The folding and solution conformation of penicillin G acylase

Abstract

The solution conformation properties of penicillin G acylase (EC 3.5.1.11) have been characterised by nearand 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 α and β 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 β peptide aggregates over a wide range of urea concentrations but that the α peptide refolds reversibly to a compact state. Physical studies showed that the refolded α peptide has a compact but asymmetric structure with more α 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 β peptide in the native enzyme. The results of these investigations indicate that the α peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase. Copyright © 1990, Wiley Blackwell. All rights reserved