Tetrameric N5-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,200 MW) from Lactococcus lactis catalyzes a NADPH-dependent reductive condensation between pyruvate and the side-chain amino group of L- ornithine or L-lysine and may be important for post-translational modification of protein lysyl residues. Guanidine-HCl (GdnHCl)-induced unfolding of this tetrameric enzyme at pH 7.2 and 25 C occurred in several phases. The enzyme was inactivated at ca. 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 ca. 15 C and yields of active tetramer increased as the protein concentration decreased. Refolding of unfolded subunits and active tetramer assembly upon 100-fold dilution from 5 M GdnHCl on ice also was increased 2- or 4-fold (to 44 or 28 per cent reactivation for 0.08 or 0.28 micromolar subunit, respectively) when incubated at 15 C, pH 7.2 for 4 h with the E. coli molecular chaperonin GroEL, ATP, Mg(II), and KCl. The unusual low-temperature requirement for refolding CEOS results from competing aggregation reactions that become dominant at temperatures higher than 15 C. - N5-(carboxyethyl)ornithine synthase, guanidine hydrochloride, inactivation, tetramer dissociation, unfolding, refolding, GroEL-Mg- ATP, chaperonin-60

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL000317-03
Application #
6290365
Study Section
Special Emphasis Panel (LB)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
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