A model for the regulation of D-3-phosphoglycerate dehydrogenase,a Vmax-type allosteric enzyme.

Escherichia coli D-3-phosphoglycerate dehydrogenase (ePGDH) is a tetramer of identical subunits that is allosterically inhibited by L-serine, the end product of its metabolic pathway. Because serine binding affects the velocity of the reaction and not the binding of substrate or cofactor, the enzyme is classified as of the Vmax type. Inhibition by a variety of amino acids and analogues of L-serine indicate that all three functional groups of serine are required for optimal interaction. Removing or altering any one functional group results in an increase in inhibitory concentration from micromolar to millimolar, and removal or alteration of any two functional groups removes all inhibitory ability. Kinetic studies indicate at least two serine-binding sites, but the crystal structure solved in the presence of bound serine and direct serine-binding studies show that there are a total of four serine-binding sites on the enzyme. However, approximately 85% inhibition is attained when only two sites are occupied. The three-dimensional structure of ePGDH shows that the serine-binding sites reside at the interface between regulatory domains of adjacent subunits. Two serine molecules bind at each of the two regulatory domain interfaces in the enzyme. When all four of the serines are bound, 100% inhibition of activity is seen. However, because the domain contacts are symmetrical, the binding of only one serine at each interface is sufficient to produce approximately 85% inhibition. The tethering of the regulatory domains to each other through multiple hydrogen bonds from serine to each subunit apparently prevents the body of these domains from undergoing the reorientation that must accompany a catalytic cycle. It is suggested that part of the conformational change may involve a hinge formed in the vicinity of the union of two antiparallel beta-sheets in the regulatory domains. The tethering function of serine, in turn, appears to prevent the substrate-binding domain from closing the cleft between it and the nucleotide-binding domain, which may be necessary to form a productive hydrophobic environment for hydride transfer. Thus, the structure provides a plausible model that is consistent with the binding and inhibition data and that suggests that catalysis and inhibition in this rare Vmax-type allosteric enzyme is based on the movement of rigid domains about flexible hinges.     

Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.     

On the structure and operation of type I DNA restriction enzymes.

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.     

Enhanced expression of the Escherichia coli serA gene in a plasmid vector. Purification, crystallization, and preliminary X-ray data of D-3 phosphoglycerate dehydrogenase

The serA gene of Escherichia coli strain K-12, which codes for the cooperative allosteric enzyme D-3-phosphoglycerate dehydrogenase, was inserted into an inducible expression vector which produced phosphoglycerate dehydrogenase as 8% of the soluble protein of E. coli. The purified protein was used to grow several different single crystal forms. One of these, with space group P2(1), appears to contain all four subunits of the tetrameric enzyme in the asymmetric unit and diffracts to sufficient resolution to allow determination of the structure of phosphoglycerate dehydrogenase.     

The first crystal structure of L-threonine dehydrogenase

L-threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.     

Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution.

The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution. The final R-factor is 0.185 for 20,307 reflections. The crystal asymmetric unit has one subunit consisting of 345 amino acid residues. The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs. The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases. A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry. Close subunit contacts are also present at two alpha-helices in the second domain. These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer. Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding. These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme.     

Structure of Plasmodium falciparum triose-phosphate isomerase-2-phosphoglycerate complex at 1.1-A resolution

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.     

The nucleotide sequence of the serA gene of Escherichia coli and the amino acid sequence of the encoded protein, D-3-phosphoglycerate dehydrogenase

The nucleotide sequence of serA, the structural gene of Escherichia coli which codes for D-3-phosphoglycerate dehydrogenase, has been determined. The structural gene contains 1233 nucleotides which code for the 409 amino acids of the subunit of the tetrameric enzyme, as well as the initiator methionine, which is cleaved from the mature protein, and the termination codon. The majority of the primary structure of the enzyme has been confirmed by automated Edman degradation of peptide fragments produced by a variety of cleavage agents. Comparison of the amino acid sequence of phosphoglycerate dehydrogenase with other NAD-dependent oxidoreductases reveals less than 20% homology, although conservation of certain specific residues in the coenzyme binding domain appears to be evident.     

