title: The nonoxidative conversion of nitroethane to ethylnitronate in Neurospora crassa 2-nitropropane dioxygenase is catalyzed by histidine 196.
abstract: The deprotonation of nitroethane catalyzed by Neurospora crassa 2-nitropropane dioxygenase was investigated by measuring the formation and release of ethylnitronate formed in turnover as a function of pH and through mutagenesis studies. Progress curves for the enzymatic reaction obtained by following the increase in absorbance at 228 nm over time were visibly nonlinear, requiring a logarithmic approximation of the initial reaction rates for the determination of the kinetic parameters of the enzyme. The pH dependence of the second-order rate constant k cat/ K m with nitroethane as substrate implicates the presence of a group with a p K a of 8.1 +/- 0.1 that must be unprotonated for nitronate formation. Mutagenesis studies suggest that this group is histidine 196 as evident from the inability of a H196N variant form of the enzyme to catalyze the formation of ethylnitronate from nitroethane. Replacement of histidine 196 with asparagine resulted in an approximately 15-fold increase in the k cat/ K m with ethylnitronate as compared to the wild-type, which results from the inability of the mutant enzyme to undergo nonoxidative turnover. The results presented herein are consistent with a branched catalytic mechanism for the enzyme in which the ethylnitronate intermediate formed from the H196-catalyzed deprotonation of nitroethane partitions between release from the active site and oxidative denitrification to yield acetaldehyde and nitrite.
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Francis
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gadda
mesh_terms: D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004980:Ethane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009492:Neurospora crassa / Q000201:enzymology* / Q000378:metabolism; D009595:Nitroparaffins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D009595:Nitroparaffins; D006639:Histidine; C014705:nitroethane; D001216:Asparagine; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; D004980:Ethane