Cleavage of carbon-hydrogen bonds is one of the most fundamental chemical reactions in biology. Despite its importance, the mechanisms by which enzymes carry out this reaction are far from being fully understood. This proposal focuses on the enzymes known as flavoprotein oxidases, which oxidize their substrates by removing a hydride equivalent and transferring it to the tightly bound flavin cofactor. The mechanism of carbon-hydrogen bond cleavage by the amino and hydroxy acid oxidases has generally been considered to involve a carbanion intermediate. However, the recent structure of D-amino acid oxidase and recent heavy isotope data suggest that the mechanisms may involve direct hydride transfer. The goal of the research here is to determine the mechanism of carbon-hydrogen bond cleavage by D-amino acid oxidase, tryptophan monooxygenase, and flavocytochrome b2. The methods rely heavily on the use of multiple kinetic isotope effects to determine transition state structure. In addition, kinetic analyses of site- specific mutants of D-amino acid oxidase will be used to determine the roles of specific amino acid residues in catalysis. Nitroalkane oxidase will be studied as an example of an enzyme likely to utilize a carbanion intermediate. The mechanism of formation of the nitrobutyl-FAD formed during turnover with nitroethane will be elucidated. Substituted nitroalkanes will be characterized as substrates; the results will be compared with model studies. The results of these studies with flavoprotein oxidases will provide insight into the mechanisms of other flavoproteins and of the general problem of cleavage of carbon-hydrogen bonds by biological systems. The increased understanding and the methods developed will be useful in developing inhibitors of other flavoproteins oxidases for biomedical applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058698-03
Application #
6343051
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
1999-01-01
Project End
2002-12-31
Budget Start
2001-01-01
Budget End
2001-12-31
Support Year
3
Fiscal Year
2001
Total Cost
$217,540
Indirect Cost
Name
Texas A&M University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
047006379
City
College Station
State
TX
Country
United States
Zip Code
77845
Fitzpatrick, Paul F (2017) Nitroalkane oxidase: Structure and mechanism. Arch Biochem Biophys 632:41-46
Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan et al. (2017) Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues. Biochemistry 56:869-875
Trimmer, Elizabeth E; Wanninayake, Udayanga S; Fitzpatrick, Paul F (2017) Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase. Biochemistry 56:2024-2030
Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan et al. (2016) Mechanism of the Flavoprotein L-Hydroxynicotine Oxidase: Kinetic Mechanism, Substrate Specificity, Reaction Product, and Roles of Active-Site Residues. Biochemistry 55:697-703
Tormos, José R; Suarez, Marina B; Fitzpatrick, Paul F (2016) 13C kinetic isotope effects on the reaction of a flavin amine oxidase determined from whole molecule isotope effects. Arch Biochem Biophys 612:115-119
Fitzpatrick, Paul F (2015) Combining solvent isotope effects with substrate isotope effects in mechanistic studies of alcohol and amine oxidation by enzymes. Biochim Biophys Acta 1854:1746-55
Roberts, Kenneth M; Tormos, José R; Fitzpatrick, Paul F (2014) Characterization of unstable products of flavin- and pterin-dependent enzymes by continuous-flow mass spectrometry. Biochemistry 53:2672-9
Gaweska, Helena M; Taylor, Alexander B; Hart, P John et al. (2013) Structure of the flavoprotein tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants. Biochemistry 52:2620-6
Gadda, Giovanni; Fitzpatrick, Paul F (2013) Solvent isotope and viscosity effects on the steady-state kinetics of the flavoprotein nitroalkane oxidase. FEBS Lett 587:2785-9
Gaweska, Helena M; Roberts, Kenneth M; Fitzpatrick, Paul F (2012) Isotope effects suggest a stepwise mechanism for berberine bridge enzyme. Biochemistry 51:7342-7

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