity of the nucleotide-binding site in the inhibited form of the enzyme. Threonine titration studies indicate that the two inhibitor-binding sites found on each subunit do not contribute equally to the fluorescence-detected conformational change. Comparison of the spectral change with the inhibition of dehydrogenase activity has revealed the exclusive involvement of the non-kinase threonine sites. No transition can be detected as a consequence of inhibitor binding at the kinase subsites. The results of the 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate study have provided further evidence for a concerted kinase-dehydrogenase conformational change which is induced by threonine interaction with the high affinity binding sites and which provides maximal inhibition of homoserine dehydrogenase and the majority of aspartokinase inhibition. The failure to observe a distinct enzyme form produced by threonine occupation of the low affinity kinase sites suggests that no large structural reorganization of the kinase active site is produced as a result of this binding event. The conformational change, suggested by the cooperativity of threonine binding, must instead involve only a subtle or highly localized alteration which does not perturb the environment of the ATP-binding cleft.  (+info)

A hybrid proteolytic fragment of Escherichia coli aspartokinase I-homoserine dehydrogenase I. Structure, inhibition pattern, dissociation properties, and generation of two homodimers. (11/18)

A hybrid dimeric fragment of Escherichia coli aspartokinase I-homoserine dehydrogenase I (Fazel, A., Muller, K., Le Bras, G., Garel, J.-R., Veron, M., and Cohen, G. N. (1983) Biochemistry 22, 158-165) has been purified and shown to possess both aspartokinase and homoserine dehydrogenase activities and is rather stable in the presence of L-threonine. Its two activities are still inhibited by threonine, but noncooperatively in contrast to the native protein. The aspartokinase activity is found to be more sensitive to threonine than the dehydrogenase activity. In the absence of threonine, the different chains of the hybrid (Mr = 89,000 + 59,000) dissociate first into monomers, this being followed by the pairing of two homologous chains to form two homodimers. In the presence of L-threonine, the two homodimers do not dissociate to re-form the hybrid fragment. The NH2-terminal analysis of different chains of the hybrid shows that the homodimers correspond, respectively, to the dimer of the native protein (Mr = 2 X 89,000) and to a dimer already described (Veron, M., Falcoz-Kelly, F., and Cohen, G. N. (1972) Eur. J. Biochem. 28, 520-527).  (+info)

Characterization of proteolysis fragments of aspartokinase I: homoserine dehydrogenase I. Fluorescence and circular dichroism studies. (12/18)

Proteolysis of native aspartokinase-homoserine dehydrogenase by chymotrypsin, subtilisin, clostripain, and V8 protease yields active dehydrogenase fragments. Fluorescence and near-UV circular dichroism measurements demonstrate that the bulk of the spectroscopic signal observed in the native protein originates in the residual fragments. Kinetic studies and far-UV CD spectra further distribute the fragments into two groups. Even though the remaining dehydrogenase activity is no longer inhibited by L-threonine, ultrafiltration binding studies and far-UV CD spectra clearly demonstrate that one of the two sets of inhibitor-binding sites is still intact. Computer analysis of the far-UV CD data of the native protein and the isolated fragments in the presence and absence of L-threonine has been used to resolve contributions from helix, beta, turn, and aperiodic components. This analysis indicates that the binding of the inhibitor induces decreases in helix content and generation of aperiodic structure within the molecule. The changes observed are similar in the native molecule and the fragments.  (+info)

The interaction between Escherichia coli aspartokinase-homoserine dehydrogenase and 3-acetylpyridine-adenine dinucleotide phosphate (reduced), an analog of NADPH. (13/18)

The interaction of 3-acetylpyridine-adenine dinucleotide phosphate, a structural analog of NADPH, with aspartokinase-homoserine dehydrogenase has been studied by fluorescence and activity measurements. This analog binds to the same site and with the same affinity as does the natural coenzyme. Also, the binding of homoserine to the dehydrogenase site or that of threonine to the regulatory site is the same whether NADPH or its analog is bound to the enzyme. So NADPH and its analog appear as equivalent in the formation of various stable enzyme-ligand(s) complexes. The analog resembles NADPH enough so that it is a substrate that the enzyme can use to reduce aspartate semialdehyde; the maximum velocity of this dehydrogenase reaction is however reduced by 90% as compared to that with NADPH. It seems as if one of the catalytic steps is affected by the replacement of a--CONH2 group by--COCH3. Another difference between the two coenzymes is that the reaction with the analog is insensitive to threonine, whereas that with NADPH is inhibited. The lack of inhibition is not due to a lack of binding, but rather to a difference in the ternary complexes composed of enzyme, coenzyme, and substrate. A possible relationship between the inhibition by threonine and the mechanism of the dehydrogenase reaction is thus suggested by this comparison between NADPH and its analog.  (+info)

Internal homologies in the two aspartokinase-homoserine dehydrogenases of Escherichia coli K-12. (14/18)

In Escherichia coli, AK I- HDH I and AK II- HDH II are two bifunctional proteins, derived from a common ancestor, that catalyze the first and third reactions of the common pathway leading to threonine and methionine. An extensive amino acid sequence comparison of both molecules reveals two main features on each of them: (i) two segments, each of about 130 amino acids, covering the first one-third of the polypeptide chain, are similar to each other and (ii) two segments, each of about 250 amino acids and covering the COOH-terminal 500 amino acids also present a significant homology. These findings suggest that these two regions may have evolved independently of each other by a process of gene duplication and fusion previous to the appearance of an ancestral aspartokinase-homoserine dehydrogenase molecule.  (+info)

Sequential folding of a bifunctional allosteric protein. (15/18)

Aspartokinase I-homoserine dehydrogenase I (EC 2.7.2.4 and EC 1.1.1.3) a bifunctional and allosteric enzyme, has been renatured from its unfolded and separated polypeptide chains. Folding was measured by the reappearance of each of the two enzymatic activities, kinase and dehydrogenase, and of their allosteric inhibition by the same effector, threonine. The various observed properties yield different kinetics of folding, which shows the presence of intermediates having only some of the functional features of the native enzyme. Apparently, three successive steps can be detected during the folding of aspartokinase I-homoserine dehydrogenase I: first, a monomolecular step leads to a monomeric species with the kinase activity; then an association step leads to a dimeric species with the kinase and dehydrogenase activities, and a threonine-sensitive dehydrogenase; finally, a second association step leads to a tetrameric species with the two activities, both sensitive to threonine. The folding of this large protein appears as a sequential process during which the functional properties are regained successively, as the protein structure becomes more complex. During this process, the two regions of each polypeptide chain respectively responsible for the kinase and dehydrogenase activities seem to acquire their native conformation rather independently of each other.  (+info)

Nucleotide sequence of the thrA gene of Escherichia coli. (16/18)

The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense codon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.  (+info)