MECHANISMS OF FLAVOPROTEIN OXIDASES

  • Fitzpatrick, Paul F, (PI)

Research project

Description

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.
StatusActive
Effective start/end date1/1/993/31/18

Funding

  • National Institutes of Health: $284,162.00
  • National Institutes of Health: $27,835.00
  • National Institutes of Health: $291,000.00
  • National Institutes of Health: $299,000.00
  • National Institutes of Health: $299,000.00
  • National Institutes of Health: $291,000.00
  • National Institutes of Health: $349,308.00
  • National Institutes of Health: $299,000.00
  • National Institutes of Health: $329,708.00
  • National Institutes of Health: $251,267.00
  • National Institutes of Health: $408,484.00
  • National Institutes of Health: $116,542.00
  • National Institutes of Health: $352,836.00
  • National Institutes of Health: $49,440.00
  • National Institutes of Health: $211,435.00
  • National Institutes of Health: $11,590.00
  • National Institutes of Health: $223,830.00
  • National Institutes of Health: $217,540.00
  • National Institutes of Health: $212,642.00

Fingerprint

Flavoproteins
Enzymes
Carbon
Hydrogen bonds
Substrates
Oxidoreductases
D-Amino-Acid Oxidase
Polyamines
Nitrogen
Isotopes
Hydrides
Proteins
Kinetics
Cells
Amines
Metabolism
Oxidation
Amino Acids
Biological systems
Oxygen

Keywords

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)