The overall objective of this project is to delineate the detailed chemical mechanism of radical generation by the Fe/S-S-adenosylmethionine (AdoMet) family of enzymes. Hypothesis: The adenosylmethionine-dependent iron-sulfur enzymes all operate by a common mechanism in which a reduced cluster interacts with AdoMet to generate an adenosyl radical intermediate which is directly involved in catalysis. These reactions represent novel chemistry for iron-sulfur clusters. To investigate this novel chemistry, biochemical, spectroscopic, mechanistic,, and structural studies of pyruvate formate-lyase activating enzyme (PFL-AE) and related enzymes will be pursued. We will also investigate the mechanism of PFL radical quenching by the iron-dependent AdhE protein.
The specific aims of this proposal are as follows: 1. To investigate the structures and properties of iron-sulfur clusters in the Fe-S/AdoMet family of enzymes, and the interactions of the clusters with AdoMet. Our work will focus primarily on PFL-AE, and will include spectroscopic (EPR, ENDOR, ESEEM, Mossbauer, resonance Raman), lectrochemical, and kinetic studies. 2. To utilize substrate analogs and mechanism-based inhibitors as probes of ES interactions and the PFL-AE catalytic mechanism.. 3. To identify and spectroscopically characterize intermediates in the radical-generation reaction catalyzed by PFL-AE by using rapid-freeze quench spectroscopy. 4. To pursue structural characterization of PFL-AE using X-ray crystallography. To date, no detailed structural information is available for any of the FE-S/AdoMet enzymes, and such structural information could greatly enhance the design and interpretation of mechanistic experiments. 5. To characterize the metal binding site of AdhE, and the role of the metal in glycyl radical quenching.
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