REHABER, V and JAENICKE, R (1992) STABILITY AND RECONSTITUTION OF D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM THE HYPERTHERMOPHILIC EUBACTERIUM THERMOTOGA-MARITIMA. JOURNAL OF BIOLOGICAL CHEMISTRY, 267 (16). pp. 10999-11006. ISSN 0021-9258,
Full text not available from this repository.Abstract
The molecular basis of thermal stability of globular proteins is a highly significant yet unsolved problem. The most promising approach to its solution is the investigation of the structure-function relationship of homologous enzymes from mesophilic and thermophilic sources. In this context, D-glyceraldehyde-3-phosphate dehydrogenase has been the most extensively studied model system. In the present study, the most thermostable homolog isolated so far is described with special emphasis on the stability of the enzyme under varying solvent conditions. D-Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima is an intrinsically thermostable enzyme with a thermal transition temperature around 110-degrees-C. The amino acid sequence, electrophoresis, and sedimentation analysis prove the enzyme to be a homotetramer with a gross structure similar to its mesophilic counterparts. The enzyme in the absence and in the presence of its coenzyme, NAD+, exhibits no drastic structural differences except for a 3% change in sedimentation velocity reflecting slight alterations in the quaternary structure of the enzyme. At low temperature, in the absence of denaturants, neither "cold denaturation" nor subunit dissociation are detectable. Guanidinium chloride and pH-dependent deactivation precede the decrease in fluorescence emission and ellipticity, suggesting a complex denaturation mechanism. An up to 3-fold activation of the enzyme at low guanidinium concentration may be interpreted in terms of a compensation of the tight packing of the thermophilic enzyme at low temperature. Under destabilizing conditions, e.g. moderate concentrations of chaotropic agents, low temperature favors denaturation. The effect becomes important in reconstitution experiments after preceding guanidinium denaturation; the reactivation yield at low temperature drops to zero, whereas between 35 and 80-degrees-C reactivation exceeds 80%. Shifting the temperature from almost-equal-to 0-degrees-C to greater-than-or-equal-to 30-degrees-C releases a trapped tetrameric intermediate in a fast reaction. Concentration-dependent reactivation experiments prove renaturation of the enzyme to involve consecutive folding and association steps. Reconstitution at room temperature yields the native protein, in spite of the fact that the temperature of the processes in vitro and in vivo differ by more than 60-degrees-C.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | YEAST GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE; AMINO-ACID-SEQUENCE; LACTIC-DEHYDROGENASE; PHYSICAL CONDITIONS; THERMAL-STABILITY; REACTIVATION; PROTEINS; DENATURATION; MECHANISM; DISSOCIATION; |
| Depositing User: | Dr. Gernot Deinzer |
| Last Modified: | 19 Oct 2022 08:44 |
| URI: | https://pred.uni-regensburg.de/id/eprint/54503 |
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