title: The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 beta-lactamase is the trans to cis isomerization of a non-proline peptide bond.
abstract: The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 beta-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166-Thr167 peptide bond, like the Glu166-Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans-->cis isomerization of the Glu166-Thr167 peptide bond.
authors.affiliation: Laboratoire d'Enzymologie, B6 Université de Liège, Sart-Tilman, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Vanhove
authors.forename: X
authors.initials: X
authors.lastname: Raquet
authors.forename: T
authors.initials: T
authors.lastname: Palzkill
authors.forename: R H
authors.initials: RH
authors.lastname: Pain
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047090:beta-Lactams
chemical_list: D000900:Anti-Bacterial Agents; D006146:Guanidines; D047090:beta-Lactams; D011392:Proline; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D019791:Guanidine