Removal of the tryptophan 139 side chain in Escherichia coli D-3-phosphoglycerate dehydrogenase produces a dimeric enzyme without cooperative effects

Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) is a homotetrameric enzyme whose activity is allosterically regulated by l-serine, the end-product of its metabolic pathway. Previous studies have shown that PGDH displays two modes of cooperative interaction. One is between the l-serine binding sites and the other is between the l-serine binding sites and the active sites. Tryptophan 139 participates in an intersubunit contact near the active site catalytic residues. Site-specific mutagenesis of tryptophan 139 to glycine results in the dissociation of the tetramer to a pair of dimers and in the loss of cooperativity in serine binding and between serine binding and inhibition. The results suggest that the magnitude of inhibition of activity at a particular active site is primarily dependent on serine binding to that subunit but that activity can be modulated in a cooperative manner by interaction with adjacent subunits. The disruption of the nucleotide domain interface in PGDH by mutating Trp-139 suggests the potential for a critical role of this interface in the cooperative allosteric processes in the native tetrameric enzyme.     

Hybrid tetramers reveal elements of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase

d-3-Phosphoglycerate dehydrogenase from Escherichia coli is a tetramer of identical subunits that is inhibited when l-serine binds at allosteric sites between subunits. Co-expression of two genes, the native gene containing a charge difference mutation and a gene containing a mutation that eliminates serine binding, produces hybrid tetramers that can be separated by ion exchange chromatography. Activity in the hybrid tetramer with only a single intact serine binding site is inhibited by approximately 58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in itself, lead to the positive cooperativity of inhibition manifest in the native enzyme. Tetramers with only two intact serine binding sites purify as a mixture that displays a maximum inhibition level that is less than that of native enzyme, suggesting the presence of a population of tetramers that are unable to be fully inhibited. Differential analysis of this mixture supports the conclusion that it contains two forms of the tetramer. One form contains two intact serine binding sites at the same interface and is not fully inhibitable. The second form is a fully inhibitable population that has one serine binding site at each interface. Overall, the hybrid tetramers show that the positive cooperativity observed for serine binding is mediated across the nucleotide binding domain interface, and the negative cooperativity is mediated across the regulatory domain interface. That is, they reveal a pattern in which the binding of serine at one interface leads to negative cooperativity of binding of a subsequent serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is propagated to subsequent binding sites in the tetramer such that the negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.     

Hybrid tetramers of porcine liver fructose-1,6-bisphosphatase reveal multiple pathways of allosteric inhibition

Fructose-1,6-bisphosphatase is a square planar tetramer of identical subunits, which exhibits cooperative allosteric inhibition of catalysis by AMP. Protocols for in vitro subunit exchange provide three of five possible hybrid tetramers of fructose-1,6-bisphosphatase in high purity. The two hybrid types with different subunits in the top and bottom halves of the tetramer co-purify. Hybrid tetramers, formed from subunits unable to bind AMP and subunits with wild-type properties, differ from the wild-type enzyme only in regard to their properties of AMP inhibition. Hybrid tetramers exhibit cooperative, potent, and complete (100%) AMP inhibition if at least one functional AMP binding site exists in the top and bottom halves of the tetramer. Furthermore, titrations of hybrid tetramers with AMP, monitored by a tryptophan reporter group, reveal cooperativity and fluorescence changes consistent with an R- to T-state transition, provided that again at least one functional AMP site exists in the top and bottom halves of the tetramer. In contrast, hybrid tetramers, which have functional AMP binding sites in only one half (top/bottom), exhibit an R- to T-state transition and complete AMP inhibition, but without cooperativity. Evidently, two pathways of allosteric inhibition of fructose-1,6-bisphosphatase are possible, only one of which is cooperative.     

