title: Exploration of two-enzyme coupled catalysis system using scanning electrochemical microscopy.
abstract: In biological metabolism, a given metabolic process usually occurs via a group of enzymes working together in sequential pathways. To explore the metabolism mechanism requires the understanding of the multienzyme coupled catalysis systems. In this paper, an approach has been proposed to study the kinetics of a two-enzyme coupled reaction using SECM combining numerical simulations. Acetylcholine esterase and choline oxidase are immobilized on cysteamine self-assembled monolayers on tip and substrate gold electrodes of SECM via electrostatic interactions, respectively. The reaction kinetics of this two-enzyme coupled system upon various separation distance precisely regulated by SECM are measured. An overall apparent Michaelis-Menten constant of this enzyme cascade is thus measured as 2.97 mM at an optimal tip-substrate gap distance of 18 μm. Then, a kinetic model of this enzyme cascade is established for evaluating the kinetic parameters of individual enzyme by using the finite element method. The simulated results demonstrate the choline oxidase catalytic reaction is the rate determining step of this enzyme cascade. The Michaelis-Menten constant of acetylcholine esterase is evaluated as 1.8 mM. This study offers a promising approach to exploring mechanism of other two-enzyme coupled reactions in biological system and would promote the development of biosensors and enzyme-based logic systems.
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China.
authors.forename: Zeng-Qiang
authors.initials: ZQ
authors.lastname: Wu
authors.forename: Wen-Zhi
authors.initials: WZ
authors.lastname: Jia
authors.forename: Kang
authors.initials: K
authors.lastname: Wang
authors.forename: Jing-Juan
authors.initials: JJ
authors.lastname: Xu
authors.forename: Hong-Yuan
authors.initials: HY
authors.lastname: Chen
authors.forename: Xing-Hua
authors.initials: XH
authors.lastname: Xia
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D004593:Electrophorus / Q000378:metabolism; D064347:Microscopy, Electrochemical, Scanning / Q000379:methods*
chemical_list: D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D000110:Acetylcholinesterase