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.
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