Effect of a single amino acid substitution on the folding of the alpha subunit of tryptophan synthase.
The urea-induced unfolding of a missense mutant of the alpha subunit of tryptophan synthase from Escherichia coli involving the replacement of Gly by Glu at position 211 has been monitored by absorbance changes at 286 nm. Like the wild-type protein, the equilibrium unfolding curve demonstrates the presence of one or more stable intermediates. Comparison of these results with those from the wild-type alpha subunit [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784] shows that the transition from the native conformation to the stable intermediates is displaced to higher urea concentration in the mutant alpha subunit; however, the transition from the intermediates to the unfolded form is unaffected. Kinetic studies show that the amino acid replacement slows the rate of unfolding by an order of magnitude. The effect on refolding rates is complex. One phase, previously assigned to proline isomerization [Crisanti, M. M., & Matthews, C. R. (1981) Biochemistry 20, 2700], is unaffected by the substitution. The rate of the second phase, which is urea dependent down to about 1 M urea, is slower than the corresponding phase in the wild-type protein by approximately a factor of 2. Below about 1 M urea, the rate of this phase becomes urea independent and identical with that of the wild-type alpha subunit. This change in urea dependence has been ascribed to a change in the nature of the rate-limiting step for this process from one involving folding to one involving proline isomerization. The results support the folding model for the alpha subunit proposed previously [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784] and clarify the role of proline isomerization in limiting the rate of folding.
D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine; D007700:Kinetics; D010446:Peptide Fragments; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase; D014508:Urea
D005971:Glutamates; D010446:Peptide Fragments; D018698:Glutamic Acid; D014508:Urea; D014367:Tryptophan Synthase; D005998:Glycine