Dynamic dissociating homo-oligomers and the control of protein function

Homo-oligomeric protein assemblies are known to participate in dynamic association/disassociation equilibria under native conditions, thus creating an equilibrium of assembly states. Such quaternary structure equilibria may be influenced in a physiologically significant manner either by covalent modification or by the non-covalent binding of ligands. This review follows the evolution of ideas about homo-oligomeric equilibria through the 20th and into the 21st centuries and the relationship of these equilibria to allosteric regulation by the non-covalent binding of ligands. A dynamic quaternary structure equilibria is described where the dissociated state can have alternate conformations that cannot reassociate to the original multimer; the alternate conformations dictate assembly to functionally distinct alternate multimers of finite stoichiometry. The functional distinction between different assemblies provides a mechanism for allostery. The requirement for dissociation distinguishes this morpheein model of allosteric regulation from the classical MWC concerted and KNF sequential models. These models are described alongside earlier dissociating allosteric models. The identification of proteins that exist as an equilibrium of diverse native quaternary structure assemblies has the potential to define new targets for allosteric modulation with significant consequences for further understanding and/or controlling protein structure and function. Thus, a rationale for identifying proteins that may use the morpheein model of allostery is presented and a selection of proteins for which published data suggests this mechanism may be operative are listed.     

Single amino acid mutations alter the distribution of human porphobilinogen synthase quaternary structure isoforms (morpheeins)

Porphobilinogen synthase (PBGS) is an obligate oligomer that can exist in functionally distinct quaternary states of different stoichiometries, which are called morpheeins. The morpheein concept describes an ensemble of quaternary structure isoforms wherein different structures of the monomer dictate different multiplicities of the oligomer (Jaffe, E. K. (2005) Trends Biochem. Sci. 30, 490-497). Human PBGS assembles into long-lived morpheeins and has been shown to be capable of forming either a high activity octamer or a low activity hexamer (Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A., and Jaffe, E. K. (2003) Nat. Struct. Biol. 10, 757-763). All PBGS monomers contain an alphabeta-barrel domain and an N-terminal arm domain. The N-terminal arm structure varies among PBGS morpheeins, and the spatial relationship between the arm and the barrel dictates the different quaternary assemblies. We have analyzed the structures of human PBGS morpheeins for key interactions that would be predicted to affect the oligomeric assembly. Examples of individual mutations that shift assembly of human PBGS away from the native octamer are R240A and W19A. The alternate morpheeins of human PBGS variants R240A and W19A are chromatographically separable from each other and kinetically distinct; their structure and dynamics have been characterized by native gel electrophoresis, dynamic light scattering, and analytical ultracentrifugation. R240A assembles into a metastable hexamer, which can undergo a reversible conversion to the octamer in the presence of substrate. The metastable nature of the R240A hexamer supports the hypothesis that octameric and hexameric morpheeins of PBGS are very close in energy. W19A assembles into a mixture of dimers, which appear to be stable.     

Substrate-induced interconversion of protein quaternary structure isoforms

Human porphobilinogen synthase (PBGS) can exist in two dramatically different quaternary structure isoforms, which have been proposed to be in dynamic equilibrium. The quaternary structure isoforms of PBGS result from two alternative conformations of the monomer; one monomer structure assembles into a high activity octamer, whereas the other monomer structure assembles into a low activity hexamer. The kinetic behavior of these oligomers led to the hypothesis that turnover facilitates the interconversion of the oligomeric structures. The current work demonstrates that the interactions of ligands at the enzyme active site promote the structural interconversion between human PBGS quaternary structure isoforms, favoring formation of the octamer. This observation illustrates that the assembly and disassembly of oligomeric proteins can be facilitated by the protein motions that accompany enzymatic catalysis.     

Structural Basis for Morpheein-type Allosteric Regulation of Escherichia coli Glucosamine-6-phosphate Synthase: EQUILIBRIUM BETWEEN INACTIVE HEXAMER AND ACTIVE DIMER*

The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.     

