title: The kinetics behavior of the reduction of formaldehyde catalyzed by Alcohol Dehydrogenase (ADH) and partial uncompetitive substrate inhibition by NADH.
abstract: Alcohol dehydrogenase (ADH) catalyzes the final step in the biosynthesis of methanol from CO2. Here, we report the steady-state kinetics for ADH, using a homogeneous enzyme preparation with formaldehyde as the substrate and nicotinamide adenine dinucleotide (NADH) as the cofactor. When changing NADH concentrations with the fixed concentrations of HCHO (more or less than NADH), kinetic studies revealed a particular zigzag phenomenon for the first time. Increasing formaldehyde concentration can weaken substrate inhibition and improve catalytic efficiency. The kinetic mechanism of ADH was analyzed using the secondary fitting method. The double reciprocal plots (1/v∼1/[HCHO] and 1/[NADH]) strongly demonstrated that the substrate inhibition by NADH was uncompetitive versus formaldehyde and partial. In the direction of formaldehyde reduction, ADH has an ordered kinetic mechanism with formaldehyde adding to enzyme first and product methanol released last. The second reactant NADH can combine with the enzyme-methanol complex and then methanol dissociates from it at a slower rate than from enzyme-methanol. The reaction velocity depends on the relative rates of the alternative pathways. The addition of NADH also accelerates the releasing of methanol. As a result, substrate inhibition and activation occurred intermittently, and the zigzag double reciprocal plot (1/v∼1/[NADH]) was obtained.
authors.affiliation: Department of Chemical Engineering and the Environment, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing, China, 100081.
authors.forename: Nuan
authors.initials: N
authors.lastname: Wen
authors.forename: Wenfang
authors.initials: W
authors.lastname: Liu
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Hou
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Zhao
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D055162:Biocatalysis; D004789:Enzyme Activation; D005557:Formaldehyde / Q000378:metabolism*; D007700:Kinetics; D000432:Methanol / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013997:Time Factors
chemical_list: D009243:NAD; D005557:Formaldehyde; D000426:Alcohol Dehydrogenase; D000432:Methanol