Crystal structure of D-erythronate-4-phosphate dehydrogenase complexed with NAD

Pyridoxal-5′-phosphate (the active form of vitamin B6) is an essential cofactor in many enzymatic reactions. While animals lack any of the pathways for de novo synthesis and salvage of vitamin B6, it is synthesized by two distinct biosynthetic routes in bacteria, fungi, parasites, and plants. One of them is the PdxA/PdxJ pathway found in the γ subdivision of proteobacteria. It depends on the pdxB gene, which encodes erythronate-4-phosphate dehydrogenase (PdxB), a member of the d-isomer specific 2-hydroxyacid dehydrogenase superfamily. Although three-dimensional structures of other functionally related dehydrogenases are available, no structure of PdxB has been reported. To provide the missing structural information and to gain insights into the catalytic mechanism, we have determined the first crystal structure of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa in the ligand-bound state. It is a homodimeric enzyme consisting of 380-residue subunits. Each subunit consists of three structural domains: the lid domain, the nucleotide-binding domain, and the C-terminal dimerization domain. The latter domain has a unique fold and is largely responsible for dimerization. Interestingly, two subunits of the dimeric enzyme are bound with different combinations of ligands in the crystal and they display significantly different conformations. Subunit A is bound with NAD and a phosphate ion, while subunit B, with a more open active site cleft, is bound with NAD and l(+)-tartrate. Our structural data allow a detailed understanding of cofactor and substrate recognition, thus providing substantial insights into PdxB catalysis.     

Quantitative relationships of site to site interaction in Escherichia coli D-3-phosphoglycerate dehydrogenase revealed by asymmetric hybrid tetramers

A set of asymmetric hybrid tetramers of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) have been made by gene co-expression and KSCN-induced dimer exchange. These tetramers contain varied numbers of active sites and effector binding sites arranged in different orientations within the tetramer. They reveal that PGDH displays half-of-the-sites activity with respect to its active sites and that the two sites that are active at any particular time lie in subunits on either side of the nucleotide binding domain interface. Half-of-the-sites functionality is also observed for the effector even though all four sites eventually bind effector. That is, only two effector sites need to be occupied for maximum inhibition. Binding of the last two effector molecules does not contribute functionally to inhibition of activity. Furthermore, positive cooperativity of inhibition of activity by the effector is completely dependent on the positive cooperativity of binding of the effector. Binding of the first effector molecule produces a conformational change that essentially completely inhibits the active site within the subunit to which it binds and produces an approximate 33% inhibition of the active site in the subunit to which it is not bound. Binding of the second effector at the opposite regulatory domain interface completes the inhibition of activity. This simple relationship defines the positional and quantitative influence of effector ligand binding on activity and can be used to predict the maximum level of inhibition of individual hybrid tetramers. In addition, the site-specific quantitative relationship of effector binding to individual active sites can be used to model the inhibition profile with relatively good agreement. These simple rules for the site to site interaction in PGDH provide significant new insight into the mechanism of allostery of this enzyme.     

Self-assembly of the isolated KCNQ2 subunit interaction domain

Mutations in the KCNQ2 gene cause myokymia and neonatal epilepsy, indicating that this K(+) channel regulates the excitability of lower motoneurons and CNS neurons. Little is known about the parameters that direct the assembly of this multimeric molecule and other KCNQ subunits. Here, we show that the carboxy-terminal subunit interaction domain of KCNQ2 autonomously folds and assembles into tetramers. This domain contains a bipartite coiled-coil motif. Whereas structural integrity of the second coiled-coil motif is crucial for tetramer formation, that of the first motif is less important. These data suggest a crucial role of coiled-coil motifs in tetrameric KCNQ channel assembly.     

The effect of hinge mutations on effector binding and domain rotation in Escherichia coli D-3-phosphoglycerate dehydrogenase

D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli contains two Gly-Gly sequences that have been shown previously to have the characteristics of hinge regions. One of these, Gly(336)-Gly(337), is found in the loop between the substrate binding domain and the regulatory domain. Changing these glycine residues to valine affected the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity was caused primarily by a decrease in the affinity of the enzyme for L-serine. These mutations also affected the domain rotation of the subunits in response to L-serine binding. A major conclusion of this study was that it defines a minimal limit on the necessary conformational changes leading to inhibition of enzyme activity. That is, some of the conformational differences seen in the native enzyme upon L-serine binding are not critical for inhibition, whereas others are maintained and may play important roles in inhibition and cooperativity. The structure of G336V demonstrates that the minimal effect of L-serine binding leading to inhibition of enzyme activity requires a domain rotation of approximately only 6 degrees in just two of the four subunits of the enzyme that are oriented diagonally across from each other in the tetramer. Moreover the structures show that both pairs of Asn190 to Asn190 hydrogen bonds across the subunit interfaces are necessary for activity. These observations are consistent with the half-the-sites activity, flip-flop mechanism proposed for this and other similar enzymes and suggest that the Asn190 hydrogen bonds may function in the conformational transition between alternate half-the-site active forms of the enzyme.     