Kinetics and thermodynamics of the interchange of the morpheein forms of human porphobilinogen synthase

A morpheein is a homo-oligomeric protein that can adopt different nonadditive quaternary assemblies (morpheein forms) with different functionalities. The human porphobilinogen synthase (PBGS) morpheein forms are a high activity octamer, a low activity hexamer, and two structurally distinct dimer conformations. Conversion between hexamer and octamer involves dissociation to dimers, conformational change at the dimer level, followed by association to the alternate assembly. The current work promotes an alternative and novel view of the physiologically relevant dimeric structures, which are derived from the crystal structures, but are distinct from the asymmetric units of their crystal forms. Using a well characterized heteromeric system (WT+F12L; Tang, L. et al. (2005) J. Biol. Chem. 280, 15786-15793), extensive study of the human PBGS morpheein reequilibration process now reveals that the intervening dimers do not dissociate to monomers. The morpheein equilibria of wild type (WT) human PBGS are found to respond to changes in pH, PBGS concentration, and substrate turnover. Notably, the WT enzyme is predominantly an octamer at neutral pH, but increasing pH results in substantial conversion to lower order oligomers. Most significantly, the free energy of activation for the conversion of WT+F12L human PBGS heterohexamers to hetero-octamers is determined to be the same as that for the catalytic conversion of substrate to product by the octamer, remarkably suggesting a common rate-limiting step for both processes, which is postulated to be the opening/closing of the active site lid.     

Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer.

Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P-450s, containing reductase and mono-oxygenase domains within one polypeptide and requiring tetrahydrobiopterin as cofactor. To investigate whether tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular dichroism, velocity sedimentation, dynamic light scattering and SDS-polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha-helix, 14% antiparallel beta-sheet, 7% parallel beta-sheet, 19% turns and 31% other structures. The secondary structure of neuronal NOS was neither modulated nor stabilized by tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS-polyacrylamide gel electrophoresis revealed that tetrahydrobiopterin and L-arginine synergistically convert neuronal NOS into an exceptionally stable, non-covalently linked homodimer surviving in 2% SDS and 5% 2-mercaptoethanol. Ligand-induced formation of an SDS-resistant dimer is unprecedented and suggests a novel role for tetrahydrobiopterin and L-arginine in the allosteric regulation of protein subunit interactions.     

Dynamic cross-talk among remote binding sites: the molecular basis for unusual synergistic allostery

Allosteric regulation of protein function is critical for metabolic control. Binding of allosteric effectors elicits a functional change in a remote ligand binding site on a protein by altering the equilibrium between different forms in the protein ensemble. 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step in the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids Trp, Phe, and Tyr. Feedback regulation by the aromatic amino acids is important for controlling the cellular levels of the aromatic amino acids, and many organisms have two or more DAH7PS isozymes that show differing sensitivities to aromatic compounds. Mycobacterium tuberculosis expresses a single DAH7PS that is insensitive to the presence of a single amino acid yet shows extraordinary synergistic inhibition by combinations of the pathway end products Trp and Phe. The Trp+Phe-bound structure for M. tuberculosis DAH7PS, showing two separate binding sites occupied by Trp and Phe for each monomer of the tetrameric protein, was obtained by cocrystallization. Comparison of this structure with the ligand-free M. tuberculosis DAH7PS demonstrates that there is no significant change in conformation upon ligand binding, suggesting that contributions from altered dynamic properties of the enzyme may account for the allosteric inhibition. Isothermal titration calorimetry experiments demonstrate that the inhibitor binding sites are in direct communication. Molecular dynamics simulations reveal different changes in dynamic fluctuations upon single ligand binding compared to dual ligand binding. These changes account for the cross-talk between inhibitor binding sites and the active site, simultaneously potentiating both dual ligand binding and diminution of catalytic function.     

Collective Dynamics Underlying Allosteric Transitions in Hemoglobin

Hemoglobin is the prototypic allosteric protein. Still, its molecular allosteric mechanism is not fully understood. To elucidate the mechanism of cooperativity on an atomistic level, we developed a novel computational technique to analyse the coupling of tertiary and quaternary motions. From Molecular Dynamics simulations showing spontaneous quaternary transitions, we separated the transition trajectories into two orthogonal sets of motions: one consisting of intra-chain motions only (referred to as tertiary-only) and one consisting of global inter-chain motions only (referred to as quaternary-only). The two underlying subspaces are orthogonal by construction and their direct sum is the space of full motions. Using Functional Mode Analysis, we were able to identify a collective coordinate within the tertiary-only subspace that is correlated to the most dominant motion within the quaternary-only motions, hence providing direct insight into the allosteric coupling mechanism between tertiary and quaternary conformation changes. This coupling-motion is substantially different from tertiary structure changes between the crystallographic structures of the T- and R-state. We found that hemoglobin''s allosteric mechanism of communication between subunits is equally based on hydrogen bonds and steric interactions. In addition, we were able to affect the T-to-R transition rates by choosing different histidine protonation states, thereby providing a possible atomistic explanation for the Bohr effect.     

