The relationship between the glucose oxidase subunit structure and its thermostability

The thermostability of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) at 60 degrees C has been studied as a function of its concentration in various media (pure water and pure deuterium oxide). In deuterium oxide, glucose oxidase is more stable than in water, and two kinds of stabilizing effect have been observed: the medium-organization effect and the enzyme-concentration effect. This effect has been related to the glucose oxidase subunit structure. This enzyme contains four forms of subunit: monomer, dimer, trimer, and tetramer, which are all composed of the identical monomer. The monomers of glucose oxidase subunits are linked by the non-covalent bond. Only dimer and trimer possess the enzymatic activity. During glucose oxidase denaturing, monomers assemble into dimer, trimer, or tetramer. This redistribution behavior depends on the enzyme concentration and the nature of the medium.     

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.     

An unusual triosephosphate isomerase from the early divergent eukaryote Giardia lamblia

Recombinant triosephosphate isomerase from the parasite Giardia lamblia (GlTIM) was characterized and immunolocalized. The enzyme is distributed uniformly throughout the cytoplasm. Size exclusion chromatography of the purified enzyme showed two peaks with molecular weights of 108 and 55 kDa. Under reducing conditions, only the 55-kDa protein was detected. In denaturing gel electrophoresis without dithiothreitol, the enzyme showed two bands with molecular weights of 28 and 50 kDa; with dithiotretitol, only the 28-kDa protein was observed. These data indicate that GlTIM may exist as a tetramer or a dimer and that, in the former, the two dimers are covalently linked by disulfide bonds. The kinetics of the dimer were similar to those of other TIMs. The tetramer exhibited half of the kcat of the dimer without changes in the Km. Studies on the thermal stability and the apparent association constants between monomers showed that the tetramer was slightly more stable than the dimer. This finding suggests the oligomerization is not related to enzyme thermostability as in Thermotoga maritima. Instead, it could be that oligomerization is related to the regulation of catalytic activity in different states of the life cycle of this mesophilic parasite.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

An ultracentrifuge study on the self-association of glucose dehydrogenase from Bacillus megaterium

The self-association of glucose dehydrogenase (beta-D-glucose:NAD(P) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium was studied by analytical ultracentrifugation. The pH and composition of the buffer used were such that, owing to a reversible partial dissociation of the tetrameric enzyme, enzyme activity was reduced. It was found that under these conditions the protein exists in a monomer/dimer/tetramer association equilibrium.     

The reconstitution of denatured phosphoglycerate mutase

The reconstitution of the tetrameric enzyme yeast phosphoglycerate mutase after denaturation in guanidine hydrochloride has been studied. Denaturation is almost completely reversible at enzyme concentrations greater than 10 micrograms/ml. Cross-linking by glutaraldehyde has been used to monitor the reassociation of the subunits; the kinetics of this process has been analyzed in terms of a model involving an equilibrium between monomer and dimer followed by a bimolecular association of two dimers to give a tetramer. Reactivation is found to parallel the appearance of tetramer. Structural changes during reconstitution have been measured by circular dichroism and fluorescence. Both methods reveal complex kinetics indicating the rapid formation of structured monomers (half-time less than 10 s), followed by slow subunit association. For comparison, preliminary reconstitution experiments were performed on the dimeric phosphoglycerate mutase from rabbit muscle.     

Urea-induced inactivation, dissociation, and unfolding of the allosteric phosphofructokinase from Escherichia coli

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.     

Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. RESULTS: We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. CONCLUSIONS: The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.     

Aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12. Concentration-dependent dissociation to dimers in the presence of L-threonine.

