Reductive half-reaction of nitroalkane oxidase: effect of mutation of the active site aspartate to glutamate.
The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the DeltaG(++) for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.
Valley
Michael P
MP
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Fitzpatrick
Paul F
PF
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D049308:Dioxygenases; D005670:Fusarium / Q000201:enzymology / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics
D001224:Aspartic Acid; D018698:Glutamic Acid; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase