Inactivation of nitroalkane oxidase upon mutation of the active site base and rescue with a deprotonated substrate

Journal of the American Chemical Society

Volumn 125, Issue 29, 2003, Pages 8738-8739

  Valley, Michael P ^a   Fitzpatrick, Paul F ^a  

^a TEXAS A AND M UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

NITROALKANE OXIDASE; OXIDOREDUCTASE; UNCLASSIFIED DRUG;

ARTICLE; ENZYME ACTIVE SITE; ENZYME ACTIVITY; ENZYME INACTIVATION; MUTATION; PH;

ASPARTIC ACID; BINDING SITES; DIOXYGENASES; ENZYME ACTIVATION; ETHANE; HYDROGEN-ION CONCENTRATION; KINETICS; MUTAGENESIS, SITE-DIRECTED; NITROPARAFFINS; OXYGENASES;

EID: 0038298092     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja036045s     Document Type: Article

References (13)

1. Kido, T.; Hashizume, K.; Soda, K. J. Bacteriol. 1978, 133, 53-58.
2. Gadda, G.; Edmondson, R. D.; Russell, D. H.; Fitzpatrick, P. F. J. Biol. Chem. 1997, 272, 5563-5570.
3. Daubner, S. C.; Gadda, G.; Valley, M. P.; Fitzpatrick, P. F. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2702-2707.
4. Valley, M. P.; Fitzpatrick, P. F. Biochemistry 2003, 42, 5850-5856.
5. Feuer, H. The Chemistry of the Nitro and Nitroso Groups, Part 1; Interscience: New York, 1969; Chapter 7.
6. Bernasconi, C. F. Acc. Chem. Res. 1992, 25, 9-16.
7. Less than one-half of the active sites of the D402A enzyme are purified with flavin bound, and the enzyme is less stable than other mutant enzymes. Because most of the characteristics of the D402A enzyme were similar to those of the D402N enzyme, a minimal amount of data was collected for this less stable variant.
8. Gadda, G.; Fitzpatrick, P. F. Biochemistry 2000, 39, 1406-1410.
9. All assays were conducted and all data were fit as previously described.
10. -1, respectively).
11. The nitroethane anion was generated by mixing concentrated neutral nitroethane with a 2-fold excess of potassium hydroxide and incubating overnight.
12. -1). No changes in the V/K values of the wild type and D402E enzymes are observed in this concentration range.
13. [N-Morpholino]-ethanesulfonic acid (MES) was used for pH 6-6.5, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES) for pH 7-8.1, and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS) for pH 7.9-9. In all cases, the concentration of the buffer was 500 mM to neutralize the hydroxide used to form the anion.