Effect of single amino acid substitutions at positions 49 and 60 on the thermal unfolding of the tryptophan synthase alpha subunit from Salmonella typhimurium.
We have used circular dichroism measurements to compare the thermal unfolding of the wild type tryptophan synthase alpha 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 Glutamic acid 49 has been replaced by phenylalanine, glutamine, or aspartic acid. Aspartic acid 60 has been replaced by alanine, aspartic acid, asparagine, or tyrosine.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 (delta G, delta H, delta S, 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 alpha subunit from 56 to 45 degrees C. The thermal unfolding of the wild type alpha 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 alpha 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 alpha/beta barrel, by a hydrophobic phenylalanine residue increases the tm from 56 to 60 degrees C. In contrast, replacement of aspartic acid 60, which is accessible to solvent, results in small reductions in the thermal stability.
Kanzaki
H
H
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
McPhie
P
P
Miles
E W
EW
D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D011487:Protein Conformation; D011489:Protein Denaturation; D012486:Salmonella typhimurium / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000737:chemistry*
D014367:Tryptophan Synthase