title: Folding of class A beta-lactamases is rate-limited by peptide bond isomerization and occurs via parallel pathways.
abstract: Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues 166 and 167 at the active site of these enzymes controls important steps in their refolding reaction. In this work, we analyzed how conformational folding, reactivation, and cis/trans peptide bond isomerizations are interrelated in the folding kinetics of beta-lactamases that differ in the nature of the cis peptide bond, which involves a Pro167 in the BS3 and TEM-1 enzyme, a Leu167 in the NMCA enzyme, and which is missing in the PER-1 enzyme. The analysis of folding by spectroscopic probes and by the regain of enzymatic activity in combination with double-mixing procedures indicates that conformational folding can proceed when the 166-167 bond is still in the incorrect trans form. The very slow trans --> cis isomerization of the Glu166-Xaa167 peptide bond, however, controls the final step of folding and is required for the regain of the enzymatic activity. This very slow phase is absent in the refolding of PER-1, in which the Glu166-Ala167 peptide bond is trans. The double-mixing experiments revealed that a second slow kinetic phase is caused by the cis/trans isomerization of prolines that are trans in the folded proteins. The folding of beta-lactamases is best described by a model that involves parallel pathways. It highlights the role of peptide bond cis/trans isomerization as a kinetic determinant of folding.
authors.affiliation: Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingenierie des Protéines, Université de Liège, Institut de Chimie B6, 4000 Liège (Sart Tilman), Belgium.
authors.forename: Julie
authors.initials: J
authors.lastname: Vandenameele
authors.forename: Annabelle
authors.initials: A
authors.lastname: Lejeune
authors.forename: Alexandre
authors.initials: A
authors.lastname: Di Paolo
authors.forename: Alain
authors.initials: A
authors.lastname: Brans
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Frère
authors.forename: Franz X
authors.initials: FX
authors.lastname: Schmid
authors.forename: André
authors.initials: A
authors.lastname: Matagne
mesh_terms: D002942:Circular Dichroism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D011392:Proline; D001618:beta-Lactamases; D007930:Leucine