Erythrina caffra trypsin inhibitor retains its native structure and function after reducing its disulfide bonds.

Erythrina trypsin inhibitor (ETI) from the seeds of Erythrina caffra is a high-affinity inhibitor of trypsin, chymotrypsin and tissue plasminogen activator. Its 172 amino acid polypeptide chain is stabilized in its compact, native state by two disulfide bonds. In spite of their conservation in all trypsin inhibitors of the soybean trypsin inhibitor (STI-Kunitz) family, their state of oxidation is essential only for protein stability but not for inhibitory function. Reduction/reoxidation of ETI in the presence of glutathione reshuffling buffer (GSH/GSSG; pH 8.3) not only allows the inhibitor to be restored in its native structure, but also does not interfere with its binding affinity; carboxymethylation or carboxamidomethylation of the free thiol groups does not affect K1 significantly (for trypsin (KI)ETIox = 2.3 nM, (KI)ETICM = 1.9 nM; for chymotrypsin (KI)ETIox = 30 microM, (KI)ETICM = 25 microM). The two cystine cross-bridges in the native ETI lead to enhanced stability toward pH and chaotropic agents. As taken from intrinsic protein fluorescence at acid pH and varying ionic strength (pH < 4, I = 0.01 to 0.15 M), the oxidized inhibitor retains its spectral properties, whereas reduced and carboxymethylated or carboxamidomethylated ETI undergo at least partial denaturation. At alkaline pH, the oxidized protein is stable up to pH 9.5, whereas the reduced protein undergoes structural alterations at pH > 7, reaching a final plateau at pH 10.0 to 10.5. In the case of urea (U) or guanidinium chloride (GdmCl) denaturation at pH 7.0, structural transitions of the oxidized inhibitor show "hysteresis" with half-concentrations (cU)1/2 approximately 10 M and (cGdmCl)1/2 approximately 4.5 M for denaturation, and (cU)1/2 = 4.7 M and (cGdmCl)1/2 = 1.5 M for renaturation. In contrast, the reduced (and chemically modified) inhibitors exhibit true equilibrium transitions at (cU)1/2 = 0.9 M and (cGdmCl)1/2 = 0.5 M, respectively. Reduction/reoxidation in the absence and in the presence of denaturants (GdmCl) can also be applied to ETI covalently attached to a solid matrix.

Folding mechanism of porcine ribonuclease.

The kinetics of unfolding and refolding of porcine ribonuclease were investigated. The unfolded state of this protein was found to consist of a fast-refolding species (UF) and two slow-refolding species (UIS and UIIS). After the rapid collapse of the structure during the N (native)----UF unfolding reaction, UIS and UIIS are produced from UF by two independent slow isomerizations of the unfolded polypeptide chain, leading ultimately to a mixture of about 10% UF, 20% UIIS and 70% UIS molecules at equilibrium. This is at variance with all other ribonucleases investigated to date, which show a distribution of 20% UF, 60 to 70% UIIS and only 10 to 20% UIS. The two isomerizations of the unfolded porcine protein differ strongly in rate. The first process with tau = 250 seconds (10 degrees C) leads to an increase in the fluorescence of Tyr92 and was identified as cis in equilibrium trans isomerization of Pro93. At equilibrium, most unfolded molecules contain an incorrect trans Pro93. The second isomerization is much slower (tau = 1300 s at 10 degrees C) and leads to a predominance of the incorrect isomer as well. Like isomerization of Pro93, it is governed by an activation enthalpy of 22 kcal/mol (92 kJ/mol) and it was tentatively assigned to the Pro114-Pro115 sequence of porcine ribonuclease. Because of the wide separation in rate between the two reactions, molecules with an incorrect isomer only at Pro93 accumulate transiently after unfolding. These are the UIIS molecules. Most of them are finally converted to UIS by the 1300 second process. All molecules that have undergone this isomerization refold very slowly, i.e. the UIS molecules. The major fraction contains two incorrect isomers. A 1300 second isomerization after unfolding and a predominant very slow refolding reaction were observed only for the porcine protein. We suggest that these changes in the folding mechanism may be correlated with the presence of the Pro114-Pro115 sequence, which occurs only in porcine ribonuclease. The refolding pathway of porcine UIIS involves the rapid formation of a native-like intermediate with an incorrect trans Pro93 as was found previously for the bovine ribonuclease, where the UIIS species predominates in the unfolded state.

Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritima.

The hyperthermophilic eubacterium Thermotoga maritima uses starch as a substrate, without releasing amylase activity into the culture medium. The enzyme is associated with the 'toga'. Its expression level is too low to allow the isolation of the pure enzyme. Using cycloheptaamylose and acarbose affinity chromatography and common chromatographic procedures, two enzyme fractions are obtained. They differ in specificity, pH-optimum, temperature dependence and stability. Substrate specificity and Ca2+ dependence indicate alpha-, beta- and gluco-amylase activity. Compared with alpha-amylase from Bacillus licheniformis (Tmax = 75 degrees C), the amylases from Thermotoga maritima show exceedingly high thermal stability with an upper temperature limit at 95 degrees C. Significant turnover occurs only between 70 and 100 degrees C, i.e. in the range of viability of the microorganism.

