Unfolding of Chondroitinase Abc Ι Is Dependent on Thermodynamic Driving Force by Kinetically Rate Constant-Amplitude Compensation: A Stopped-Flow Fluorescence Study Publisher Pubmed



Shirdel SA¹ ; Khalifeh K¹ ; Ranjbar B² ; Golestani A³ ; Khajeh K⁴ Show Affiliations
Source: Enzyme and Microbial Technology Published:2016

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 Asp689 was 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 2 min 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. © 2016 Elsevier Inc.