The folding and solution conformation of penicillin G acylase.
The solution conformation properties of penicillin G acylase (EC 3.5.1.11) have been characterised by near- and far-ultraviolet circular dichroism, steady-state and time-resolved fluorescence spectroscopy and differential sedimentation velocity. The enzyme (86 kDa) was found to be spherical and stable unfolding over a narrow range of urea concentrations in an apparently cooperative fashion with a mid-point of 4.5 M urea. Separation of its constituent alpha and beta peptides (23.8 kDa and 62.2 kDa, respectively) was accompanied by loss of enzyme activity and unfolding, the kinetics of unfolding being highly dependent upon urea concentration. Urea gradient gel electrophoresis showed that the separated beta peptide aggregates over a wide range of urea concentrations but that the alpha peptide refolds reversibly to a compact state. Physical studies showed that the refolded alpha peptide has a compact but asymmetric structure with more alpha helix than the native enzyme, but is more sensitive to denaturant. The latter is suggested to be due to a hydrophobic patch detected by 8-anilino-1-naphthalene sulfonic acid binding and which is normally covered by the beta peptide in the native enzyme. The results of these investigations indicate that the alpha peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase.
Department of Biochemistry and Genetics, School of Biomedical and Biomolecular Sciences, University of Newcastle upon Tyne, England.
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D000581:Amidohydrolases; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005453:Fluorescence; D007700:Kinetics; D010399:Penicillin Amidase; D010400:Penicillin G; D010455:Peptides; D011487:Protein Conformation; D012996:Solutions; D014364:Tryptophan / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D010455:Peptides; D012996:Solutions; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D000581:Amidohydrolases; D010399:Penicillin Amidase; D010400:Penicillin G