Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.
Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 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 degrees C. Refolding and reconstitution of the enzyme were optimal at </=15 degrees 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 degrees C also was increased two- or fourfold (to 44 or 28% reactivation for 0.08 or 0.28 microM subunit, respectively) when incubated at 15 degrees C, pH 7.2, for 4 h with the Escherichia coli molecular chaperonin GroEL, ATP, MgCl(2), and KCl.
Ruvinov
S B
SB
Laboratory of Biochemistry, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, USA.
Thompson
J
J
Sackett
D L
DL
Ginsburg
A
A
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli; D019791:Guanidine / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology*; D046911:Macromolecular Substances; D011489:Protein Denaturation / Q000187:drug effects*; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004228:Dithionitrobenzoic Acid; D000594:Amino Acid Oxidoreductases; C064774:N(5)-(carboxyethyl)ornithine synthase; D019791:Guanidine