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EC number: 201-188-9
CAS number: 79-24-3
Steady-State Kinetics with Nitroethane.
An initial steadystate kinetic analysis was previously described for nitroalkane oxidase (10). In that case, the V/K value for oxygen was independent of whether nitroethane, 1-nitropropane, or 1-nitropentane was the substrate. On the basis of that result, Heasley and Fitzpatrick (10) concluded that the steady-state kinetic mechanism for nitr oalkane oxidase is ping-pong. The nitroalkane substrate would react with the oxidized enzyme to form the reduced enzyme. After product dissociation, the reduced FAD would be reoxidized by oxygen, forming hydrogen peroxide. TheV/K value for oxygen is independent of the specific nitroalkane substrate because oxygen reacts with the enzyme after product dissociation. Consistent with this chemistry, nitroethane readily reduces the enzyme in the absence of oxygen (11). The previous analysis used enzyme that was predominantly inactive due to the majority of the flavin being in the form of a 5-nitrobutyl adduct, as indicated by the very low specific activities (10). Methods have recently been developed to convert the 5-nitrobutyl-FAD in nitroalkane oxidase to FAD and thereby convert the inactive enzyme to the fully active form (11). As a first step in the analysis of the kinetic mechanism of the enzyme, the steady-state kinetic parameters have been determined for the fully active enzyme using nitroethane as substrate, by varying the concentrations of both nitroethane and oxygen at pH 7 and 30 °C. The results were fit well by eq 1, consistent with the proposed ping-pong mechanism (Table 1). If the data were fit to the equation for a sequential mechanism, eq 2, no improvement was seen in the quality of the fit as reflected in the ó value, and the Kia value was not significantly different from zero. The Km values in Table 1 are in agreement with the values previously reported for the unactivated enzyme (10). In contrast, the Vmax value is 11 times the previous number determined at pH 8, consistent with the earlier enzyme being at least 90% inactive.
Table 1: Steady-State Kinetic Parameters for Nitroalkane Oxidase with Nitroethane as Substratea
3.3 + 0.6
1700 + 220
23 + 7
0.24 + 0.06
5.5 + 0.3
2.7 + 0.6
1960 + 360
15 + 9
0.34 + 0.18
5.3 + 0.3
5 + 6
a The enzyme activity was measured in 0.5 mM FAD and 50 mM potassium phosphate buffer, pH 7, at 30 °C.
Product Inhibition Studies.
As a separate approach, the inhibition patterns were determined for products. Critically, the mechanism predicts that the products will be uncompetitive versus nitroethane and competitive versus oxygen. As shown in Table 2, the data were fit best with acetaldehyde being noncompetitive versus nitroethane. However, the quality of the fit was not substantially better when the results were fit to eq 4, which describes an uncompetitive inhibition pattern. Consequently, the inhibition pattern versus nitroethane was also determined with butyraldehyde. In this case, the results were clearly fit best by eq 5, consistent with the aldehyde product being noncompetitive versus nitroethane. Acetaldehyde is an uncompetitive inhibitor versus oxygen (Table 2). If the data were fit to the equation for noncompetitive inhibition, no improvement was seen in the quality of the fit as judged by the value of about 1018 for the Kis value. The inhibition pattern for nitrite was fit best with nitrite being competitive versus nitroethane, with a Kis value of 124 ( 23 mM (data not shown). These results are consistent with nitrite binding to E.FADox but provide no insight into when nitrite release occurs.
The nitroalkane substrate would react with E.FADox to irreversibly form the reduced enzyme with the aldehyde product bound (E.FADred.P). The irreversibility of the oxidation of nitroethane is supported by primary kinetic isotope effects studies with [1,1-2H2]nitroethane (17). After product dissociation, the reduced enzyme irreversibly isomerizes to yield the species E'.FADred. E'.FADred can be reoxidized by oxygen with production of hydrogen peroxide. The noncompetitive inhibition pattern observed for aldehydes versus nitroethane is consistent with the product binding to both E.FADox and E.FADred. The uncompetitive inhibition pattern seen for acetaldehyde versus oxygen is due to the irreversible isomerization of E.FADred after product release from E.FADred.P.
