Comparison of the structures of these two enzymes has revealed one major difference: the structure of the hyperthermophilic enzyme contains a striking series of ion-pair networks on the surface of the protein subunits and buried at both interdomain and intersubunit interfaces. We propose that the formation of such extended networks may represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures.

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The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pl of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100°C was 12 h), totally independent of enzyme concentration.P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P..furiosus.Recently we reported the purification of an NAD(P)-dependent glutamate dehydrogenase from the thermophilic archaebacterium Sulfolobus so@ztaricus [l, 21. The enzyme of this microorganism is probably involved in the first step of ammonia assimilation by the following reaction: glutamate + NAD(P)+ + H 2 0 + 2-oxoglutarate + NH,' + NAD(P)H + H + .The glutamate dehydrogenase from S . solfutaricus presents some interesting properties; it has a double coenzyme specificity, is strictly specific for L-glutamate and 2-oxoglutarate and its thermal stability is a function of the enzyme concentration [2]. This latter property is in good agreement with the observed self-associating behaviour of the purified enzyme.Among archaebacterial glutamate dehydrogenases, the only data available for comparison are restricted to the halophilic phenotype [3]. Therefore, it is difficult to extrapolate from our observations on S. solfituricus glutamate dehydrogenase to obtain general rules for thermal adaptation in this class of enzymes and general knowledge regarding nitrogen metabolism in archaebacteria.To provide more information about the enzymological properties of archaebacterial glutamate dehydrogenases and to extend our investigations towards more thermostable forms of this enzyme, we attampted to purify glutamate dehydrogenase from the so-called 'hyperthermophiles'. These remarkable microorganisms, recently discovered, grow optimally at temperatures near 100 "C [4]. They all are archaebacteria and most of them are strict anaerobes and depend on the reduction of elemental sulfur for growth. So far, hyperthermophiles -___ have been classified into three distinct genera, Pyrodiictium, Pyrobaculurn and Pyrococcus. As a source of hyperthermophilic glutamate dehydrogenase we used Pyrococcus furiosus [5], one of the most interesting members of this last genus because of its relatively high cell yield and rapid growth rate. The microorganism grows optimally at 10O'C by a fermentative-type metabolism and is a strict heterotroph which utilizes both simple and complex carbohydrates and produces only H2 and C 0 2 as detectable products. P. furiosus reduces eleme...

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme. The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.

Background: ␤-Amyloid aggregates are at the basis of Alzheimer disease development. Short synthetic peptides are seen to inhibit polymerization. Results: Various synthetic peptides have been studied by MD simulations and tested experimentally. Conclusion:Combined results indicate Ac-LPFFN-NH 2 as an effective lead compound able to slow down A␤ 1-40 aggregation. Significance: Designing potential A␤ aggregation inhibitors will help fight Alzheimer disease.

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The stability of the dodecameric Listeria innocua ferritin at low pH values has been investigated by spectroscopic methods and size-exclusion chromatography. The dodecamer is extremely stable in comparison to the classic ferritin tetracosamer and preserves its quaternary assembly at pH 2.0, despite an altered tertiary structure. Below pH 2.0, dissociation into dimers occurs and is paralleled by the complete loss of tertiary structure and a significant decrease in secondary structure elements. Dissociation of dimers into monomers occurs only at pH 1.0. Addition of NaCl to the protein at pH 2.0 induces structural changes similar to those observed upon increasing the proton concentration, although dissociation proceeds only to the dimer stage. Addition of sulfate at pH values $ 1.5 prevents the dissociation of the dodecamer. The role played by hydrophilic and hydrophobic interactions in determining the resistance to dissociation of L. innocua ferritin at low pH is discussed in the light of its three-dimensional structure.Keywords: acid dissociation; dodecameric assembly; ferritin; Listeria innocua; pH stability.The effect of low pH on protein structure is related to the electrostatic forces that originate from the changes in net charge and charge distribution attendant with the addition of protons. In monomeric proteins the progressive protonation of amino acid residues on decreasing the pH gives rise to repulsive interactions that lead to loss of secondary and tertiary structure and hence can be exploited to study protein stability [1]. Typically, a relatively fully unfolded conformation is attained in the vicinity of pH 2 because stabilizing salt-bridges are removed and buried side chains become protonated. Several proteins do not unfold completely, however, and adopt relatively compact, soluble conformational states that differ from the native structure as indicated by their spectroscopic properties [2]. In oligomeric proteins the acid denaturation is not limited to the destruction of secondary and tertiary structure of each subunit, but entails the perturbation of intersubunit interactions. In oligomeric proteins, therefore, the structural changes induced by low pH provide additional information on the forces involved in subunit recognition and association, and on the intermediates that may be formed during the assembly/disassembly processes.The mechanism of acid-induced denaturation has been studied extensively for several monomeric [2,3] and a few oligomeric proteins [4±9]. Most oligomeric proteins were found to be fully disassembled at pH 2.6 [10]. However, two systems of extreme pH stability have been described that undergo dissociation at pH values lower than 2.0, namely the B subunit pentamer of Escherichia coli heat-labile enterotoxin [8] and the glutamate dehydrogenase hexamer from the hyperthermophile Pyrococcus furiosus [11]. In both systems the molecular basis for the extreme stability of the quaternary assemblage has been ascribed to the presence of specific intersubunit interactions revealed by the...

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