Sequencing, expression and properties of triosephosphate isomerase from Entamoeba histolytica.

We have isolated a cDNA clone of the glycolytic enzyme, triosephosphate isomerase (TPI) from Entamoeba histolytica. Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences, derived from TPIs of other organisms, were used to amplify a 450-bp fragment using E. histolytica cDNA as a template. The fragment was used to screen a cDNA library. The isolated cDNA, encoding a protein of 261 amino acids, shares 43-52.6% positional identity with other known protozoan TPIs. The catalytic residues were conserved; nevertheless, several indels occurred at other regions in the protein sequence. The complete coding sequence of the E. histolytica TPI gene was cloned into the expression vector pRSET and expressed as a wild-type TPI enzyme (E. histolytica TPI) and as a fusion protein with an N-terminal tail of six histidine residues E. histolytica TPI-His6); both recombinant proteins were purified. Molecular modeling of E. histolytica TPI showed an identical topology to the known structures of other TPI molecules, but with a remarkable feature; more than 10 inserted residues are located in the same region of the molecular surface. Studies were performed to detect possible changes that might be caused by the inserted amino acids. The catalytic activity and oligomeric state of the purified protein were similar to that reported for TPI from other sources. In contrast, stability towards dilution, as well as thermal inactivation and unfolding assays, showed that E. histolytica TPI is significantly more stable towards denaturation than Trypanosoma brucei TPI.

Water requirements in monomer folding and dimerization of triosephosphate isomerase in reverse micelles. Intrinsic fluorescence of conformers related to reactivation.

The possibility of using reverse micelles to stabilize monomers prior to formation of dimeric triosephosphate isomerase (TPI) from rabbit muscle was studied. TPI denatured with guanidine hydrochloride undergoes reactivation in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide, and water. Reactivation of around 80% is observed at TPI concentrations of about 2 micrograms/mL of reverse micelles and water concentrations above 4.0%. With 3.0% water, reactivation is about 10%. If denatured TPI is incubated for a few seconds in reverse micelles with 5.0% water (or higher) followed by incubation in 3.0% water, reactivation is between 35% and 50%. That is, a brief exposure of denatured TPI to reverse micelles with a relatively high water concentration yielded a significant amount of structures competent for formation of catalytically active dimers. As evidenced by kinetic data, these structures correspond to monomers of TPI [Garza-Ramos, G. Tuena de Gómez-Puyou, M., Gómez-Puyou, A., & Gracy R. W. (1992) Eur. J. Biochem. 208, 389-395]. After a 5-2.0% water transition, competent monomers were stabilized for at least 30 min; a subsequent rise in water concentration led to dimerization and appearance of activity. By changes in the amount of water, it was possible to determine in reverse micelles the amount of water required for monomer folding and dimerization; i.e., less water was required in the dimerization step. Experiments with a model system, trypsin and the soybean inhibitor, showed that, in reverse micelles with 2.0% water, protein-protein interactions readily take place.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of heart and muscle lactate dehydrogenases in all-aqueous systems and in organic solvents with low amounts of water. Effect of guanidine chloride.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of lactate dehydrogenases from heart and muscle was studied in standard water mixtures and in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide and water in a concentration that ranged over 2.5-6.0% (by vol.). In all water mixtures GdmCl (0.15-0.75 M) and urea (0.5-3.0 M) inhibited the activity of the enzymes at non-saturating pyruvate concentrations. At concentrations of pyruvate that proved inhibitory for enzyme activity due to the formation of a ternary enzyme-NAD-pyruvate complex, GdmCl and urea increased the activity of the enzymes. This increase correlated with a decrease of the ternary complex, as evidenced by its absorbance at 320-325 nm. In the low-water system it was found that: (a) at all concentrations of pyruvate tested (0.74-30 mM), GdmCl enhanced the activity of the heart enzyme to a similar extent; (b) in the muscle enzyme, GdmCl inhibited or increased the activity through a process that depended on the concentration of pyruvate and GdmCl; (c) under optimal conditions, the activation by GdmCl was about two times lower in the muscle than in the heart enzyme, although in all-water media the activity of the muscle enzyme was twice as high. The expression of lactate dehydrogenase activity in the low-water system was higher with the heart than with the muscle enzyme compared to their activities in all-water media (about 260 and 600 mumol min-1 mg-1 in the heart and muscle enzymes respectively). Apparently for catalysis, the water requirement in the heart enzyme is lower than in the muscle enzyme. It is likely that the different response of the two enzymes to solvent is due to their distinct structural features.

Enzyme activation by denaturants in organic solvent systems with a low water content.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of heart lactate dehydrogenase, glycerol-3-phosphate dehydrogenase, hexokinase, inorganic pyrophosphatase, and glyceraldehyde-3-phosphate dehydrogenase was studied in low-water systems. Most of the experiments were made in a system formed with toluene, phospholipids, Triton X-100, and water in a range that varied over 1.0-6.5% (by vol.) [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1990) Biochemistry 29, 751-757]. In such conditions at saturating substrate concentrations, the activity of the enzymes was more than 10 times lower than in all-water media. However the activity of the first four aforementioned enzymes was increased between 4 and 20 times by the denaturants. The most marked activating effect was found with lactate dehydrogenase; with 3.8% (by vol.) water maximal activation was observed with 1.5 M GdmCl (about 20-fold); 4 M urea activated, but to a lower extent. Activation by guanidine thiocyanate was lower than with GdmCl. The activating and inactivating effects of GdmCl on lactate dehydrogenase depended on the amount of water; as the amount of water was increased from 2.0% to 6.0% (by vol.), activation and inactivation took place with progressively lower GdmCl concentrations. When activity was measured as a function of the volume of 1.5 M GdmCl solution, a bell-shaped activation curve was observed. In a low-water system formed with n-octane, hexanol, cetyltrimethylammonium bromide and 3.0% water, a similar activation of lactate dehydrogenase by GdmCl and urea was observed. The water solubility diagrams were modified by GdmCl and urea, and this could reflect on enzyme activity. However, from a comparison of denaturant concentrations on the activity of the enzymes studied, it would seem that, independently of their effect on the characteristics of the low-water systems, denaturants bring about activation through their known mechanism of action on the protein. It is suggested that the effect of denaturants is due to the release of constraints in enzyme catalysis imposed by a low-water environment.