Inactivation of Nitroalkane Oxidase upon Mutation of the Active Site Base and Rescue with a Deprotonated Substrate

• Published in: Journal of the American Chemical Society Vol. 125; no. 29; pp. 8738 - 8739
• Main Authors: Valley, Michael P, Fitzpatrick, Paul F
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 23.07.2003
• Subjects: Analytical, structural and metabolic biochemistry; Aspartic Acid - chemistry; Aspartic Acid - metabolism; Binding Sites; Biological and medical sciences; Dioxygenases; Enzyme Activation; Enzymes and enzyme inhibitors; Ethane - analogs & derivatives; Ethane - chemistry; Ethane - metabolism; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Kinetics; Mutagenesis, Site-Directed; Nitroparaffins - chemistry; Nitroparaffins - metabolism; Oxidoreductases; Oxygenases - chemistry; Oxygenases - genetics; Oxygenases - metabolism; 2-Nitropropane dioxygenase; Oxidoreductases; Mutation; Inactivation; Enzyme; Active site
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja036045s

Mutation of Asp402 in nitroalkane oxidase to Asn or Ala inactivates the enzyme with neutral nitroethane as substrate, but the activity can be rescued with the nitroethane anion. The V/K values of the D402N and D402A enzymes with the nitroethane anion are independent of pH, whereas the V/K values of the wild-type and D402E enzymes are pH dependent with both the protonated and the deprotonated forms of nitroethane. Moreover, although the V/K value of the D402E enzyme with neutral nitroethane is 20-fold less than that of the wild-type enzyme, there is only a 2-fold difference in the V/K values with the nitroethane anion. These results are fully consistent with a primary role for Asp402 as the active site base in nitroalkane oxidase which abstracts the substrate α-proton.