ALAD porphyria is a conformational disease

ALAD porphyria is a rare porphyric disorder, with five documented compound heterozygous patients, and it is caused by a profound lack of porphobilinogen synthase (PBGS) activity. PBGS, also called "delta-aminolevulinate dehydratase," is encoded by the ALAD gene and catalyzes the second step in the biosynthesis of heme. ALAD porphyria is a recessive disorder; there are two common variant ALAD alleles, which encode K59 and N59, and eight known porphyria-associated ALAD mutations, which encode F12L, E89K, C132R, G133R, V153M, R240W, A274T, and V275M. Human PBGS exists as an equilibrium of functionally distinct quaternary structure assemblies, known as "morpheeins," in which one functional homo-oligomer can dissociate, change conformation, and reassociate into a different oligomer. In the case of human PBGS, the two assemblies are a high-activity octamer and a low-activity hexamer. The current study quantifies the morpheein forms of human PBGS for the common and porphyria-associated variants. Heterologous expression in Escherichia coli, followed by separation of the octameric and hexameric assemblies on an ion-exchange column, showed that the percentage of hexamer for F12L (100%), R240W (80%), G133R (48%), C132R (36%), E89K (31%), and A274T (14%) was appreciably larger than for the wild-type proteins K59 and N59 (0% and 3%, respectively). All eight porphyria-associated variants, including V153M and V275M, showed an increased propensity to form the hexamer, according to a kinetic analysis. Thus, all porphyria-associated human PBGS variants are found to shift the morpheein equilibrium for PBGS toward the less active hexamer. We propose that the disequilibrium of morpheein assemblies broadens the definition of conformational diseases beyond the prion disorders and that ALAD porphyria is the first example of a morpheein-based conformational disease.     

Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing

The bacterial Sm-like protein Hfq forms a ring-shaped homo-hexamer that is necessary for Hfq to bind nucleic acids and to act in small noncoding RNA regulation. Using semi-native gels and fluorescence anisotropy, we show that Hfq undergoes a cooperative conformational change from monomer to hexamer around 1 μM protein, which is comparable to the in vivo concentration of Hfq and above the dissociation constant of the Hfq hexamer from many RNA substrates. Above 2 μM protein, Hfq hexamers associate in high-molecular-weight complexes. Mutations that impair RNA binding to the proximal face strongly destabilize the hexamer, while the mutation R16A near the outer rim prevents hexamer association. Stopped-flow fluorescence resonance energy transfer experiments showed that Hfq subunits interact within a few seconds, suggesting that Hfq monomers, hexamers and multi-hexamer complexes are in dynamic equilibrium. Finally, we show that Hfq is most active in RNA annealing when the hexamer is present. These results suggest that RNA binding is coupled to hexamer assembly and that the biochemical activity of Hfq reflects the equilibrium between different quaternary structures.     

Crystal structures of Escherichia coli glycerol kinase variant S58-->W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits. However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol. This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.     

Evolution of allosteric models for hemoglobin

We compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)- and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond - millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.     

UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces

UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132) to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters revealed structurally dependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium to function efficiently and preserve regulated activity in the cell.     

Dynamic Allostery Controls Coat Protein Conformer Switching during MS2 Phage Assembly

Previously, an RNA stem-loop (TR) encompassing 19 nt of the genome of bacteriophage MS2 was shown to act as an allosteric effector of conformational switching in the coat protein during in vitro capsid assembly. TR RNA binding to symmetric coat protein dimers results in conformational changes, principally at the FG-loop connecting the F and G β-strands in each subunit, yielding an asymmetric structure. The FG-loops define the quasi-equivalent conformers of the coat protein subunit (A, B, and C) in the T = 3 capsid. Efficient assembly of this capsid in vitro requires that both symmetrical and asymmetrical forms of the coat protein dimer be present in solution, implying that they closely resemble the quasi-equivalent dimers (A/B and C/C) seen in the final capsid. Experiments show that assembly can be triggered by a number of RNA stem-loops unrelated to TR in sequence and detailed secondary structure, suggesting that there is little sequence specificity to the allosteric effect. Since the stem-loop binding site on the coat protein dimer is distal to the FG-loops the mechanism of this switching effect needs to be investigated. We have analyzed the vibrational modes of both TR-bound and RNA-free coat protein dimers using an all-atom normal-mode analysis. The results suggest that asymmetric contacts between the A-duplex RNA phosphodiester backbone and the EF-loop in one coat protein subunit result in the FG-loop of that subunit becoming more dynamic, whilst the equivalent loop on the other monomer decreases its mobility. The increased dynamic behaviour occurs in the FG-loop of the subunit required to undergo the largest conformational change when adopting the quasi-equivalent B conformation. The free energy barrier on the pathway to form this new structure would consequently be reduced compared to the unbound subunit. Our results also imply that the allosteric effect should be independent of the base sequence of the bound stem-loop, as observed experimentally. As a test of this model, we also examined the vibrational modes of a known assembly mutant, W82R, which cannot assemble beyond dimer. This mutation leads to an increased mobility of the DE-loop rather than the FG-loop after TR binding, consistent with the non-assembling phenotype of this mutant protein.     

