MULTIPLE REPLACEMENTS AT POSITION-211 IN THE ALPHA-SUBUNIT OF TRYPTOPHAN SYNTHASE AS A PROBE OF THE FOLDING UNIT ASSOCIATION REACTION

Equilibrium and kinetic studies on the folding of a series of amino acid replacements at position 211 in the α subunit of tryptophan synthase from Escherichia coli were performed in order to determine the role of this position in the rate-limiting step in folding. Previous studies [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T., Onuffer, J. J., & Matthews, C. R. (1986) Biochemisty 25, 2965-2974] have shown that the rate-limiting step corresponds to the association/dissociation of the amino (residues 1-188) and carboxy (residues 189-268) folding units. In terms of the secondary structure, the amino folding unit consists of the first six strands and five α helices of this α/β barrel protein. The carboxy folding unit comprises the remaining two strands and three α helices; position 211 is in strand 7. Replacement of the wild-type glycine at position 211 with serine, valine, and tryptophan at most alters the rate of dissociation of the folding units; association is not changed significantly. In contrast, glutamic acid and arginine dramatically decelerate and accelerate, respectively, both association and dissociation. The difference in effects is attributed to long-range electrostatic interactions for these charged side chains; steric effects and/or hydrogen bonding play lesser roles. When considered with previous data on replacements at other positions in the α subunit [Hurle, M. R., Tweedy, N. B., & Matthews, C. R. (1986) Biochemistry 25, 6356-6360], it is clear that β strands 6 (in the amino folding unit) and 7 (in the carboxy folding unit and containing position 211) dock late in the folding process. © 1990, American Chemical Society. All rights reserved.