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

The flavoprotein nitroalkane oxidase catalyzes the oxidation of a broad ran ge of primary and secondary nitroalkanes to the respective aldehydes or ket ones, with production of hydrogen peroxide and nitrite. The VIR values for primary nitroalkanes increase with increasing chain length, reaching a maxi mum with 1-nitrobutane [Gadda, G., and Fitzpatrick, P. F. (1999) Arch. Bioc hem. Biophys. 363, 369-313]. In the present report, pH and deuterium kineti c isotope effects with a series of primary nitroalkanes and phenylnitrometh ane 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 un protonated 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 1 with phenylnitromethane. These results show that i ncreasing the size of the substrate results in an increased partitioning fo rward to catalysis. The (D)(V/K) and V-D(max) values at pH 5.5 have been us ed 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 substra te, with a Delta Delta G(binding) Of 1.7 kcal mol(-1) for each additional m ethylene group. Such a value is less than the value of 2.6 kcal mol(-1) pre viously determined for the effect of a methylene group on the V/K value [Ga dda, G., and Fitzpatrick, P. F. (1999) Arch. Biochem. Biophys. 363, 309-313 ], suggesting that the total energy available per methylene group is used n ot only to enhance binding but also to increase the rate of catalysis. (C) 2000 Academic Press.