title: New insights into the effects of thermal treatment on the catalytic activity and conformational structure of glucose oxidase studied by electrochemistry, IR spectroscopy, and theoretical calculation.
abstract: Protein conformational changes may be associated with particular properties such as its function, transportation, assembly, tendency to aggregate, and potential cytotoxicity. In this study, the mechanism of the effects of thermal unfolding of proteins on their catalytic activities and conformational structures were studied by utilizing glucose oxidase (GOx) as a model protein. The characteristic kinetic constants for the enzymatic reaction were evaluated by the use of an electrochemical approach under a substrate-saturated condition. A combination of quantitative second-derivative infrared analysis, two-dimensional infrared correlation spectroscopy (2D IR), and theoretical calculation was used to elucidate the conformational structures that were responsible for inactivation and denaturation of GOx induced by heat. The IR analysis demonstrated that the conformational structures of GOx, especially the α-helix and unordered structures, were greatly dependent on the system temperature. Thermal treatment resulted in the increase of the unordered structure accompanied by the loss of the α-helical structure in GOx conformation. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations revealed that thermal treatment could significantly alter the electronic characteristics and the intramolecular electron transfer ability of FAD (flavin adenine dinucleotide), and hydrogen bond networks formed between FAD and the amino acid residues around the cofactor, leading to the change of the secondary structure and the catalytic activity of GOx. The study essentially paves an effective approach to investigation of the mechanism of protein unfolding.
authors.affiliation: Jiangsu Key Laboratory of Biofunctional Materials, Laboratory of Electrochemistry, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Wang
authors.initials: W
authors.lastname: Xu
authors.forename: Ping
authors.initials: P
authors.lastname: Wu
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Chenxin
authors.initials: C
authors.lastname: Cai
authors.forename: Bo
authors.initials: B
authors.lastname: Zhao
mesh_terms: D055162:Biocatalysis; D055664:Electrochemical Techniques; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008962:Models, Theoretical; D056004:Molecular Dynamics Simulation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011789:Quantum Theory; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D005949:Glucose Oxidase; D005947:Glucose