Structural basis for retinoic X receptor repression on the tetramer

Retinoic X receptor (RXR) is a master nuclear receptor in the processes of cell development and homeostasis. Unliganded RXR exists in an autorepressed tetramer, and agonists can induce RXR dimerization and coactivator recruitment for activation. However, the molecular mechanisms involving the corepressor recruitment and antagonist-mediated repression of RXR are still elusive. Here we report the crystal structure of RXRα ligand-binding domain (LBD) complexed with silencing mediator for retinoid and thyroid hormone receptors (SMRT) corepressor motif. As the first structural report on the unliganded nuclear receptor bound to the corepressor motif, RXRαLBD-SMRT exhibits a significant structural rearrangement, compared with apoRXRαLBD tetramer. To elucidate further the molecular determinants for RXR repression by its antagonist, we also determine the crystal structure of RXRαLBD-SMRT complexed with the identified antagonist rhein. In the structure, two rhein molecules and two SMRT peptides are in the RXRαLBD tetramer, different from the case in RXRαLBD-SMRT structure, where four SMRT peptides bind to RXRαLBD tetramer. It seems that rhein induces a displacement of SMRT motif by activation function 2 (AF-2) motif binding to the receptor. Combining our current work with the published results, structural superposition of RXRαLBDs in different states reveals that RXR uses an overlapped binding site for coactivator, corepressor, and AF-2 motifs, whereas the AF-2 motif adopts different conformations for agonist or antagonist interaction and coactivator or corepressor recruitment. Taken together, we thus propose a molecular model of RXR repression on the tetramer.     

FDH: an aldehyde dehydrogenase fusion enzyme in folate metabolism

FDH (10-formyltetrahydrofolate dehydrogenase, Aldh1L1, EC 1.5.1.6) converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate and CO(2) in a NADP(+)-dependent reaction. It is a tetramer of four identical 902 amino acid residue subunits. The protein subunit is a product of a natural fusion of three unrelated genes and consists of three distinct domains. The N-terminal domain of FDH (residues 1-310) carries the folate binding site and shares sequence homology and structural topology with other enzymes utilizing 10-formyl-THF as a substrate. In vitro it functions as 10-formyl-THF hydrolase, and evidence indicate that this activity is a part of the overall FDH mechanism. The C-terminal domain of FDH (residues 400-902) originated from an aldehyde dehydrogenase-related gene and is capable of oxidation of short-chain aldehydes to corresponding acids. Similar to classes 1 and 2 aldehyde dehydrogenases, this domain exists as a tetramer and defines the oligomeric structure of the full-length enzyme. The two catalytic domains are connected by an intermediate linker (residues 311-399), which is a structural and functional homolog of carrier proteins possessing a 4'-phosphopantetheine prosthetic group. In the FDH mechanism, the intermediate linker domain transfers a formyl, covalently attached to the sulfhydryl group of the phosphopantetheine arm, from the N-terminal domain to the C-terminal domain. The overall FDH mechanism is a coupling of two sequential reactions, a hydrolase and a formyl dehydrogenase, bridged by a substrate transfer step. In this mechanism, one domain provides the folate binding site and a hydrolase catalytic center to remove the formyl group from the folate substrate, another provides a transfer vehicle between catalytic centers and the third one contributes the dehydrogenase machinery further oxidizing formyl to CO(2).     

Construction of molecular assemblies via docking: modeling of tetramers with D2 symmetry

Comparative modeling methods are commonly used to construct models of homologous proteins or oligomers. However, comparative modeling may be inapplicable when the number of subunits in a modeled oligomer is different than in the modeling template. Thus, a dimer cannot be a template for a tetramer because a new monomer-monomer interface must be predicted. We present in this study a new prediction approach, which combines protein-protein docking with either of two tetramer-forming algorithms designed to predict the structures of tetramers with D2 symmetry. Both algorithms impose symmetry constraints. However, one of them requires identification of two of the C2 dimers within the tetramer in the docking step, whereas the other, less demanding algorithm, requires identification of only one such dimer. Starting from the structure of one subunit, the procedures successfully reconstructed 16 known D2 tetramers, which crystallize with either a monomer, a dimer or a tetramer in the asymmetric unit. In some cases we obtained clusters of native-like tetramers that differ in the relative rotation of the two identical dimers within the tetramer. The predicted structural pliability for concanavalin-A, phosphofructokinase, and fructose-1,6-bisphosphatase agrees semiquantitatively with the observed differences between the several experimental structures of these tetramers. Hence, our procedure identifies a structural soft-mode that allows regulation via relative rigid-body movements of the dimers within these tetramers. The algorithm also predicted three nearly correct tetramers from model structures of single subunits, which were constructed by comparative modeling from subunits of homologous tetrameric, dimeric, or hexameric systems.     

