title: Inactivation of nitroalkane oxidase upon mutation of the active site base and rescue with a deprotonated substrate.
abstract: 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 alpha-proton.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Valley
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D049308:Dioxygenases; D004789:Enzyme Activation; D004980:Ethane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009595:Nitroparaffins / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D009595:Nitroparaffins; D001224:Aspartic Acid; C014705:nitroethane; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; D004980:Ethane