Grättinger, Mira and Dankesreiter, Arnd and Schurig, Hartmut and Jaenicke, Rainer (1998) Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima: Catalytic, spectral and thermodynamic properties. JOURNAL OF MOLECULAR BIOLOGY, 280 (3). pp. 525-533. ISSN 0022-2836, 1089-8638
Full text not available from this repository.Abstract
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 degrees C, 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 degrees C. The difference in the free energies of stabilization for the enzymes from yeast and Thermotoga amounts to Delta Delta G = 85 kJ/mol. The extrapolated temperatures of cold and heat-denaturation are about - 10 and +85 degrees C. 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 similar to 1.8 or similar to 0.9 M are required. Due to a kinetic intermediate on the pathway of cold denaturation, equilibration in the transition range takes exceedingly long. (C) 1998 Academic Press.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | HINGE-BENDING ENZYME; EVOLUTIONARY CONSERVATION; GUANIDINE-HYDROCHLORIDE; DENATURATION; STABILITY; SEQUENCE; PROTEINS; DOMAINS; STEP; HEAT; glycolytic enzymes; hyperthermophiles; phosphoglycerate kinase; protein stability; Thermotoga maritima |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 23 Feb 2023 11:01 |
| Last Modified: | 23 Feb 2023 11:01 |
| URI: | https://pred.uni-regensburg.de/id/eprint/49667 |
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