Disruption of an ionic network leads to accelerated thermal denaturation of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima.
The role of an ionic network of four charged amino acid side-chains in the thermostability of the enzyme D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima (TmGAPDH) has been assessed by site-directed mutagenesis, replacing the central residue of the ionic network, arginine 20, by either alanine (R20A) or asparagine (R20N). The purified mutant enzymes display no differences to the wild-type enzyme regarding spectroscopic properties and enzymatic activity. However, denaturation kinetics reveal that the resistance towards thermal denaturation is strongly diminished in the mutant enzymes. This is reflected by a decrease in free energy of activation for thermal unfolding of about 4 kJ/mol at 100 degrees C and a shift of temperature of half denaturation after one hour incubation from 96 to 89 degrees C for both mutant enzymes. Due to a large decrease in activation enthalpy, the effects of the mutations are temperature dependent and become even more significant at the physiological temperature of Thermotoga maritima (approximately 80 degrees C). The importance of the arginine 20 side-chain for kinetic thermal stability is plausible in the light of its key role in the ionic network and the strategic positioning of this ionic network in the context of the overall protein structure.
Pappenberger
G
G
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Schurig
H
H
Jaenicke
R
R
D000409:Alanine; D001216:Asparagine; D002942:Circular Dichroism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D007477:Ions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature
D007477:Ions; D001216:Asparagine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000409:Alanine