title: Unfolding of chondroitinase ABC Ι is dependent on thermodynamic driving force by kinetically rate constant-amplitude compensation: A stopped-flow fluorescence study.
abstract: We had previously investigated the role of a loop on the activity and conformational stability of chondroitinase ABC Ι (cABC Ι) by constructing some representative mutants in which a network interaction around Asp689was manipulated. Here we extended our study by measuring the proteolytic resistance, long term and thermal stability as well as unfolding kinetics of these variants. Long term stability data at 4 and 25°C for 3 weeks indicates that all mutants remain considerably active at 4°C. Thermoinactivation rates for all variants shows that the wild type (WT) enzyme retained 50% of its activity after 2min keeping at 40°C, while L701T, H700N and H700N/L701T as conformationally stabilized variants, have slower inactivation rate. It was also found that compact and thermodynamically stabilized variants are more resistant to tryptolytic digestion. Also, kinetic curves of chemical unfolding of the enzyme variants from stopped-flow fluorescence measurements were best fitted into a three-exponential function with three rate constants and corresponding amplitudes. We found that the energy barrier of the fast unfolding phase is lower in stabilized variants; while the amplitude of this phase to the whole amplitude of the unfolding reaction is lower than that of destabilized variants, indicating more population of stabilized mutants unfold via slower unfolding phase. We concluded that the rate of local conformational change alone is not the same that is expected from global thermodynamic stability; however the corresponding amplitude can compensate the rate constant toward thermodynamic stability.
authors.affiliation: Department of Biology, Faculty of Sciences, University of Zanjan, Zanjan, Iran.
authors.forename: S Akram
authors.initials: SA
authors.lastname: Shirdel
authors.affiliation: Department of Biology, Faculty of Sciences, University of Zanjan, Zanjan, Iran.
authors.forename: Khosrow
authors.initials: K
authors.lastname: Khalifeh
authors.affiliation: Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
authors.affiliation: Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Abolfazl
authors.initials: A
authors.lastname: Golestani
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: khajeh@modares.ac.ir.
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
mesh_terms: D019943:Amino Acid Substitution; D019765:Chondroitin ABC Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D056811:Unfolded Protein Response
chemical_list: D011994:Recombinant Proteins; D019765:Chondroitin ABC Lyase
keywords: Chondroitinase ABC Ι; Conformational change; Limited proteolysis; Stopped-flow; Thermal stability; Unfolding kinetics