Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity.

The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.     

Properties of Threonine Deaminase From a Bacterium Able to Use Threonine as Sole Source of Carbon

A threonine deaminase susceptible to inhibition by isoleucine was purified over 3,000-fold from extracts of Pseudomonas multivorans, a bacterium able to use threonine or α-ketobutyrate as sole source of carbon and energy. The enzyme was characterized with respect to molecular weight, dissociation to subunits, and apparent affinities for threonine, isoleucine, and several other ligands. Certain features of the enzyme including its reversible dissociation to subunits, its high constitutive activity, its marked stability, and high apparent orders of binding for threonine and isoleucine were unusual compared to those of isoleucine-inhibitable enzymes from other bacteria. These findings suggested some relationship between properties of the enzyme and the ability of P. multivorans to use threonine as sole carbon source. However, mutant studies ruled out a direct role of the enzyme in threonine catabolism and indicated that another enzyme, threonine dehydrogenase, is essential for growth on threonine.     

Threonine in the selectivity filter of the acetylcholine receptor channel.

The acetylcholine receptor (AChR) is a cation selective channel whose biophysical properties as well as its molecular composition are fairly well characterized. Previous studies on the rat muscle alpha-subunit indicate that a threonine residue located near the cytoplasmic side of the M2 segment is a determinant of ion flow. We have studied the role of this threonine in ionic selectivity by measuring conductance sequences for monovalent alkali cations and bionic reversal potentials of the wild type (alpha beta gamma delta channel) and two mutant channels in which this threonine was replaced by either valine (alpha T264V) or glycine (alpha T264G). For the wild type channel we found the selectivity sequence Rb greater than Cs greater than K greater than Na. The alpha T264V mutant channel had the sequence Rb greater than K greater than Cs greater than Na. The alpha T264G mutant channel on the other hand had the same selectivity sequence as the wild type, but larger permeability ratios Px/PNa for the larger cations. Conductance concentration curves indicate that the effect of both mutations is to change both the maximum conductance as well as the apparent binding constant of the ions to the channel. A difference in Mg2+ sensitivity between wild-type and mutant channels, which is a consequence of the differences in ion binding, was also found. The present results suggest that alpha T264 form part of the selectivity filter of the AChR channel were large ions are selected according to their dehydrated size.     

Cobalt(III) affinity-labeled aspartokinase. Formation of substrate and inhibitor adducts.

The kinase active site of the aspartokinase-homoserine dehydrogenase enzyme complex of Excherichia coli has been affinity labeled both with substrates aspartate and adenosine triphosphate and feedback inhibitor threonine. Co(III) exchange-inert adducts of aspartokinase and inhibitor or substrates were produced in situ by oxidation of Co(II) with H2O2. Emzyme-Co(III)-adenosine 5'-triphosphate (ATP), enzyme-Co(III)-aspartate, and enzyme-Co(III)-threonine ternary adducts were produced in this manner. The formation of the enzyme-Co(III)-threonine adduct leads us to conclude that threonine inhibits the kinase activity of this enzyme complex by binding in the first coordination sphere of the catalytic metal ion cofactor, a conclusion which is consistent with evidence derived from previous nuclear magnetic resonance data obtained in this laboratory. The quaternary adducts formed by H2O2 oxidation in the presence of aspartokinase, Co(II), ATP, aspartate, and threonine comprised a mixture of both ezyme-Co(III)-ATP-aspartate and enzyme-Co(III)-ATP-threonine adducts. The formation of the quaternary aspartate-containing adduct was unexpected, since the presence of threonine was expected to prevent access of the aspartate to the active site; most significantly however, the the sum of the numbers of aspartate plus threonine molecules incorporated per active site is one. We believe that this shows direct steric overlap between the metal-adjacent binding sites for aspartate and threonine. Aspartate or threonine can not occupy the kinase active site simultaneously; this conclusion is consistent with the direct competitive inhibition of aspartate by threonine observed in steady-state kinetic studies.     

Threonine phosphorylation sites in the beta 2 and beta 7 leukocyte integrin polypeptides

The cytoplasmic domains of integrins play a key role in a variety of integrin-mediated events including adhesion, migration, and signaling. The molecular mechanisms that enhance integrin function are still incompletely understood. Because protein kinases are known to be involved in the signaling and the activation of integrins, the role of phosphorylation has been studied by several groups. The beta(2) leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758-760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the beta(2) integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, beta(7), has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused beta(7) integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the beta(7) subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in beta(2) phosphorylation.     

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

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.     

Threonine degradation by Serratia marcescens.

