title: Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni.
abstract: Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.
authors.affiliation: Division of Molecular and Life Sciences, Center for Biofunctional Molecules, and Department of Chemistry, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea.
authors.forename: D H
authors.initials: DH
authors.lastname: Kim
authors.forename: D S
authors.initials: DS
authors.lastname: Jang
authors.forename: G H
authors.initials: GH
authors.lastname: Nam
authors.forename: S
authors.initials: S
authors.lastname: Yun
authors.forename: J H
authors.initials: JH
authors.lastname: Cho
authors.forename: G
authors.initials: G
authors.lastname: Choi
authors.forename: H C
authors.initials: HC
authors.lastname: Lee
authors.forename: K Y
authors.initials: KY
authors.lastname: Choi
mesh_terms: D002850:Chromatography, Gel; D002942:Circular Dichroism; D020604:Comamonas testosteroni / Q000201:enzymology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D013251:Steroid Isomerases / Q000737:chemistry / Q000378:metabolism*; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
chemical_list: D014508:Urea; D013251:Steroid Isomerases; C022660:steroid delta-isomerase