Section 43
Chapter 42,517

Characterization of the slow steps in the folding of the alpha subunit of tryptophan synthase

Crisanti, M.M.; Matthews, C.R.

Biochemistry 20(9): 2700-2706


ISSN/ISBN: 0006-2960
PMID: 7016182
DOI: 10.1021/bi00512a052
Accession: 042516260

The 2 slow phases detected by UV spectroscopy in the refolding of the urea-unfolded .alpha. subunit of tryptophan synthase (EC of Escherichia coli were analyzed for their dependence on final urea concentration, pH and temperature. The decay time of the slower of the 2 phases is independent of final urea concentration and pH and has an activation energy of 17.6 .+-. 2.5 kcal/mol. This behavior is consistent with the explanation that proline isomerization is the rate-limiting process in the step corresponding to this phase. The decay time for the faster phase depends on both the final urea concentration and the pH; the variation with pH is well described by a single protonation event with a pK of 7.6 .+-. 0.3. The activation energy for this phase is 14.5 .+-. 2.6 kcal/mol. The step corresponding to this phase is apparently not simply related to proline isomerization. The binding of a competitive inhibitor for the .alpha. subunit, indolepropanol phosphate, during refolding displays biphasic kinetics; the decay times for the 2 phases are identical with those observed for the folding of the protein in the absence of inhibitor. All of the results can be explained in terms of a folding model in which the unfolded protein exists in 2 kinetically different forms. These 2 species rapidly collapse to 2 intermediate forms, 1 of which proceeds directly to the native conformation by the pH-dependent step, with the intermediate decay time. The 2nd intermediate must first execute a proline isomerization before proceeding to the native form.

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