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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

1981


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 4.2.1.20) 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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