The wild strain of Serratia marcescens rapidly degraded threonine and formed aminoacetone in a medium containing glucose and urea. Extracts of this strain showed high threonine dehydrogenase and "biosynthetic" threonine deaminase activities, but no threonine aldolase activity. Threonine dehydrogenase-deficient strain Mu-910 was selected among mutants unable to grow on threonine as the carbon source. This strain did not form aminoacetone from threonine, but it slowly degraded threonine. Strain D-60, deficient in both threonine dehydrogenase and threonine deaminase, was derived from strain Mu-910 and barely degraded threonine. A glycine-requiring strain derived from the wild strain grew in minimal medium containing threonine as the glycine source, whereas a glycine-requiring strain derived from strain Mu-910 did not grow. This indicates that threonine dehydrogenase participates in glycine formation from threonine (via alpha-amino-beta-ketobutyrate) as well as in threonine degradation to aminoacetone.     

The amino-terminal cyclic nucleotide binding site of the type II cGMP-dependent protein kinase is essential for full cyclic nucleotide-dependent activation

For the type I cGMP-dependent protein kinases (cGKIalpha and cGKIbeta), a high affinity interaction exists between the C2 amino group of cGMP and the hydroxyl side chain of a threonine conserved in most cGMP binding sites. To examine the effect of this interaction on ligand binding and kinase activation in the type II isozyme of cGMP-dependent protein kinase (cGKII), alanine was substituted for the conserved threonine or serine. cGKII was found to require the C2 amino group of cGMP and its cognate serine or threonine hydroxyl for efficient cGMP activation. Of the two binding sites, disruption of cGMP-specific binding in the NH(2)-terminal binding site had the greatest effect on cGMP-dependent kinase activation, like cGKI. However, ligand dissociation studies showed that the location of the rapid and slow dissociation sites of cGKII was reversed relative to cGKI. Another set of mutations that prevented cyclic nucleotide binding demonstrated the necessity of the NH(2)-terminal, rapid dissociation binding site for cyclic nucleotide-dependent activation of cGKII. These findings suggest distinct mechanisms of activation for cGKII and cGKI isoforms. Because cGKII mediates the effects of heat-stable enterotoxins via the cystic fibrosis transmembrane regulator Cl(-) channel, these findings define a structural target for drug design.     

Isoleucine Auxotrophy Due to Feedback Hypersensitivity of Biosynthetic Threonine Deaminase

Isoleucine-deficient mutants of Salmonella typhimurium were isolated. Three groups of mutants can be discerned by their nutritional requirements and enzyme patterns. (i) Mutants which grow with isoleucine alone are devoid of biosynthetic threonine deaminase (TD). (ii) Mutants growing with isoleucine and valine are devoid of transaminase B. (iii) Mutants growing with either isoleucine or threonine have normal levels of TD. However, the sensitivity of this enzyme to feedback inhibition by isoleucine is greatly enhanced. The inhibitory effect of isoleucine can be counterbalanced by high concentrations of threonine. These results indicate that the production of isoleucine in the mutants is restricted to a low level not sufficient to support the growth of the cells. This hypothesis is confirmed by studies with revertants of an isoleucine-threonine mutant. In nine revertants, wild-type properties of TD have been restored. In four revertants, the hypersensitivity of TD is unchanged, but the strains produce a greatly enhanced quantity of threonine, which is excreted into the culture medium. It follows, that hypersensitivity of TD to inhibition by isoleucine is the cause of the nutritional requirement of isoleucine-threonine mutants.     

Nutrition of Threonine

The threonine content in blood and urine increased and threonine decomposition ability in liver decreased by feeding lower level of lysine, whereas threonine content in blood and urine decreased and the ability of liver increased gradually with increasing lysine content in diet. These phenomena were owing to the increase of threonine dehydratase activity of liver, which was measured from produced α-ketobutyric acid amount, by excess administration of lysine. The phenomena that threonine content in urine decreased and threonine decomposition ability of liver increased with increasing threonine content in diet when adequate amount of lysine was fed, were also ascribed to the increase of the dehydratase activity.

One m mole of threonine was incubated with liver homogenate in presence of PALP*** at pH 8.2 for 20 and 30 min and α-ketobutyric acid produced was introduced to its 2,4-dinitrophenylhydrazone, which was chromatographed on silica-gel thin-layer plate and determined spectrophotometrically at 395 mμ under N,N-dimethylformamide.

Other enzyme systems relating to threonine catabolism were also investigated, including threonine aldolase, threonine dehydrogenase and ornithine transaminase, showing no significant changes in activities by excess administration of lysine and/or threonine.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Glycosylated Threonine but not 4-Hydroxyproline Dominates the Triple Helix Stabilizing Positions in the Sequence of a Hydrothermal Vent Worm Cuticle Collagen