The relationship between effector binding and inhibition of activity in D-3-phosphoglycerate dehydrogenase

The binding of L-serine to phosphoglycerate dehydrogenase from Escherichia coli displays elements of both positive and negative cooperativity. At pH 7.5, approximately 2 mol of serine are bound per mole of tetrameric enzyme. A substantial degree of positive cooperativity is seen for the binding of the second ligand, but the binding of the third and fourth ligand display substantial negative cooperativity. The data indicate a state of approximately 50% inhibition when only one serine is bound and approximately 80-90% inhibition when two serines are bound. This is consistent with the tethered domain hypothesis that has been presented previously. Comparison of the data derived directly from binding stoichiometry to the binding constants determined from the best fit to the Adair equation, produce a close agreement, and reinforce the general validity of the derived binding constants. The data also support the conclusion that the positive cooperativity between the binding to the first and second site involves binding sites at opposite interfaces over 110 A apart. Thus, an order of binding can be envisioned where the binding of the first ligand initiates a conformational transition that allows the second ligand to bind with much higher affinity at the opposite interface. This is followed by the third ligand, which binds with lesser affinity to one of the two already occupied interfaces, and in so doing, completes a global conformational transition that produces maximum inhibition of activity and an even lower affinity for the fourth ligand, excluding it completely. Thus, maximal inhibition is accomplished with less than maximal occupancy of effector sites through a mechanism that displays strong elements of both positive and negative cooperativity.     

Binding of AMP to two of the four subunits of pig kidney fructose-1,6-bisphosphatase induces the allosteric transition.

To study the allosteric transition in pig kidney fructose 1,6-bisphosphatase (FBPase), we constructed hybrids in which subunits have either their active or regulatory sites rendered nonfunctional by specific mutations. This was accomplished by the coexpression of the enzyme from a plasmid that contained two slightly different copies of the cDNA. To resolve and purify each of the hybrid enzymes, six aspartic acid codons were added before the termination codon of one of the cDNAs. The addition of these Asp residues to the protein did not alter the kinetic or allosteric properties of the resulting FBPase. Expression of the enzyme from a dual-gene plasmid resulted in the production of a set of five different enzymes (two homotetramers and three hybrid tetramers) that could be purified by a combination of affinity and anion-exchange chromatography because of the differential charge on each of these species. The hybrid with one subunit that only had a functional regulatory site (R) and three subunits that only had a functional active site (A) exhibited biphasic AMP inhibition. Analysis of these data suggest that the binding of AMP to the R subunit is able to globally alter the activity of the other three A subunits. The hybrid composed of two R and two A subunits is completely inhibited at an AMP concentration of approximately 0.5 mM, 100-fold less than the concentration required to fully inhibit the A(4) enzyme. The monophasic nature of this cooperative inhibition suggests that the AMP binding to the two R subunits is sufficient to completely inhibit the enzyme and suggests that the binding of AMP to only two of the four subunits of the enzyme induces the global allosteric transition from the R to the T state.     

Specific interactions at the regulatory domain-substrate binding domain interface influence the cooperativity of inhibition and effector binding in Escherichia coli D-3-phosphoglycerate dehydrogenase

The crystal structure of d-3-phosphoglycerate dehydrogenase reveals a limited number of contacts between the regulatory and substrate binding domains of each subunit in the tetrameric enzyme. These occur between the side chains of Arg-339, Arg-405, and Arg-407 in the regulatory domain and main chain carbonyls in the substrate binding domain. In addition, Arg-339 participates in a hydrogen bonding network within the regulatory domain involving Arg-338 and Tyr-410, the C-terminal residue of the enzyme subunit. Mutagenic analysis of these residues produce profound effects on the enzyme's sensitivity to serine, the cooperativity of serine inhibition, and in some cases, the apparent overall conformation of the enzyme. Mutations of Arg-405 and Arg-407, which span the interface where the two domains come together, reduce the cooperativity of inhibition and increase the sensitivity of the enzyme to serine concentration. Serine binding studies with Arg-407 converted to Ala demonstrate that cooperativity of serine binding is also significantly reduced in a manner similar to the reduction in the cooperativity of inhibition. Mutations of Tyr-410 and Arg-338 decrease the sensitivity to serine without an appreciable effect on the cooperativity of inhibition. In the case of Tyr-410, a deletion mutant demonstrates that this effect is due to the loss of the C-terminal carboxyl group rather than the tyrosine side chain. All mutations of Arg-339, with the exception of its conversion to Lys, had profound effects on the stability of the enzyme. In general, those mutants that decrease sensitivity to serine are those that participate mainly in intradomain interactions and may also directly affect the serine binding sites themselves. Those mutants that decrease cooperativity are those that participate in interdomain interaction within the subunit. The observation that the mutants that decrease cooperativity also increase sensitivity to serine suggests a potential separation of pathways between how the simple act of serine binding results in noncooperative active site inhibition in the first place and how serine binding also leads to cooperativity between sites in the native enzyme.     

