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Chapter 5,216

Effect of a single amino acid substitution on the folding of the alpha subunit of tryptophan synthase

Matthews, C.R.; Crisanti, M.M.; Manz, J.T.; Gepner, G.L.

Biochemistry 22(6): 1445-1452

1983


ISSN/ISBN: 0006-2960
PMID: 6132619
DOI: 10.1021/bi00275a019
Accession: 005215932

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, was monitored by absorbance changes at 286 nm. Like the wild-type protein, the equilibrium unfolding curve demonstrates the presence of .gtoreq. 1 stable intermediates. Comparison of these results with those from the wild-type .alpha. subunit 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, is unaffected by the substitution. The rate of the 2nd phase, which is urea dependent down to .apprx. 1 M urea, is slower than the corresponding phase in the wild-type protein by a factor of .apprx. 2. Below .apprx. 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 was ascribed to 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 and clarify the role of proline isomerization in limiting the rate of folding.

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