Evidence for an essential arginine in the flavoprotein nitroalkane oxidase.
The flavoprotein nitroalkane oxidase from the fungus Fusarium oxysporum catalyzes the oxidative denitrification of primary or secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. The enzyme is inactivated in a time-dependent fashion upon treatment with the arginine-directed reagents phenylglyoxal, 2,3-butanedione, and cyclohexanedione. The inactivation shows first order kinetics with all reagents. Valerate, a competitive inhibitor of the enzyme, fully protects the enzyme from inactivation, indicating that modification is active site directed. The most rapid inactivation is seen with phenylglyoxal, with a k(inact) of 14.3 +/- 1.1 M(-1) min(-1) in phosphate buffer at pH 7.3 and 30 degrees C. The lack of increase in the enzymatic activity of the phenylglyoxal-inactivated enzyme after removing the unreacted reagent by gel filtration is consistent with inactivation being due to covalent modification of the enzyme. A possible role for an active site arginine in substrate binding is discussed.
Gadda
G
G
Department of Biochemistry and Biophysics; Texas A & M University, College Station, Texas 77843-2128, USA.
Banerjee
A
A
Fleming
G S
GS
Fitzpatrick
P F
PF
D001120:Arginine; D001667:Binding, Competitive; D003512:Cyclohexanones / Q000494:pharmacology; D003931:Diacetyl / Q000494:pharmacology; D049308:Dioxygenases; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005420:Flavoproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D005670:Fusarium / Q000201:enzymology*; D007700:Kinetics; D010105:Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D010421:Pentanoic Acids / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology; D013379:Substrate Specificity
D003512:Cyclohexanones; D004791:Enzyme Inhibitors; D005420:Flavoproteins; D010421:Pentanoic Acids; C018031:1,2-cyclohexanedione; D001120:Arginine; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; C038780:n-pentanoic acid; D003931:Diacetyl; D010658:Phenylglyoxal