The concentration-dependent association-dissociation equilibrium of the bifunctional enzyme aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12 has been investigated at pH 7.6 in the presence of 10 mM L-threonine and 0.1 M KCl by equilibrium gel permeation monitored by a single-photon counting spectrophotometer. The results obtained are consistent with the existence of a dimer-tetramer equilibrium with the association constant of 2.6 X 10(7) M-1 (deltaG0 = -9.9 kcal/mol of dimer). The limiting partition cross-sections estimated by a three-parameter least squares minimization procedure indicate that the molecular radii of the dimer and tetramer are 53.8 A and 70 A, respectively. Both the dimeric and tetrameric forms of the enzyme possess dehydrogenase activity. Treatment of the enzyme with the chaotropic salts, potassium thiocyanate or potassium trichloroacetate, generates a monomeric form that is devoid of dehydrogenase activity. The catalytically inactive monomeric form of the enzyme has a molecular radius between 43 and 45.5 A and a molecular weight of approximately 80,000 as determined by small zone gel chromatography and sedimentation equilibrium studies.     

Human platelet factor 4 subunit association/dissociation thermodynamics and kinetics

Platelet factor 4 (PF4) monomers (7800 daltons) form dimers and tetramers in varying molar ratios under certain solution conditions [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469]. The presence of a simplified aromatic region (one Tyr and two His) and resolved monomer, dimer, and tetramer Y60 3,5 ring proton resonances makes study of PF4 aggregate association/dissociation thermodynamics and kinetics possible. PF4 protein subunit association/dissociation equilibrium thermodynamic parameters have been derived by 1H NMR (500MHz) resonance line-fitting analysis of steady-state Y60 3,5 ring proton resonance monomer-dimer-tetramer populations as a function of temperature from 10 to 40 degrees C. Below 10 degrees C and above 40 degrees C, resonance broadening and overlap severely impaired analysis. Enthalpic and entropic contributions to dimer association Gibb's free energy [-5.1 kcal/mol (30 degrees C)] are +2.5 +/- 1 kcal/mol and +26 +/- 7 eu, respectively, and for tetramer association Gibb's free energy [-5.7 kcal/mol (30 degrees C)], they are -7.5 +/- 1 kcal/mol and -7 +/- 3 eu, respectively. These thermodynamic parameters are consistent with low dielectric medium electrostatic/hydrophobic interactions governing dimer formation and hydrogen bonding governing tetramer formation. Association/dissociation kinetic parameters, i.e., steady-state jump rates, have been derived from exchange-induced line-width increases and from 1H NMR (500 MHz) saturation-transfer and spin-lattice (Tl) relaxation experiments. From dissociation jump rates and equilibrium constants, association rate constants were estimated. For dimer and tetramer equilibria at 30 degrees C, unimolecular dissociation rate constants are 35 +/- 10 s-1 for dimer dissociation and 6 +/- 2 s-1 for tetramer dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitor stabilization of human immunodeficiency virus type-2 proteinase dimer formation

We report the first direct observation of the subunit self-association behavior of highly purified recombinant human immunodeficiency virus type-2 (HIV-2) proteinase. Multiple samples of enzyme were subjected to sedimentation equilibrium analytical ultracentrifugation sequentially at 8.8 degrees C and two pH values in the presence and absence of a C2 symmetric, peptidomimetic inhibitor. At both pH values the enzyme exhibited sedimentation equilibrium behavior which fit a monomer-dimer-tetramer model. In the absence of inhibitor, the apparent Kd for dimer formation was less than approximately 100 microM and the apparent Kd for the weaker dimer-tetramer association was greater than approximately 100 microM. In the presence of inhibitor, at either pH, dimer formation was more strongly favored as indicated by a approximately 5-14-fold decrease in the apparent Kd for dimer formation and a approximately 1.2-4-fold increase in the apparent Kd for tetramer formation. The enhanced formation of dimer and decrease in higher order self-associated forms in the presence of an inhibitor is consistent with inhibitor stabilization of an active dimer. The inhibitor-induced stabilization of the dimeric species is consistent with a model for substrate-induced formation of active proteinase dimers in virion assembly.     