The cuticle collagen of the vestimentiferanRiftia pachyptila, an organism which is endemic to deep-sea hydrothermal vents, has several unusual properties including an extraordinary length (1.5 μm), a high thermal stability (37°C) in spite of a low 4-hydroxyproline content and an atypically high threonine content (20 mol%). We have now purified the constituent chain of cuticle collagen and show that it contains about 40% carbohydrate, which is mainly galactose, indicating that the chain has a molecular mass of approximately 750 kDa. Several large (30 to 150 kDa) fragments, which all contained carbohydrate, could be produced by cleavage with endoproteinase Lys-C, bacterial collagenase and cyanogen bromide (CNBr). Edman degradation of these and several smaller fragments was used to determine about 3000 sequence positions comprising 60% of the total triple-helical sequence. This demonstrated mainly typical Gly-X-Y triplet repeats with a few imperfections and a longer N-terminal non-triplet sequence. Most of the 4-hydroxyproline was found in triplet position X, where it decreases the stability of the triple helix. About 40% of the Y positions could not be identified, which correlated with a low abundance of threonine in the sequence and the demonstration of threonine in these positions after deglycosylation of several peptides by treatment with hydrofluoric acid. Matrix-assisted laser desorption ionisation mass spectrometry of selected peptides indicated that the blocked threonine residues are occupied by chains of one, two or three hexoses (presumably galactose). These glycosylated threonine residues in Y positions are therefore likely to replace 4-hydroxyproline as the major contributor to triple helix stabilization. Studies with a synthetic (Gly-Pro-Thr)₁₀oligopeptide demonstrated a low thermal stability of its triple helix which emphasizes a crucial role of glycosylation for stabilization.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

In Vivo Regulation of Threonine and Isoleucine Biosynthesis in Lemna paucicostata Hegelm. 6746

Little, if any, regulation of threonine synthesis was observed in Lemna paucicostata Hegelm. 6746 supplemented with concentrations of threonine and/or isoleucine that allow for uptake of these amino acids in amounts sufficient for total plant requirements, and that increase tissue concentrations of soluble threonine manyfold. High tissue concentrations of soluble threonine generated endogenously in isoleucine-supplemented plants were no more effective in regulation than a similar concentration of threonine accumulated from the medium. These studies exclude also major regulation of threonine biosynthesis by bivalent repression by threonine plus isoleucine. Isoleucine biosynthesis was severely inhibited by supplementation with isoleucine, but not with threonine or methionine. The fivefold increase in soluble threonine in isoleucine-supplemented plants suggests that threonine dehydratase is a major locus for feedback regulation of isoleucine synthesis. It is concluded that regulation of threonine biosynthesis differs from that of the other amino acids of the aspartate family (isoleucine, methionine, and lysine), each of which strongly feedback regulates its own synthesis. Methionine supplementation had a negligible effect on the tissue concentration of soluble threonine, indicating that threonine is not important in balancing changes of flux into methionine by equivalent changes of flux through the step catalyzed by aspartokinase.     

WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.     

Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.     

A kinetic method for distinguishing whether an enzyme has one or two active sites for two different substrates. Rat liver L-threonine dehydratase has a single active site for threonine and serine

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 L-threonine 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.     

A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.     

The binding of divalent cations to myosin.

Centrifuge transport, equilibrium dialysis, and electron paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of 10(6) M-1 (Class I) and 10(3) M-1 (Class II), respectively. At 0.6 M KCl concentration, the affinity of Mn2+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca greater than Mg = Zn = Co greater than Sr greater than Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37 degrees partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PPi, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.     

Interaction of substrates and inhibitors with the homoserine dehydrogenase of kinase-inactivated aspartokinase I.

The aspartokinase activity of the aspartokinase-homoserine dehydrogenase complex of Escherichia coli was affinity labeled with substrates ATP, aspartate, and feedback inhibitor threonine. Exchange-inert ternary adducts of Co(III)-aspartokinase and either ATP, aspartate or threonine were formed by oxidation of corresponding Co(II) ternary complexes with H2O2. The ternary enzyme-Co(III)-threonine adduct (I) had 3.8 threonine binding sites per tetramer, one-half that of the native enzyme. The binding of threonine to I was still cooperative as determined by equilibrium dialysis (nH = 2.2) or by studying inhibition of residual dehydrogenase activity (nH = 2.7). Threonine still protected the SH groups of I against 5,5'-dithiobis(2-nitrobenzoate) (DTNB) reaction but the number of SH groups reacting with thiol reagents (DTNB) was reduced by 1-2 per subunit in the absence of threonine. This suggests either that Co(III) is bound to the enzyme via sulfhydryl groups or that 1-2SH groups are buried or rendered inaccessible in I. The binding of threonine to sites not blocked by the affinity labeling produced changes in the circular dichroism of the complex comparable to changes produced by threonine binding to native enzyme and also protected against proteolytic digestion. The major conformational changes produced by threonine are thus ascribable to binding at this one class of regulatory sites. The interactions of kinase substrates with various aspartokinase-Co(III) complexes containing ATP, aspartate, or threonine and a threonine-insensitive homoserine dehydrogenase produced by mild proteolysis were studied. The inhibition of homoserine dehydrogenase by kinase substrates is not due to binding of these inhibitors at the kinase active site but was shown to be due to binding to sites within the dehydrogenase domain of the enzyme. L-alpha-Aminobutyrate, a presumed threonine analogue, also inhibits the dehydrogenase by binding at the same or similar sites in the dehydrogenase domain and not at threonine regulatory site.