Changes In Quaternary Structure Research Articles

Substrate Occupancy at the Onset of Oligomeric Transitions of DegP

The protease-chaperone DegP undergoes secondary through quaternary structural changes, regulating function and preventing indiscriminate proteolysis. Several structures of DegP oligomers have been observed, including the resting state 6-mer and the 12-mer and 24-mer active states. However, the precise events of the transition between the resting and active states still need to be elucidated. We used native mass spectrometry to demonstrate that binding of multiple substrate-mimicking peptide ligands to the DegP resting state occurs prior to the transition to an active conformation. This transition occurred at a 6-mer occupancy of 40% for each peptide ligand. We observed ligand-specific 9-mer formation with a maximum load of 9 peptides, whereas other substrates led to 12-mers accommodating 24 peptides. Based on these data, we present a model for the initial steps of substrate-induced transitions from the resting to active states of DegP.

Crystal Structures of Human Muscle Fructose-1,6-Bisphosphatase: Novel Quaternary States, Enhanced AMP Affinity, and Allosteric Signal Transmission Pathway

Fructose-1,6-bisphosphatase, a key enzyme in gluconeogenesis, is subject to metabolic regulation. The human muscle isozyme is significantly more sensitive towards the allosteric inhibitor, AMP, than the liver isoform. Here we report crystal structures and kinetic studies for wild-type human muscle Fru-1,6-Pase, the AMP-bound (1.6 Å), and product-bound complexes of the Q32R mutant, which was firstly introduced by an error in the cloning. Our high-resolution structure reveals for the first time that the higher sensitivity of the muscle isozyme towards AMP originates from an additional water-mediated, H-bonded network established between AMP and the binding pocket. Also present in our structures are a metaphosphate molecule, alternate conformations of Glu97 coordinating Mg2+, and possible metal migration during catalysis. Although the individual subunit is similar to previously reported Fru-1,6-Pase structures, the tetrameric assembly of all these structures deviates from the canonical R- or T-states, representing novel tetrameric assemblies. Intriguingly, the concentration of AMP required for 50% inhibition of the Q32R mutant is increased 19-fold, and the cooperativity of both AMP and Mg2+ is abolished or decreased. These structures demonstrate the Q32R mutation affects the conformations of both N-terminal residues and the dynamic loop 52–72. Also importantly, structural comparison indicates that this mutation in helix α2 is detrimental to the R-to-T conversion as evidenced by the absence of quaternary structural changes upon AMP binding, providing direct evidence for the critical role of helix α2 in the allosteric signal transduction.

Switching between the Alternative Structures and Functions of a 2-Cys Peroxiredoxin, by Site-Directed Mutagenesis

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.

PH-induced quaternary assembly of Vitreoscilla hemoglobin: The monomer exhibits better peroxidase activity

pH-dependent (pH6.0–8.0) quaternary structural changes of ferric Vitreoscilla hemoglobin (VHb) have been investigated using dynamic light scattering. The VHb exhibits a monomeric state under neutral conditions at pH7.0, while the protein forms distinct homodimeric species at pH6.0 and 8.0, respectively. The dissociation constant obtained using the Bio-Layer Interferometry technology indicates that, at pH7.0, the monomer–monomer dissociation of VHb is about 6-fold or 5-fold higher (KD=6.34μM) compared with that at slightly acidic pH (KD=1.05μM) or slightly alkaline pH (KD=1.22μM). The pH-dependent absorption spectra demonstrate that the heme microenvironment of VHb is sensitive to the changes of pH value. The maximum absorption band of heme group of VHb shifts from 402nm to 407nm when pH changes from 6.0 to 8.0. In addition, the fluorescence emission spectra of VHb, taken at excitation wavelength of 295nm, suggest that the single Trp122 fluorescence quantum yields in VHb are decreased due to the formation of the homodimeric species. However, the circular dichroism spectra data display that the secondary structures of VHb are little affected by pH transitions. The pH-dependent peroxidase activity of VHb was also investigated in this study. The optimum pH for VHb using 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) as substrate is 7.0, which implies that the monomer state of VHb would exhibit better peroxidase activity than the homodimeric species of VHb at pH6.0 and 8.0.