Substrate activation of insulin-degrading enzyme (insulysin). A potential target for drug development

The rate of the insulin-degrading enzyme (IDE)-catalyzed hydrolysis of the fluorogenic substrate 2-aminobenzoyl-GGFLRKHGQ-ethylenediamine-2,4-dinitrophenyl is increased 2-7-fold by other peptide substrates but not by peptide non-substrates. This increased rate is attributed to a decrease in Km with little effect on Vmax. An approximately 2.5-fold increase in the rate of amyloid beta peptide hydrolysis is produced by dynorphin B-9. However, with insulin as substrate, dynorphin B-9 is inhibitory. Immunoprecipitation of differentially tagged IDE and gel filtration analysis were used to show that IDE exists as a mixture of dimers and tetramers. The equilibrium between dimer and tetramer is concentration-dependent, with the dimer the more active form. Bradykinin shifted the equilibrium toward dimer. Activation of substrate hydrolysis is not seen with a mixed dimer of IDE containing one active subunit and one subunit that is catalytically inactive and deficient in substrate binding. On the other hand, a mixed dimer containing one active subunit and one subunit that is catalytically inactive but binds substrate with normal affinity is activated by peptides. These findings suggest that peptides bind to one subunit of IDE and induce a conformational change that shifts the equilibrium to the more active dimer as well as activates the adjacent subunit. The selective activation of IDE toward amyloid beta peptide relative to insulin suggests the potential for development of compounds that increase IDE activity toward amyloid beta peptide as a therapeutic intervention for the treatment of Alzheimer's disease.     

Subunit interactions in yeast glyceraldehyde-3-phosphate dehydrogenase.

The spontaneous inactivation of yeast glyceraldehyde-3-phosphate dehydrogenase was found to fit a simple two-state model at pH 8.5 and 25 degrees. The first step is a relatively rapid dissociation of the tetramer to dimers with the equilibrium largely in favor of the tetramer. In the absence of NAD+ the dimer inactivates irreversibly. The apoenzyme is quite stable with a half-life for complete activity loss proportional to the square root of the enzyme concentration. Perturbances of the protein structure (by pH, ionic strength, and specific salts), which have no effect on the tetrameric state of the molecule, result in an alteration of the cooperativity of NAD+ binding, the reactivity of the active-site sulfhydryl group, and the catalytic activity of the enzyme. Covalent modification of two of the four active-site sulfhydryl groups has profound effects on the enzymic activity which are mediated by changes in the subunit interactions. Sedimentation analysis and hybridization studies indicate that the interaction between subunits remains strong after covalent modification. Under normal physiological and equilibrium dialysis conditions the protein is a tetramer. Equilibrium dialysis studies of NAD+ binding to the enzyme at pH 8.5 and 25 degrees reveal a mixed cooperativity pattern. A model consistent with these observations and the observed half-of-the-sites reactivity is that of ligand induced sequential conformational changes which are transferred across strongly interacting subunit domains. Methods for distinguishing negatively cooperative binding patterns from mixtures of denatured enzyme and multiple species are discussed.     

Subunit dissociation in the allosteric regulation of Glycerol kinase from Escherichia coli. 3. Role in desensitization.

The mechanism of desensitization of glycerol kinase to allosteric inhibition by fructose 1,6-bisphosphate caused by salt, urea, and high pH has been examined in the light of the model proposed in an earlier paper [de Riel, J. K., and Paulus H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding. KCl (0.4 M) causes a tenfold decrease in the affinity of tetrameric glycerol kinase for fructose, 1,6-bisphosphate but has no significant effect on the dissociation process itself. Urea (2 M) causes a large increase in the equilibrium constant for the dissociation of the glycerol kinase tetramer to dimer but has no effect on the affinity of the tetramer for the allosteric inhibitor. High pH (9--10) has only a small effect on the subunit dissociation constant but greatly reduces the rates of subunit association and dissociation. Desensitization of glycerol kinase to allosteric inhibition can thus occur by three different mechanisms, two of which are directly related to the polysteric nature of the enzyme.     

ATP-sensitive and ATP-insensitive phosphofructokinase in Escherichia coli K-12.

