Tetrameric N5-(l-1-Carboxyethyl)-l-Ornithine Synthase: Guanidine · HCl-Induced Unfolding and a Low Temperature Requirement for Refolding

Guanidine · HCl (GdnHCl)-induced unfolding of tetrameric N5-(l-1-carboxyethyl)-l-ornithine synthase (CEOS; 141,300 Mr) from Lactococcus lactis at pH 7.2 and 25°C occurred in several phases. The enzyme was inactivated at ∼1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5–1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25°C. Refolding and reconstitution of the enzyme were optimal at ≤15°C and yields of active tetramer increased as the concentration of unfolded subunits decreased. Refolding of unfolded subunits and active tetramer assembly upon 100-fold dilution from 5 M GdnHCl at 0°C also was increased two- or fourfold (to 44 or 28% reactivation for 0.08 or 0.28 μM subunit, respectively) when incubated at 15°C, pH 7.2, for 4 h with the Escherichia coli molecular chaperonin GroEL, ATP, MgCl2, and KCl.