Assembly-Directed Antivirals Differentially Bind Quasiequivalent Pockets to Modify Hepatitis B Virus Capsid Tertiary and Quaternary Structure

Hepatitis B virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized heteroaryldihydropyrimidine compounds but favors a unique quasiequivalent location on the capsid surface. Thus, this pocket is a promiscuous drug-binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses.

Change in single cystathionine β-synthase domain-containing protein from a bent to flat conformation upon adenosine monophosphate binding

Cystathionine β-synthase (CBS) domains are small intracellular modules that can act as binding domains for adenosine derivatives, and they may regulate the activity of associated enzymes or other functional domains. Among these, the single CBS domain-containing proteins, CBSXs, from Arabidopsis thaliana, have recently been identified as redox regulators of the thioredoxin system. Here, the crystal structure of CBSX2 in complex with adenosine monophosphate (AMP) is reported at 2.2Å resolution. The structure of dimeric CBSX2 with bound-AMP is shown to be approximately flat, which is in stark contrast to the bent form of apo-CBSXs. This conformational change in quaternary structure is triggered by a local structural change of the unique α5 helix, and by moving each loop P into an open conformation to accommodate incoming ligands. Furthermore, subtle rearrangement of the dimer interface triggers movement of all subunits, and consequently, the bent structure of the CBSX2 dimer becomes a flat structure. This reshaping of the structure upon complex formation with adenosine-containing ligand provides evidence that ligand-induced conformational reorganization of antiparallel CBS domains is an important regulatory mechanism.

Membrane Mimetics Induce Helix Formation and Oligomerization of the Chloride Intracellular Channel Protein 1 Transmembrane Domain

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The transmembrane domain (TMD), implicated in membrane penetration and pore formation, comprises helix α1 and strand β2 of the N-domain of soluble CLIC1. The mechanism by which the TMD binds, inserts, and oligomerizes in membranes to form a functional chloride channel is unknown. Here we report the secondary, tertiary, and quaternary structural changes of the CLIC1 TMD as it partitions between an aqueous and membrane-mimicking environment. A synthetic 30-mer peptide comprising the TMD was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate (SDS) micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes using far-ultraviolet circular dichroism and fluorescence spectroscopy. Data obtained in the presence of SDS micelles and POPC liposomes show that Trp35 and Cys24 have reduced solvent accessibility, indicating that the peptide adopts an inserted orientation. The peptide assumes a helical structure in the presence of these mimetics, consistent with its predicted membrane conformation. This acquisition of secondary structure is concentration-dependent, suggesting an oligomerization event. Stable dimeric and trimeric species were subsequently identified using SDS-polyacrylamide gel electrophoresis. We propose that, in the vicinity of membranes, the mixed α/β TMD in CLIC1 rearranges to form a helix that then likely dimerizes via noncovalent helix-helix interactions to form a membrane-competent protopore complex. Such oligomerization would be essential for forming a functional ion channel, given that each CLIC1 monomer possesses only a single TMD. This work highlights the central role of the TMD in CLIC1 function: It is capable of promoting membrane insertion and dimerization in the absence of the C-domain and large portions of the N-domain.

Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain

Glutamate receptor ion channels are membrane proteins that mediate excitatory synaptic transmission in the central nervous system of vertebrates. Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states. Here, we report the use of single-particle cryoelectron tomography to determine the structures, at ∼21 Å resolution, of full-length GluK2 kainate receptors trapped in antagonist-bound resting and agonist-bound desensitized states. The resting state, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2, with well-resolved proximal and distal subunits exhibiting cross-over between the twofold symmetric amino terminal domain and a twofold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD undergoes a major rearrangement, resulting in a separation of the four subunits by ∼25 Å. However, the amino terminal domain, transmembrane, and cytoplasmic regions of the receptor have similar conformations in the resting and desensitized states. The LBD rearrangement was not anticipated in prior models based on crystal structures for soluble LBD dimer assemblies, and we speculate that subunit separation allows a better match to the fourfold symmetric ion channel domain. From fits of the amino terminal domain and LBD domains into the density map of the desensitized state we have derived a structural model for differences in quaternary conformation between the resting and desensitized states.

