Abstract
The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG‡ for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 5850-5856 |
| Number of pages | 7 |
| Journal | Biochemistry |
| Volume | 42 |
| Issue number | 19 |
| DOIs | |
| State | Published - May 20 2003 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Reductive half-reaction of nitroalkane oxidase : Effect of mutation of the active site aspartate to glutamate. / Valley, Michael P.; Fitzpatrick, Paul F.
In: Biochemistry, Vol. 42, No. 19, 20.05.2003, p. 5850-5856.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Reductive half-reaction of nitroalkane oxidase
T2 - Effect of mutation of the active site aspartate to glutamate
AU - Valley, Michael P.
AU - Fitzpatrick, Paul F
PY - 2003/5/20
Y1 - 2003/5/20
N2 - The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG‡ for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.
AB - The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG‡ for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.
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UR - http://www.scopus.com/inward/citedby.url?scp=0038613012&partnerID=8YFLogxK
U2 - 10.1021/bi034061w
DO - 10.1021/bi034061w
M3 - Article
C2 - 12741843
AN - SCOPUS:0038613012
VL - 42
SP - 5850
EP - 5856
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 19
ER -