Characterization of proteolysis fragments of aspartokinase I: homoserine dehydrogenase I. Fluorescence and circular dichroism studies

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.     

Regulation of aspartokinase and homoserine dehydrogenase in acetic acid bacteria

The regulation of aspartokinase and homoserine dehydrogenase has been studied in three Acetobacter and two Gluconobacter species. Both enzymes were regulated by feedback inhibition. Aspartokinase was inhibited by L-threonine and concertedly inhibited by L-threonine plus L-lysine. The homoserine dehydrogenase was NADP-specific and was inhibited by L-threonine. Separation of the two enzymes by ammonium sulphate fractionation was possible in Acetobacter peroxydans, A. rancens and Gluconobacter melanogenus but not in A. liquefaciens or G. oxydans.     

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

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.     

Bifunctional protein in carrot contains both aspartokinase and homoserine dehydrogenase activities

We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product aspartokinase I-homoserine dehydrogenase I.     

New phenolic inhibitors of yeast homoserine dehydrogenase

A relatively unexploited potential target for antimicrobial agents is the biosynthesis of essential amino acids. Homoserine dehydrogenase, which reduces aspartate semi-aldehyde to homoserine in a NAD(P)H-dependent reaction, is one such target that is required for the biosynthesis of Met, Thr, and Ile from Asp. We report a small molecule screen of yeast homoserine dehydrogenase that has identified a new class of phenolic inhibitors of this class of enzyme. X-ray crystal structural analysis of one of the inhibitors in complex with homoserine dehydrogenase reveals that these molecules bind in the amino acid binding region of the active site and that the phenolic hydroxyl group interacts specifically with the backbone amide of Gly175. These results provide the first nonamino acid inhibitors of this class of enzyme and have the potential to be exploited as leads in antifungal compound design.     

Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I.

The nucleotide sequence of the Serratia marcescens threonine operon (thrA1A2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrA(1)1A(2)1 and thrA(1)5A(2)5 mutations of threonine-excreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A(2)2 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).     

Proteolysis of the bifunctional methionine-repressible aspartokinase II-homoserine dehydrogenase II of Escherichia coli K12. Production of an active homoserine dehydrogenase fragment.

The dimeric bifunctional enzyme aspartokinase II-homoserine dehydrogenase II (Mr = 2 X 88,000) of Escherichia coli K12 can be cleaved into two nonoverlapping fragments by limited proteolysis with subtilisin. These two fragments can be separated under nondenaturing conditions as dimeric species, which indicates that each fragment has retained some of the association areas involved in the conformation of the native protein. The smaller fragment (Mr = 2 X 24,000) is devoid of aspartokinase and homoserine dehydrogenase activity. The larger fragment (Mr = 2 X 37,000) is endowed with full homoserine dehydrogenase activity. These results show that the polypeptide chains of the native enzyme are organized in two different domains, that both domains participate in building up the native dimeric structure, and that one of these domains only is responsible for homoserine dehydrogenase activity. A model of aspartokinase II-homoserine dehydrogenase II is proposed, which accounts for the present results.     

Isolation of the aspartokinase domain of bifunctional aspartokinase I-homoserine dehydrogenase I from E.coli K12

A proteolytic fragment (Mr approximately 25 000) carrying only the aspartokinase activity has been purified by chromatofocusing after limited proteolysis of aspartokinase I-homoserine dehydrogenase I from E.coli K12. The NH2-terminal sequence shows that it corresponds to the amino terminal peptide of the native enzyme. The results confirm a previous hypothesis about the organization of native aspartokinase I-homoserine dehydrogenase I.     

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Specific inactivation of the homoserine dehydrogenase activity by the affinity label, 2-amino-4-oxo-5-chloropentanoic acid.

2-Amino-4-oxo-5-chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I--homoserine dehydrogenase I. The aspartokinase activity remains essentially untouched and retains its threonine sensitivity. The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit. Alkylation does not affect the tetrameric state of the protein. The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label.     

Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli.

The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies. Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant. The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates. In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates. This was unexpected since the beta-carboxyl group is the usual site of phosphorylation. The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy. These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor. Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway. This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway.     

Preferred order random kinetic mechanism for homoserine dehydrogenase of Escherichia coli (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I: equilibrium isotope exchange kinetics.

Isotope exchange kinetics at chemical equilibrium have been used to investigate the kinetic mechanism of homoserine dehydrogenase (EC 1.1.1.3) of the (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I multifunctional enzyme from E. coli. For the reaction (L-ASA + NADPH + H+ = L-Hse + NADP+), at pH 9.0, 37 degrees C, Keq = 100 (+/- 20). Under these conditions, the rate for exchange of [14C]-L-homoserine (Hse) in equilibrium L-aspartate-beta-semialdehyde (ASA) is nearly twice that for the [3H]-NADP+ in equilibrium NADPH exchange. This indicates that covalent interconversion between reactants and products bound in the active site cannot be rate-limiting. Upon variation of the concentrations of all four substrates in constant ratio at equilibrium (to minimize dead-end complex formation), the Hse in equilibrium ASA exchange increased smoothly toward a maximum. In contrast, the NADP+ in equilibrium NADPH exchange rate increased to a maximum value at partial saturation, then decreased to approximately half the maximum rate. These data are consistent with a preferred-order random kinetic mechanism in which the dominant pathway involves association of NADPH prior to L-ASA and dissociation of L-Hse prior to NADP+.     

A preliminary immunochemical study of E. coli aspartokinase I-homoserine dehydrogenase I

The preparation of immunoadsorbents against aspartokinase I-homoserine dehydrogenase I from E.coli is described. In the presence of aspartate, considerably less enzyme is bound by the fixed antibodies. The fixed protein can be displaced by a protein extracted from a nonsense mutant.     

15-Hydroxy-prostanoate dehydrogenase. Prostaglandins as substrates and inhibitors

Determination of Michaelis constnats and maximum reaction rates for a series of prostaglandin dehydrogenase substrates showed that the enzyme is stereospecific with regard to configuration at C-15. Substituents of the cyclopentane ring did not markedly affect the properties as a substrate, whereas the nature of the carboxyl side chain proved important. A noncompetitive inhibition was produced between prostaglandin B compounds and a synthetic epimer of prostaglandin E1, 15-R-prostaglandin E1.