Flexibility of the rings: structural asymmetry in the DnaB hexameric helicase

DnaB is the primary replicative helicase in Escherichia coli and the hexameric DnaB ring has previously been shown to exist in two states in the presence of nucleotides. In one, all subunits are equivalent, while in the other, there are two different subunit conformations resulting in a trimer of dimers. Under all conditions that we have used for electron microscopy, including the absence of nucleotide, some rings exist as trimers of dimers, showing that the symmetry of the DnaB hexamer can be broken prior to nucleotide binding. Three-dimensional reconstructions reveal that the N-terminal domain of DnaB makes two very different contacts with neighboring subunits in the trimer of dimers, but does not form a predicted dimer with a neighboring N-terminal domain. Within the trimer of dimers, the helicase domain exists in two alternate conformations, each of which can form symmetrical hexamers depending upon the nucleotide cofactor used. These results provide new information about the modular architecture and domain dynamics of helicases, and suggest, by comparison with the hexameric bacteriophage T7 gp4 and SV40 large T-antigen helicases, that a great structural and mechanistic diversity may exist among the hexameric helicases.     

Ligand-induced asymmetry between active sites of cytoplasmic malate dehydrogenase: a chemical modification study

The iodoacetate-dependent and iodoacetamide-dependent inhibition of cytoplasmic malate dehydrogenase (s-MDH) has been examined. We have confirmed previous reports that iodoacetate inhibits this dimeric enzyme by modifying a single active site methionine per s-MDH subunit. Time courses for the inactivation of the solution-state enzyme with both reagents indicate each s-MDH subunit is modified with equal rapidity in the absence of substrate or cofactor. However, the subunits react with distinctly different rates in the presence of cofactor or cofactor/substrate combinations, indicating some conformational asymmetry between subunits occurs when these ligands are bound. This is consistent with solution-state s-MDH behaving as a cooperative enzyme. Apo and holo crystalline s-MDH are also inhibited by iodoacetic acid. However, subunits of the crystalline enzyme are inhibited with different rates in the presence or absence of active site ligands. This suggests subunit conformations of the dimeric enzyme are not identical in crystalline s-MDH preparations regardless of ligand binding. Furthermore, by the criterion of inhibition rate constants, subunit conformations of the crystalline enzyme are not rigid but are perturbed by ligand binding. Comparisons of inactivation time courses for solution- and crystalline-state s-MDH suggest crystalline s-MDH exhibits at least some of the subunit asymmetry associated with the solution-state enzyme.     

Structure of betaine aldehyde dehydrogenase at 2.1 A resolution.

The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.     

Crystal structure of prephenate dehydrogenase from Aquifex aeolicus. Insights into the catalytic mechanism

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyper-thermophile Aquifex aeolicus in complex with NAD+. This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg250. In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser126 and Lys246 and the catalytic histidine, His147. Analysis of the structure further reveals that two secondary structure elements, beta3 and beta7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.     

Rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase: half-of-the-sites reactivity of the enzyme modified at arginine residues.

Modification of a single arginine residue per subunit of rabbit muscle apo-D-Glyceraldehyde-3-phosphate dehydrogenase does not affect the rate of hydrolysis of p-nitrophenyl acetate catalyzed by the enzyme, but locks the tetramer in a conformation wherein only two active sites are functioning. The modified enzyme also exhibits half-of-the sites reactivity towards iodoacetate and iodoacetamide. On the other hand, its NAD(+)-binding characteristics remain unchanged. Evidence is presented supporting the view that mechanisms of half-of-the-sites reactivity and negative cooperativity in coenzyme binding are different. The results are consistent with a built-in asymmetry of the tetramer and suggest that the arginine residue (probably Arg-231) controls the conformational transition between the asymmetric and symmetric states of the tetramer.