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Related Experiment Videos

Substrate specificity of a nitroalkane-oxidizing enzyme.

G Gadda1, P F Fitzpatrick

  • 1Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas, 77843-2128, USA.

Archives of Biochemistry and Biophysics
|March 9, 1999
PubMed
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Nitroalkane oxidase from Fusarium oxysporum oxidizes nitroalkanes to aldehydes. Its substrate specificity reveals a hydrophobic active site, preferring shorter, unbranched chains for efficient binding.

Area of Science:

  • Biochemistry
  • Enzymology
  • Fungal Metabolism

Background:

  • Nitroalkane oxidase is a flavoprotein from Fusarium oxysporum.
  • It catalyzes the oxidation of nitroalkanes to aldehydes, producing hydrogen peroxide and nitrite.

Purpose of the Study:

  • To determine the substrate specificity of nitroalkane oxidase.
  • To use specificity as a probe for understanding the enzyme's active site structure.

Main Methods:

  • Enzyme kinetics studies were performed using various primary and secondary nitroalkanes.
  • The Michaelis constant (K) and maximum velocity (V) were measured to calculate V/K values.
  • Competitive inhibition was assessed using 3-nitro-1-propionate.

Main Results:

Related Experiment Videos

  • The enzyme exhibits activity on both primary and secondary nitroalkanes, with a preference for unbranched primary substrates.
  • V/K values increase with alkyl chain length up to 1-nitrobutane, indicating a hydrophobic binding pocket accommodating four carbons.
  • Substrates with hydroxyl groups showed significantly lower V/K values, further supporting a hydrophobic active site.
  • 3-Nitro-1-propionate acted as a competitive inhibitor with a Kis of 3.1 mM.

Conclusions:

  • The active site of Fusarium oxysporum nitroalkane oxidase is predominantly hydrophobic.
  • The binding pocket can accommodate alkyl chains up to four carbons, with each methylene group contributing to binding energy.
  • The enzyme's specificity is finely tuned for efficient catalysis of nitroalkane oxidation.