Phosphate ion partially relieves the cooperativity of effector binding in D-3-phosphoglycerate dehydrogenase without altering the cooperativity of inhibition

The binding of L-serine to phosphoglycerate dehydrogenase from E. coli displays elements of both positive and negative cooperativity. In addition, the inhibition of enzymatic activity by L-serine is also cooperative with Hill coefficients greater than 1. However, phosphate buffer significantly reduces the cooperative effects in serine binding without affecting the cooperativity of inhibition of activity. The maximal degree of inhibition and fluorescence quenching in Tris buffer occurs when an average of two serine binding sites out of four are occupied. This value increases to three out of the four sites at maximal levels of inhibition and quenching in phosphate buffer. The increase from two to three sites appears to be due to the ability of phosphate to reduce the site to site cooperative effects and render each ligand binding site less dependent on each other. The correlation between the level of inhibition and the fractional site occupancy indicates that in Tris buffer, one serine is bound to each interface at maximal effect. In the presence of phosphate, the order of binding appears to change so that both sites at one interface fill before the first site at the opposite interface is occupied. In each case, there is a good correlation between serine binding, conformational change at the regulatory site interfaces, and inhibition of enzyme activity. The observation that phosphate does not appear to have a similar effect on the cooperativity of inhibition of enzymatic activity suggests that there are two distinct cooperative pathways at work: one path between the four serine binding sites, and one path between the serine binding sites and the active sites.     

A quantitative model for allosteric control of purine reduction by murine ribonucleotide reductase

The reduction of purine nucleoside diphosphates by murine ribonucleotide reductase requires catalytic (R1) and free radical-containing (R2) enzyme subunits and deoxynucleoside triphosphate allosteric effectors. A quantitative 16 species model is presented, in which all pertinent equilibrium constants are evaluated, that accounts for the effects of the purine substrates ADP and GDP, the deoxynucleoside triphosphate allosteric effectors dGTP and dTTP, and the dimeric murine R2 subunit on both the quaternary structure of murine R1 subunit and the dependence of holoenzyme (R1(2)R2(2)) activity on substrate and effector concentrations. R1, monomeric in the absence of ligands, dimerizes in the presence of substrate, effectors, or R2(2) because each of these ligands binds R1(2) with higher affinity than R1 monomer. This leads to apparent positive heterotropic cooperativity between substrate and allosteric effector binding that is not observed when binding to the dimeric protein itself is evaluated. Allosteric activation results from an increase in k(cat) for substrate reduction upon binding of the correct effector, rather than from heterotropic cooperativity between effector and substrate. Neither the allosteric site nor the active site displays nucleotide base specificity: dissociation constants for dGTP and dTTP are nearly equivalent and K(m) and k(cat) values for both ADP and GDP are similar. R2(2) binding to R1(2) shows negative heterotropic cooperativity vis-à-vis effectors but positive heterotropic cooperativity vis-à-vis substrates. Binding of allosteric effectors to the holoenzyme shows homotropic cooperativity, suggestive of a conformational change induced by activator binding. This is consistent with kinetic results indicating full dimer activation upon binding a single equivalent of effector per R1(2)R2(2).     

Multiconformational states in phosphoglycerate dehydrogenase

Phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in the serine biosynthetic pathway. In lower plants and bacteria, the PGDH reaction is regulated by the end-product of the pathway, serine. The regulation occurs through a V(max) mechanism with serine binding and inhibition occurring in a cooperative manner. The three-dimensional structure of the serine inhibited enzyme, determined by previous work, showed a tetrameric enzyme with 222 symmetry and an unusual overall toroidal appearance. To characterize the allosteric, cooperative effects of serine, we identified W139G PGDH as an enzymatically active mutant responsive to serine but not in a cooperative manner. The position of W139 near a subunit interface and the active site cleft suggested that this residue is a key player in relaying allosteric effects. The 2.09 A crystal structure of W139G-PGDH, determined in the absence of serine, revealed major quaternary and tertiary structural changes. Contrary to the wildtype enzyme where residues encompassing residue 139 formed extensive intersubunit contacts, the corresponding residues in the mutant were conformationally flexible. Within each of the three-domain subunits, one domain has rotated approximately 42 degrees relative to the other two. The resulting quaternary structure is now in a novel conformation creating new subunit-to-subunit contacts and illustrates the unusual flexibility in this V(max) regulated enzyme. Although changes at the regulatory domain interface have implications in other enzymes containing a similar regulatory or ACT domain, the serine binding site in W139G PGDH is essentially unchanged from the wildtype enzyme. The structural and previous biochemical characterization of W139G PGDH suggests that the allosteric regulation of PGDH is mediated not only by changes occurring at the ACT domain interface but also by conformational changes at the interface encompassing residue W139.     

Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site

Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.     

D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis is a link between the Escherichia coli and mammalian enzymes

D-3-Phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis has been isolated to homogeneity and displays an unusual relationship to the Escherichia coli and mammalian enzymes. In almost all aspects investigated, the M. tuberculosis enzyme shares the characteristics of the mammalian PGDHs. These include an extended C-terminal motif, substrate inhibition kinetics, dependence of activity levels and stability on ionic strength, and the inability to utilize alpha-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by L-serine, with an I(0.5) = 30 microm. The mammalian enzymes are not inhibited by L-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis.     

Identification of amino acid residues contributing to the mechanism of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase

L-Serine inhibits the catalytic activity of Escherichia coli D-3-phosphoglycerate dehydrogenase (PGDH) by binding to its regulatory domain. This domain is a member of the ACT domain family of regulatory domains that are modulated by small molecules. A comparison of the phi and psi torsional angle differences between the crystal structures of PGDH solved in the presence and in the absence of L-serine demonstrated a clustering of significant angle deviations in the regulatory domain. A similar clustering was not observed in either of the other two structural domains of PGDH. In addition, significant differences were also observed at the active site and in the Trp-139 loop. To determine if these residues were functionally significant and not just due to other factors such as crystal packing, mutagenic analysis of these residues was performed. Not unexpectedly, this analysis showed that residues that affected the kcat/Km were grouped around the active site and those that affected the serine sensitivity were grouped in the regulatory domain. However, more significantly, residues that affected the cooperativity of inhibition of activity were identified at both locations. These latter residues represent structural elements that participate in both the initial and the ultimate events of the transfer of cooperative behavior from the regulatory domain to the active site. As such, their identification will assist in the elucidation of the pathway of cooperative interaction in this enzyme as well as in the elucidation of the regulatory mechanism of the ACT domain in general.     

The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase

D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway. Previous results have shown that inhibition by serine has a large effect on Vmax and only a small or negligible effect on Km. PGDH is thus classified as a V-type allosteric enzyme. In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis. These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue. Interestingly, Trp-139' and Lys-141' are part of the polypeptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site. Mutants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subunits.     

Mechanism of substrate inhibition in Escherichia coli phosphofructokinase

Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites, which bind the substrates fructose-6-phosphate (Fru-6-P) and MgATP. In the presence of low concentrations of Fru-6-P, MgATP displays substrate inhibition. Previous proposals to explain this substrate inhibition have included both kinetic and allosteric mechanisms. We have isolated hybrid tetramers containing one wild type subunit and three mutated subunits (1:3). The mutated subunits contain mutations that decrease affinity for Fru-6-P (R243E) or MgATP (F76A/R77D/R82A) allowing us to systematically simplify the possible allosteric interactions between the two substrates. In the absence of a rate equation to explain the allosteric effects in a tetramer, the data have been compared to simulated data for an allosteric dimer. Since the apparent substrate inhibition caused by MgATP binding is not seen in hybrid tetramers with only a single native MgATP binding site, the proposed kinetic mechanism is not able to explain this phenomenon. The data presented are consistent with an allosteric antagonism between MgATP in one active site and Fru-6-P in a second active site.     

Importance of domain closure for the catalysis and regulation of Escherichia coli aspartate transcarbamoylase

Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.     

Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe-4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and β domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron-sulfur center while the β domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 β domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni-Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with K(i) values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron-sulfur complex.     

Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus

A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described. The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP). Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site. Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification. The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 A apart, respectively. The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy. Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH. Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme. The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding. The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.     

Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta2 crystallin

Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.     

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.     

Characterization of a second ligand binding site of the insulin receptor

Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the alpha subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K(d) of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site.