Flavoproteins catalyze a wide range of biologically critical reactions. Many of these involve the two electron oxidation of a carbon-oxygen or carbon-nitrogen bond. Despite the importance of such reactions, the mechanisms of these enzymes continue to be controversial. In the next grant period we propose to focus on flavoenzymes which oxidize bonds between carbon and nitrogen. We propose to study two enzymes involved in metabolism of polyamines: polyamine oxidase, which preferentially oxidizes acetylated spermine and spermidine, and spermine oxidase, which prefers unacetylated spermine. Polyamines are critical for the growth of cells, and alternate substrates for polyamine oxidase and spermine oxidase prevent growth of cancer cells. The catalytic mechanisms and structural bases for substrate specificity in these two enzymes will be studied. We will also continue study of nitroalkane oxidase. This enzyme is an unusual flavoprotein which oxidizes the carbon-nitrogen bond of nitroalkanes by forming a carbanion intermediate. The enzyme serves as a model for flavoprotein-catalyzed proton removal from carbon. The approaches to all three enzymes will combine steady-state and rapid-reaction kinetics, including kinetic isotope effects, with structural approaches. In the case of nitroalkane oxidase, the nonenzymatic reaction will also be studied to gain insight into the mechanism by which the enzyme increases the rate of proton abstraction.
Polyamine oxidase and spermine oxidase are both involved in recycling of polyamines. Polyamines are needed for the growth of cells, and compounds which disrupt}polyamine metabolism have been shown to prevent the growth of cancer cells. Nitroalkane oxidase is a model enzyme for studying the mechanisms of proteins which remove a proton from carbon.
|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|
|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; 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|
|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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