Table 2: Inhibition of Nitroalkane Oxidase by Aldehydesa
0.04 + 0.01
1.26 + 0.06
0.22 + 0.05
0.16 + 0.07
-0.06 + 0.8
0.18 + 0.02
0.18 + 0.03
(7 + 3) x 10 18
13.5 + 1.8
417.6 + 3.0
43.5 + 5.1
28.3 + 3.1
a The enzyme activity was measured in 0.5 mM FAD, 100 mM ACES, 52 mM Tris, and 52 mM ethanolamine buffer, pH 7, at 30 °C. When kept fixed, the concentration of nitroethane was 17 mM; that of oxygen was 230 mM.
b Activity was measured in 0.5 mM FAD and 50 mM potassium phosphate buffer, pH 7, at 30 °C.
Substrate Inhibition Studies.
At concentrations of nitroethane above 25 mM, significant inhibition of the enzyme is observed. In general, this type of inhibition is observed when the substrate binds an enzyme form other than that which catalyzes the conversion of the substrate to the product. The most likely candidate for such an enzyme form is E.FADred. If this is the case, the apparent inhibition constant (Kai) for nitroethane should be independent of the concentration of oxygen. By varying the concentration of nitroethane over the range 1-200 mM, Kai values of 32 ( 14 mM and 30 (6 mM were determined at 54 and 230 uM oxygen, respectively. These values are not significantly different from one another, consistent with nitroethane binding the free reduced enzyme. The formation of the E.FADred.S and E.FADox.P complexes suggests that the substrate/product binding pocket is not significantly affected by the oxidation state of the bound flavin.
Effect of Imidazole on the Activity of Nitroalkane Oxidase.
In the course of this study we observed that the activity of nitroalkane oxidase was severalfold higher when imidazole was present in the reaction assay mixture. This increase in activity showed saturation kinetics, suggesting that imidazole binds to the enzyme.
10. Heasley, C. J., and Fitzpatrick, P. F. (1996) Biochem. Biophys. Res. Commun. 225, 6-10.
11. Gadda, G., and Fitzpatrick, P. F. (1998) Biochemistry 37, 6154-6164.
17. Gadda, G., and Fitzpatrick, P. F. (2000), Biochemistry 39, 1406-1410.
The flavoprotein nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to aldehydes and ketones, respectively, transferring electrons to oxygen to form hydrogen peroxide. The steady-state kinetic mechanism of the active flavin adenine dinucleotide-(FAD-) containing form of the enzyme has been determined with nitroethane at pH 7 to be bi-ter ping-pong, with oxygen reacting with the free reduced enzyme after release of the aldehyde product. The Vmax value is 5.5 ( 0.3 s-1 and the Km values for nitroethane and oxygen are 3.3 ( 0.6 and 0.023 ( 0.007 mM, respectively. The free reduced enzyme forms a dead-end complex with nitroethane, with a Kai value of 30 ( 6 mM. Acetaldehyde and butyraldehyde are noncompetitive inhibitors versus nitroethane due to formation of a dead-end complex between the oxidized enzyme and the product. Acetaldehyde is an uncompetitive inhibitor versus oxygen, indicating that an irreversible isomerization of the free reduced enzyme occurs before the reaction with oxygen. Addition of unprotonated imidazole results in a 5-fold increase in the Vmax value, while the V/K values for nitroethane and oxygen are unaffected. A 5-fold increase in the Kai value for nitroethane and a 6.5-fold increase in the Kii value for butyraldehyde are observed in the presence of imidazole. These results are consistent with the isomerization of the free reduced enzyme being about 80% rate-limiting for catalysis and with a model in which unprotonated imidazole accelerates the rate of isomerization.
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