The effect of ion pairs on the thermal stability of D-glyceraldehyde 3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima.
D-Glyceraldehyde 3-phosphate dehydrogenase from Thermotoga maritima (TmGAPDH) is intrinsically thermostable, exhibiting a thermal transition beyond 105 degrees C. Neither the amino-acid composition nor homology modelling, based on sequence alignment and known 3-D structures of the enzyme from meso- and thermophiles, provide an explanation of the anomalous stability. Recent X-ray data suggest that an increased number of ion pairs is involved. To prove this hypothesis, a number of charged residues contributing to ion pairs in TmGAPDH, but absent in the moderately thermophilic enzyme were altered. Elimination of peripheral ion pairs (E103-K104, E261-R266) was found to be ineffective. Altering a central charge cluster (R10-D47, E314, D*186) led to a drastic decrease in coenzyme binding. As a consequence, guanidine-dependent deactivation is shifted to significantly lower guanidinium chloride (GdmCl) concentrations without altering the denaturation/dissociation profile of the wild type enzyme. Mutants in the S loop (R195D, R195D-D181K) lead to a biphasic profile in the GdmCl-dependent denaturation transition and significant destabilization; at room temperature no subunit dissociation could be observed.
Tomschy
A
A
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Böhm
G
G
Jaenicke
R
R
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry*; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D016415:Sequence Alignment
D009243:NAD; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008239:Lysine