Effects of the phenylalanine-22----leucine, glutamic acid-49----methionine, glycine-234----aspartic acid, and glycine-234----lysine mutations on the folding and stability of the alpha subunit of tryptophan synthase from Escherichia coli.
The effects of four single amino acid replacements on the stability and folding of the alpha subunit of tryptophan synthase from Escherichia coli have been investigated by ultraviolet differences spectroscopy. In previous studies [Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586], it had been shown that the urea-induced unfolding at pH 7.8, 25 degrees C, proceeds by the initial unfolding of the less stable carboxyl domain (residues 189-268) followed by the unfolding of the more stable amino domain (residues 1-188). The effects of the Phe-22----Leu, Glu-49----Met, Gly-234----Asp, and Gly-234----Lys mutants on the equilibrium unfolding process can all be understood in terms of the domain unfolding model. With the exception of the Glu-49----Met replacement, the effects on stability are small. In contrast, the effects of three of the four mutations on the kinetics of interconversion of the native form and one of the stable partially folded intermediates are dramatic. The results for the Phe-22----Leu and Gly-234----Asp mutations indicate that these residues play a key role in the rate-limiting step. The Glu-49----Met mutation increases the stability of the native form with respect to that of the intermediate but does not affect the rate-limiting step. The Gly-234----Lys mutation does not affect either the stability or the kinetics of folding for the transition between native and intermediate forms. The changes in stability calculated from the unfolding and refolding rate constants agree quantitatively with those obtained from the equilibrium data. When considered with the results from a previous study on the Gly-211----Glu replacement [Matthews, C. R., Crisanti, M. M., Manz, J. T., & Gepner G. L. (1983) Biochemistry 22, 1445], it can be concluded that the rate-limiting step in the conversion of the intermediate to the native conformation involves either domain association or some other type of molecule-wide phenomenon.
Beasty
A M
AM
Hurle
M R
MR
Manz
J T
JT
Stackhouse
T
T
Onuffer
J J
JJ
Matthews
C R
CR
D001224:Aspartic Acid; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine; D007700:Kinetics; D007930:Leucine; D008239:Lysine; D046911:Macromolecular Substances; D008715:Methionine; D009154:Mutation; D010649:Phenylalanine; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000235:genetics* / Q000378:metabolism
D005971:Glutamates; D046911:Macromolecular Substances; D001224:Aspartic Acid; D018698:Glutamic Acid; D010649:Phenylalanine; D008715:Methionine; D014367:Tryptophan Synthase; D007930:Leucine; D008239:Lysine; D005998:Glycine