Conformational equilibrium of an enzyme catalytic site in the allosteric transition.

The dynamic equilibrium of a catalytic site between active and inactive conformations, the missing link between the structure and function of allosteric enzymes, was identified using protein engineering and NMR techniques. Kinetic analyses of the wild-type and three mutants of Thermus L-lactate dehydrogenase established that the allosteric property of the enzyme is associated with a concerted transition between the high-affinity (R) and low-affinity (T) states. By introducing mutations, we prepared an enzyme in which the R and T states were balanced. The conformation of the enzyme-bound coenzyme, NAD+, which interacts directly with the substrate, was analyzed using NMR spectroscopy. NAD+ bound to the mutant enzyme was in a conformational mixture of the active and inactive forms, while NAD+ took on predominantly one of the two forms when it was bound to the other enzymes we had analyzed. We interpret this to mean that the catalytic site is in equilibrium between the two conformations. The ratio of the conformers of each enzyme agreed with the [T]/[R] ratio as determined by kinetic analyses. Therefore, it is the identified conformational equilibrium of the catalytic site that governs the allosteric regulation of the enzyme activity.     

Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes

A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non‐polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild‐type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate‐analog bound conformation and the third hinge angle is intermediate to the other two. The active‐site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active‐site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active‐site conformations.     

Allosteric regulation and communication between subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus

Uracil phosphoribosyltransferase (UPRTase) catalyzes the conversion of 5-phosphate-alpha-1-diphosphate (PRPP) and uracil to uridine 5'-monophosphate (UMP) and diphosphate. The UPRTase from Sulfolobus solfataricus has a unique regulation by nucleoside triphosphates compared to UPRTases from other organisms. To understand the allosteric regulation, crystal structures were determined for S. solfataricus UPRTase in complex with UMP and with UMP and the allosteric inhibitor CTP. Also, a structure with UMP bound in half of the active sites was determined. All three complexes form tetramers but reveal differences in the subunits and their relative arrangement. In the UPRTase-UMP complex, the peptide bond between a conserved arginine residue (Arg80) and the preceding residue (Leu79) adopts a cis conformation in half of the subunits and a trans conformation in the other half and the tetramer comprises two cis-trans dimers. In contrast, four identical subunits compose the UPRTase-UMP-CTP tetramer. CTP binding affects the conformation of Arg80, and the Arg80 conformation in the UPRTase-UMP-CTP complex leaves no room for binding of the substrate PRPP. The different conformations of Arg80 coupled to rearrangements in the quaternary structure imply that this residue plays a major role in regulation of the enzyme and in communication between subunits. The ribose ring of UMP adopts alternative conformations in the cis and trans subunits of the UPRTase-UMP tetramer with associated differences in the interactions of the catalytically important Asp209. The active-site differences have been related to proposed kinetic models and provide an explanation for the regulatory significance of the C-terminal Gly216.     

The structural basis for allosteric inhibition of a threonine-sensitive aspartokinase

The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK ( Angeles, T. S., Hunsley, J. R., and Viola, R. E. (1992) Biochemistry 31, 799-805 ). Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation.     

Structure and function of malic enzymes, a new class of oxidative decarboxylases

Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interface is more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composed of four structural domains, which show a different folding topology from those of the other oxidative decarboxylases. The active center is located at the interface between domains B and C. For human mitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and II, which is mediated by the movements of domains A and D. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analogue at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-112 and lysine-183 as the general acid and base, respectively. In addition, a divalent metal ion (Mn(2+) or Mg(2+)) is essential in helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternary structural integrity of the enzyme.