The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coli exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150,000 (subunit mol. wt 73,000), whereas the anaerobic culture of E. coli revealed only one type, which is a tetramer of mol. wt 350,000 (subunit mol. wt 90,000). Type 1 of the aerobic enzyme, representing approximately 70% of the total enzyme activity, is ATP-insensitive, whereas type II and the anaerobic enzyme are ATP-sensitive. The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type II dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type II of the aerobic dimer. The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect.     

Unique oligomeric intermediates of bovine liver catalase

Catalases, although synthesized from single genes and built up from only one type of subunit, exist in heterogeneous form with respect to their conformations and association states in biological systems. This heterogeneity is not of genetic origin, but rather reflects the instability of this oligomeric heme enzyme. To understand better the factors that stabilize the various association states of catalase, we performed studies on the multimeric intermediates that are stabilized during guanidine-hydrochloride- and urea-induced unfolding of bovine liver catalase (BLC). For the first time, we have observed an enzymatically active, folded dimer of native BLC. This dimer has slightly higher enzymatic activity and altered structural properties compared to the native tetramer. Comparative studies of the effect of NaCl, GdmCl, and urea on BLC show that cation binding to negatively charged groups present in amino acid side chains of the enzyme leads to stabilization of an enzymatically active, folded dimer of BLC. Besides the folded dimer, an enzymatically active expanded tetramer and a partially unfolded, enzymatically inactive dimer of BLC were also observed. A complete recovery of native enzyme was observed on refolding of expanded tetramers and folded dimers; however, a very low recovery (maximum of approximately 5%) of native enzyme was observed on refolding of partially unfolded dimers and fully unfolded monomers.     

Regulation of Phosphoenolpyruvate Carboxylase from Crassula argentea: Further Evidence on the Dimer-Tetramer Interconversion

Phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants during the day exists in dimeric form the activity of which is strongly inhibited by malate. Enzyme purified from Crassula leaves collected during the day and stored at −70°C for 49 days shows a steady progression of change from dimer to tetramer, and this change in oligomeric state is accompanied by a decrease in the sensitivity of the enzyme to inhibition by malate. At 10 minutes preincubation of enzyme after 11 days storage—which is composed of an equilibrium mixture of dimer and tetramer—with malate causes most of the enzyme to be converted to dimer and increases the sensitivity of the enzyme to malate inhibition during assay. Preincubation with phosphoenolpyruvate shifts the equilibrium toward the tetrameric form and reduces the maximal inhibition produced by 5 millimolar malate to less than 20%. However, none of the treatments used resulted in shifting the oligomerization equilibrium completely in either direction. Thus the question of whether some covalent modification of the enzyme, such as phosphorylation, is required to permit complete changes in equilibrium remains open.     

Tandem dimerization of the human p53 tetramerization domain stabilizes a primary dimer intermediate and dramatically enhances its oligomeric stability

Tetramerization of the human p53 tumor suppressor protein is required for its biological functions. However, cellular levels of p53 indicate that it exists predominantly in a monomeric state. Since the oligomerization of p53 involves the rate-limiting formation of a primary dimer intermediate, we engineered a covalently linked pair of human p53 tetramerization (p53tet) domains to generate a tandem dimer (p53tetTD) that minimizes the energetic requirements for forming the primary dimer. We demonstrate that p53tetTD self-assembles into an oligomeric structure equivalent to the wild-type p53tet tetramer and exhibits dramatically enhanced oligomeric stability. Specifically, the p53tetTD dimer exhibits an unfolding/dissociation equilibrium constant of 26 fM at 37 degrees C, or a million-fold increase in stability relative to the wild-type p53tet tetramer, and resists subunit exchange with monomeric p53tet. In addition, whereas the wild-type p53tet tetramer undergoes coupled (i.e. two-state) dissociation/unfolding to unfolded monomers, the p53tetTD dimer denatures via an intermediate that is detectable by differential scanning calorimetry but not CD spectroscopy, consistent with a folded p53tetTD monomer that is equivalent to the p53tet primary dimer. Given its oligomeric stability and resistance against hetero-oligomerization, dimerization of p53 constructs incorporating the tetramerization domain may yield functional constructs that may resist exchange with wild-type or mutant forms of p53.