Up-regulation of neutrophil activating protein in Helicobacter pylori under high-salt stress: Structural and phylogenetic comparison with bacterial iron-binding ferritins

It is generally accepted that most gastrointestinal diseases are probably caused by the bacterial pathogen Helicobacter pylori (H. pylori). In this study we have focused on the comparison of protein expression profiles of H. pylori grown under normal and high-salt conditions by a proteomics approach. We have identified about 190 proteins whose expression levels changed after growth at high salt concentration. Among these proteins, neutrophil-activating protein (NapA) was found to be consistently up-regulated under osmotic stress brought by high salts. We have investigated the effect of high salt on secondary and tertiary structures of NapA by circular dichroism spectroscopy followed by analytical ultracentrifugation to monitor the change of quaternary structure of recombinant NapA with increasing salt concentration. The loss of iron-binding activity of NapA coupled with noticeable energetic variation in protein association of NapA as revealed by isothermal titration calorimetry was found under high salt condition. The phylogenetic tree analysis based on sequence comparison of 16 protein sequences encompassing NapA proteins and ferritin of H. pylori and other prokaryotic organisms pointed to the fact that all H. pylori NapA proteins of human origin are more homologous to NapA of Helicobacter genus than to other bacterial NapA. Based on computer modeling, NapA proteins from H. pylori of human isolates are found more similar to ferritin from H. pylori than to NapA from other species of bacteria. Taken together, these results suggested that divergent evolution of NapA and ferritin possessing dissimilar and diverse sequences follows a path distinct from that of convergent evolution of NapA and ferritin with similar dual functionality of iron-binding and ferroxidase activities.

Ligand-Controlled Assembly of Hexamers, Dihexamers, and Linear Multihexamer Structures by the Engineered Acylated Insulin Degludec

Insulin degludec, an engineered acylated insulin, was recently reported to form a soluble depot after subcutaneous injection with a subsequent slow release of insulin and an ultralong glucose-lowering effect in excess of 40 h in humans. We describe the structure, ligand binding properties, and self-assemblies of insulin degludec using orthogonal structural methods. The protein fold adopted by insulin degludec is very similar to that of human insulin. Hexamers in the R(6) state similar to those of human insulin are observed for insulin degludec in the presence of zinc and resorcinol. However, under conditions comparable to the pharmaceutical formulation comprising zinc and phenol, insulin degludec forms finite dihexamers that are composed of hexamers in the T(3)R(3) state that interact to form an R(3)T(3)-T(3)R(3) structure. When the phenolic ligand is depleted and the solvent condition thereby mimics that of the injection site, the quaternary structure changes from dihexamers to a supramolecular structure composed of linear arrays of hundreds of hexamers in the T(6) state and an average molar mass, M(0), of 59.7 × 10(3) kg/mol. This novel concept of self-assemblies of insulin controlled by zinc and phenol provides the basis for the slow action profile of insulin degludec. To the best of our knowledge, this report for the first time describes a tight linkage between quaternary insulin structures of hexamers, dihexamers, and multihexamers and their allosteric state and its origin in the inherent propensity of the insulin hexamer for allosteric half-site reactivity.

