Use of pH and kinetic isotope effects to dissect the effects of substrate size on binding and catalysis by nitroalkane oxidase

Giovanni Gadda, Damon Y. Choe, Paul F. Fitzpatrick

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The flavoprotein nitroalkane oxidase catalyzes the oxidation of a broad range of primary and secondary nitroalkanes to the respective aldehydes or ketones, with production of hydrogen peroxide and nitrite. The V/K values for primary nitroalkanes increase with increasing chain length, reaching a maximum with 1-nitrobutane [Gadda, G., and Fitzpatrick, P. F. (1999) Arch. Biochem. Biophys. 363, 309-313]. In the present report, pH and deuterium kinetic isotope effects with a series of primary nitroalkanes and phenylnitromethane as substrates have been used to dissect the effects of chain length on binding and catalysis. The apparent pK(a) value for a group that must be unprotonated for catalysis decreases from about 7 to 5.3 with increasing size of the substrate. The (D)(V/K) values for these substrates decrease from 7.5 with nitroethane to I with phenylnitromethane. These results show that increasing the size of the substrate results in an increased partitioning forward to catalysis. The (D)(V/K) and (D)V(max) values at pH 5.5 have been used to calculate the effect of substrate size on the K(d) values for primary nitroalkanes. The K(d) values decrease with increasing length of the substrate, with a ΔΔG(binding) of 1.7 kcal mol-1 for each additional methylene group. Such a value is less than the value of 2.6 kcal mol-1 previously determined for the effect of a methylene group on the V/K value [Gadda, G., and Fitzpatrick, P. F. (1999) Arch. Biochem. Biophys. 363, 309-313], suggesting that the total energy available per methylene group is used not only to enhance binding but also to increase the rate of catalysis. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)138-144
Number of pages7
JournalArchives of Biochemistry and Biophysics
Issue number1
Publication statusPublished - Oct 1 2000



  • Activation energy
  • Binding
  • Commitments to catalysis
  • Flavoprotein
  • Fusarium oxysporum
  • Kinetic isotope effects
  • Nitroalkane
  • Nitroalkane oxidase
  • pH dependence

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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