Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni

摘要: Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The KSI was reversible since activity as well fluorescence CD spectra almost completely recovered after refolding. equilibrium unfolding transitions monitored by measurements were coincident with each other, transition midpoint increased increasing protein concentration. This suggests that follows a simple two-state consisting native dimer unfolded monomer without any thermodynamically stable intermediates. Sedimentation analysis size-exclusion chromatography at different urea concentrations supported model evidence folded monomeric Consistent model, (1)H-(15)N HSQC obtained for in region could be reproduced addition KSI. refolding kinetics intensity described fast first-order process followed second-order subsequent slow processes rate constants 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), 0.017 respectively, 0.62 M urea, suggesting there may intermediate. After burst phase accounts >83% total amplitude, negative molar ellipticity 225 nm slowly single comparable bimolecular intermediate step. recovery denatured state markedly dependent upon concentration, implying monomers are not fully active. Taken together, our results demonstrate dimerization induces fold into complete structure is crucial maintaining tertiary perform efficient catalysis.