Structural and Evolutionary Dynamics Facilitate Ordered Protein Complex Assembly

Most proteins can interact with other proteins to form complexes. Is the dynamic assembly of a protein complex connected to the intrinsic flexibility of its component subunits? Can the order of this assembly process be related to evolutionary changes in quaternary structure? Two methodological advances have allowed us to address these questions. First, we introduced a simple method for predicting the free-state flexibility of protein subunits from the structures of protein complexes based on accessible surface area calculations. Second, we showed that we can predict assembly pathways through analysis of intersubunit interface areas, and validated this method by experimentally characterizing the assembly of several heteromers with macromolecular mass spectrometry. using these new methods, we demonstrate that the intrinsic flexibility of protein subunits is crucial for assembly, as it facilitates the conformational changes that enable both the packing together of multiple distinct subunits and the formation of asymmetric interfaces required for cyclic symmetries. We also find that protein complex subunits have a strong tendency to assemble in an order corresponding to their flexibility and magnitude of conformational change, thus suggesting a possible mechanism for control of ordered assembly. Moreover, we reveal that changes in quaternary structure driven by evolutionary gene fusion events tend to occur in such a way so that they mimic the existing order of subunit assembly. This shows that there is selective pressure in the evolution of protein complexes to conserve assembly pathways. Finally, we observe that evolutionarily more recent subunits tend to be more flexible than more ancient subunits within a complex. Together, these results reveal the influence of both evolution and intrinsic structural dynamics on protein assembly and demonstrate, for the first time on a genome-wide scale, the biological importance of ordered assembly pathways.

Glutamate Receptor Desensitization Mediated by Changes in Quaternary Structure of the Ligand Binding Domain

Glutamate receptor ion channels are membrane proteins that mediate excitatory synaptic transmission in the central nervous system of vertebrates. Insight into the molecular mechanisms underlying their gating is limited by lack of structural information for receptors trapped in different conformational states. Here, we report the use of single particle cryo-electron tomography to determine the structures, at ∼ 25 A resolution, of full-length GluK2 kainate receptors trapped in resting and desensitized states. The resting state structure, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2, with well-resolved proximal and distal subunits exhibiting cross over between the amino terminal (ATD) and a 2-fold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD regions undergo major rearrangements resulting in a remarkable separation of the four subunits by ∼ 30 A. From fits of the ATD and LBD domains into the density map of the desensitized state, we have derived a structural model for the differences in quaternary conformation between resting and desensitized states of glutamate receptor ion channels.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Under Pressure That Splits a Family in Two. The Case of Lipocalin Family

The lipocalin family is typically composed of small proteins characterized by a range of different molecular recognition properties. Odorant binding proteins (OBPs) are a class of proteins of this family devoted to the transport of small hydrophobic molecules in the nasal mucosa of vertebrates. Among OBPs, bovine OBP (bOBP) is of great interest for its peculiar structural organization, characterized by a domain swapping of its two monomeric subunits. The effect of pressure on unfolding and refolding of native dimeric bOBP and of an engineered monomeric form has been investigated by theoretical and experimental studies under pressure. A coherent model explains the pressure-induced protein structural changes: i) the substrate-bound protein stays in its native configuration up to 330 MPa, where it loses its substrate; ii) the substrate-free protein dissociates into monomers at 200 MPa; and iii) the monomeric substrate-free form unfolds at 120 MPa. Molecular dynamics simulations showed that the pressure-induced tertiary structural changes that accompany the quaternary structural changes are mainly localized at the interface between the monomers. Interestingly, pressure-induced unfolding is reversible, but dimerization and substrate binding can no longer occur. The volume of the unfolding kinetic transition state of the monomer has been found to be similar to that of the folded state. This suggests that its refolding requires relatively large structural and/or hydrational changes, explaining thus the relatively low stability of the monomeric form of this class of proteins.

Structures of haemoglobin from woolly mammoth in liganded and unliganded states

The haemoglobin (Hb) of the extinct woolly mammoth has been recreated using recombinant genes expressed in Escherichia coli. The globin gene sequences were previously determined using DNA recovered from frozen cadavers. Although highly similar to the Hb of existing elephants, the woolly mammoth protein shows rather different responses to chloride ions and temperature. In particular, the heat of oxygenation is found to be much lower in mammoth Hb, which appears to be an adaptation to the harsh high-latitude climates of the Pleistocene Ice Ages and has been linked to heightened sensitivity of the mammoth protein to protons, chloride ions and organic phosphates relative to that of Asian elephants. To elucidate the structural basis for the altered homotropic and heterotropic effects, the crystal structures of mammoth Hb have been determined in the deoxy, carbonmonoxy and aquo-met forms. These models, which are the first structures of Hb from an extinct species, show many features reminiscent of human Hb, but underline how the delicate control of oxygen affinity relies on much more than simple overall quaternary-structure changes.