Folding kinetics of mammalian ribonucleases.

The folding kinetics of seven different pancreatic ribonucleases are compared both under native conditions and within the unfolding transition. In general, the folding kinetics of these proteins are similar despite numerous amino acid substitutions. Ribonucleases with 4-6 proline residues show 80% slow-folding species. For three ribonucleases with 7 prolines this number increases to 90%. Porcine ribonuclease with a unique Pro 114-Pro 115 sequence folds significantly slower than other ribonucleases which do not show this sequence.

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui.

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.

Lactate dehydrogenase from the extreme thermophile Thermotoga maritima.

Lactate dehydrogenase was isolated from the extreme thermophilic eubacterium Thermotoga maritima. The enzyme is stereospecific for L(+)-lactate. It represents a homotetramer of 144 kDa molecular mass, with a sedimentation coefficient of s20,w approximately 7 S. Under physiological temperature conditions, the enzyme shows high catalytic efficiency with a broad pH optimum at pH 7.0 +/- 1.0, and long-term stability up to 80 degrees C. The coenzyme, NAD+, and the effector fructose 1,6-bisphosphate [Fru(1,6)P2] increase the thermal stability: at 90 degrees C (pH 6.0), the liganded enzyme exhibits a half-life of thermal inactivation of 150 min. The enhanced rigidity of the enzyme at ambient temperature is reflected by an anomalously high stability toward guanidine denaturation: the midpoint of the equilibrium transition being 1.6 M guanidine hydrochloride. Under optimum conditions of the enzyme assay, the Michaelis constants (Km) for NADH, NAD+, pyruvate and L(+)-lactate at 55 degrees C, and in the absence of Fru(1,6)P2, are 0.03 mM, 0.09 mM, 3.7 mM and 410 mM, respectively; Fru(1,6)P2 as a positive effector shifts the Km values for pyruvate and L(+)-lactate to 0.06 mM and 25 mM, respectively. The Km values for the coenzyme are not affected. Neither Mn2+ nor other divalent cations have any activating effect. In contrast to lactate dehydrogenases from eukaryotes, the N-terminus of the enzyme from Th. maritima is not acetylated. Comparison of the 30 N-terminal amino acid residues with lactate dehydrogenase from Thermus aquaticus shows a high degree of similarity. This also holds if the two lactate dehydrogenases are compared with the glyceraldehyde-3-phosphate dehydrogenases from the same organisms.

Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima.

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Thermotoga maritima, a hyperthermophilic eubacterium, has been isolated in pure crystalline form. The enzyme is a homotetramer with a subunit molecular mass of 37 kDa. The sedimentation coefficient of the native enzyme is 7.3 X 10(-13)s, the isoelectric point is 4.6, and the specific absorption coefficient A1%, 1cm 280nm = 8.4. The enzyme shows extreme thermal stability: differential scanning calorimetry yields a transition temperature (Tm) of 109 degrees C for the NAD-saturated enzyme. Thermal deactivation occurs at T greater than 90 degrees C. The physicochemical characteristics of the enzyme suggest that its gross structure must be very similar to the structure of GAPDHs from mesophilic sources. The amino acid composition does not confirm the known "traffic rules" of thermal adaptation, apart from the Lys----Arg exchange. One reactive and at least two buried SH groups can be titrated with 5,5'-dithiobis(2-nitrobenzoate). The highly reactive SH group is probably the active-site cysteine residue common to all known GAPDHs. The activation energy of the glyceraldehyde 3-phosphate oxidation reaction decreases with increasing temperature. This functional behavior can be correlated with the temperature-dependent changes of both the intrinsic fluorescence and the near-UV circular dichroism; both indicate a temperature-dependent structural reorganization of the enzyme. Hydrogen-deuterium exchange reveals significantly increased rigidity of the thermophilic enzyme if compared to mesophilic GAPDHs at 25 degrees C, thus indicating that the conformational flexibility is similar at the corresponding physiological temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of proline peptide bond isomerization in unfolding and refolding of ribonuclease.

The isomerization of the proline peptide bond between tyrosine-92 and proline-93 in bovine pancreatic ribonuclease A has been investigated in the unfolded protein as well as during the slow refolding process. This bond is in the cis state in the native protein. By comparison of various homologous ribonucleases we show that isomerization of proline-93 is associated with a change in fluorescence of tyrosine-92. This provides a spectroscopic probe to monitor this process in the disordered chain after unfolding as well as its reversal in the course of slow refolding. In unfolded ribonuclease incorrect trans isomers of proline-93 are found in both slow-folding species. trans----cis reversal of isomerization of this proline peptide bond during refolding shows kinetics that are identical with the time course of formation of native protein. Isomerization of proline-93 is slower than the formation of a native-like folded intermediate that accumulates on the major slow refolding pathway. Models to explain these results are discussed.