We have used circular dichroism measurements to compare the thermal unfolding of the wild type tryptophan synthase α subunit from Salmonella typhimurium with that of seven mutant forms with single amino acid replacements at two active site residues. Glutamic acid 49 has been replaced by phenylalanine, glutamine, or aspartic acid. Aspartic acid 60 has been replaced by alanine, aspartic acid, asparagine, or tyrosine. Thermodynamic properties (ΔG, ΔH, ΔS, and Tm) of the wild type and mutant forms have been determined experimentally by measuring the free energy of unfolding as a function of temperature. Increasing the pH from 7.0 to 8.8 decreases the tm of the wild type α subunit from 56 to 45 °C. The thermal unfolding of the wild type α subunit and of six of the seven mutant forms can be described as reversible, two-state transitions. In contrast, the melting curve of a mutant α subunit in which aspartic acid 60 is replaced by tyrosine indicates the presence of a folding intermediate which may correspond to a "molten globule". Correlations between our observations and previous folding studies and the X-ray crystallographic structure are presented. Substitution of glutamic acid 49, which is located in the hydrophobic "pit" of an eight-fold α β barrel, by a hydrophobic phenylalanine residue increases the tm from 56 to 60 °C. In contrast, replacement of aspartic acid 60, which is accessible to solvent, results in small reductions in the thermal stability.
ASJC Scopus subject areas
- Molecular Biology