Improved data visualization techniques for analyzing macromolecule structural changes

The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color-blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three-index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique.

Role of the Subunit Interactions in the Conformational Transitions in Adult Human Hemoglobin: An Explicit Solvent Molecular Dynamics Study

Hemoglobin exhibits allosteric structural changes upon ligand binding due to the dynamic interactions between the ligand binding sites, the amino acids residues and some other solutes present under physiological conditions. In the present study, the dynamical and quaternary structural changes occurring in two unligated (deoxy-) T structures and two fully ligated (oxy-) R, R2 structures of adult human hemoglobin were investigated with molecular dynamics. It is shown that, in the submicrosecond time scale, there is no marked difference in the global dynamics of the amino acid residues in both the oxy- and the deoxy-forms of the individual structures. In addition, the R, R2 are relatively stable and do not present quaternary conformational changes within the time scale of our simulations, while the T structure is dynamically more flexible and exhibited the T → R quaternary conformational transition, which is propagated by the relative rotation of the residues at the α(1)β(2) and α(2)β(1) interface.

Effect of quaternary structure change on the low-lying electronic states of the ferrous heme in deoxy-Hb studied by multi-frequency EPR

The effect of the quaternary structure change in deoxy-Hb on the electronic states of the ferrous high-spin heme was studied by multi-frequency electron paramagnetic resonance (MFEPR). We used a single-pass transmission-type EPR at 70–220GHz and at 1.5K. We compared the EPR spectra of deoxy-Hb at high pH with those at low pH and in the presence of the allosteric effectors, such as inositol hexaphosphate (IHP). Our EPR results imply that the low-lying electronic states of the ferrous heme in deoxy-Hb could be altered by the changes in the heme geometry, which might be caused by the quaternary structure change in Hb. We also measured EPR spectra of deoxy FeMg hybrid Hb to clarify the difference between α and β hemes at low pH with IHP. The additional complicated resonance branches appeared in deoxy-Hb as well as in deoxy FeMg hybrid Hbs at low pH with IHP may reflect the energy states of the ferrous high-spin heme correlated with the low oxygen affinity state. The magnetic moment of the lowest excited state of the α heme seems to be different from that of the β heme in the low oxygen affinity state.

Conversion of a Dodecahedral Protein Capsid into Pentamers via Minimal Point Mutations

Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure-function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS's) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids. To improve our understanding of the basis for this difference in assembly, we attempted to convert the capsid-forming LS from Aquifex aeolicus (AaLS) into pentamers through a small number of rationally designed amino acid substitutions. Our mutations targeted side chains at ionic (R40), hydrogen bonding (H41), and hydrophobic (L121 and I125) interaction sites along the interfaces between pentamers. We found that substitutions at two or three of these positions could reliably generate pentameric variants of AaLS. Biophysical characterization indicates that this quaternary structure change is not accompanied by substantial changes in secondary or tertiary structure. Interestingly, previous homology-based studies of the assembly determinants in LS's had identified only one of these four positions. The ability to control assembly state in protein capsids such as AaLS could aid efforts in the development of new systems for drug delivery, biocatalysis, or materials synthesis.

Ordered Self-Assembly Mechanism of a Spherical Oncoprotein Oligomer Triggered by Zinc Removal and Stabilized by an Intrinsically Disordered Domain

BackgroundSelf-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism.Methodology/Principal FindingsUsing various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric “Z-nucleus” after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 µM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a “conformation editing” mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route.ConclusionWe described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions.

Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase

UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its Km values with Glc-1-P and UTP were 0.33 and 0.25mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the Km for Gal-1-P was very high (>10mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5–8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.