Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima: catalytic, spectral and thermodynamic properties.
Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima (TmPGK) has been expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity applying heat incubation of the crude extract at 80 degreesC, ion exchange chromatography and gel filtration. The biochemical, catalytic and spectral properties were compared with those of the natural enzyme and found to be identical. As shown by SDS-PAGE, ultracentrifugal analysis and gel filtration chromatography, the enzyme is a 43 kDa monomer. At neutral pH, the guanidinium chloride (GdmCl) and temperature-induced denaturation transitions reveal two-state behaviour with high cooperativity. As taken from the temperature dependence of the free energy of unfolding at zero GdmCl concentration and pH 7, optimum stability is observed at approximately 30 degreesC. The difference in the free energies of stabilization for the enzymes from yeast and Thermotoga amounts to Delta DeltaG=85 kJ/mol. The extrapolated temperatures of cold and heat-denaturation are about -10 and +85 degreesC. This indicates that the stability profile of TmPGK is shifted to higher free energy values and broadened over a wider temperature range, compared to that observed for PGKs from mesophiles or moderately thermophiles. In order to achieve cold or heat-denaturation, GdmCl concentrations of approximately 1.8 or approximately 0.9 M are required. Due to a kinetic intermediate on the pathway of cold denaturation, equilibration in the transition range takes exceedingly long.
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Regensburg, D-93040, Germany.
M
M
Grättinger
A
A
Dankesreiter
H
H
Schurig
R
R
Jaenicke
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry*; D013057:Spectrum Analysis; D013816:Thermodynamics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010735:Phosphoglycerate Kinase