title: Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme.
abstract: The activation of pyruvate formate-lyase (PFL) by pyruvate formate-lyase activating enzyme (PFL-AE) involves formation of a specific glycyl radical on PFL by the PFL-AE in a reaction requiring S-adenosylmethionine (AdoMet). Surface plasmon resonance experiments were performed under anaerobic conditions on the oxygen-sensitive PFL-AE to determine the kinetics and equilibrium constant for its interaction with PFL. These experiments show that the interaction is very slow and rate-limited by large conformational changes. A novel AdoMet binding assay was used to accurately determine the equilibrium constants for AdoMet binding to PFL-AE alone and in complex with PFL. The PFL-AE bound AdoMet with the same affinity (∼6 μM) regardless of the presence or absence of PFL. Activation of PFL in the presence of its substrate pyruvate or the analog oxamate resulted in stoichiometric conversion of the [4Fe-4S](1+) cluster to the glycyl radical on PFL; however, 3.7-fold less activation was achieved in the absence of these small molecules, demonstrating that pyruvate or oxamate are required for optimal activation. Finally, in vivo concentrations of the entire PFL system were calculated to estimate the amount of bound protein in the cell. PFL, PFL-AE, and AdoMet are essentially fully bound in vivo, whereas electron donor proteins are partially bound.
authors: From the Department of Chemistry and Biochemistry and the Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, Montana 59717.
authors: Adam V
authors: AV
authors: Crain
authors: Joan B
authors: JB
authors: Broderick
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D019289:Pyruvic Acid / Q000737:chemistry* / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004798:Enzymes; D029968:Escherichia coli Proteins; D012436:S-Adenosylmethionine; D019289:Pyruvic Acid; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme; C022560:formate C-acetyltransferase
keywords: Circular Dichroism (CD); Electron Paramagnetic Resonance (EPR); Radicals; S-Adenosylmethionine (AdoMet); Surface Plasmon Resonance (SPR)