title: Stereoselective ligand interactions of chicken liver phosphoenolpyruvate carboxykinase with fluorophosphoenolpyruvate.
abstract: The stereospecific interactions of chicken liver phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) with the two geometric isomers of 3-fluorophosphoenolpyruvate (F-P-enolpyruvate) were examined. Previous studies have shown that the Z isomer of F-P-enolpyruvate is a substrate for P-enolpyruvate carboxykinase but the E isomer is a competitive inhibitor [T. H. Duffy and T. Nowak (1984) Biochemistry 23, 661-670]. The reasons for this substrate selectivity were investigated. Studies of the 1H, 19F, and 31P relaxation rates of the ligands in the binary Mn-ligand complexes indicate the formation of direct coordination complexes. The temperature and frequency dependence of the proton relaxation rates (PRR) of the respective enzyme-Mn-ligand complexes demonstrates that the perturbation of the electronic environment at the Mn(II) site on the enzyme is different upon binding of the inhibitor (E-F-P-enolpyruvate) in contrast to the binding of substrates (P-enolpyruvate or Z-F-P-enolpyruvate). Structural studies demonstrate that Z-F-P-enolpyruvate forms a second sphere coordination complex with enzyme-bound Mn(II). E-F-P-enolpyruvate exchanges slowly from the ternary complex and binds less than or equal to 10 A from the bound Mn(II). CD studies in the far-uv region demonstrate that the alpha-helical content of P-enolpyruvate carboxykinase is increased at the expense of antiparallel and parallel beta-sheet structure upon binding of Mn(II) and substrate (P-enolpyruvate or Z-F-P-enolpyruvate) to the apoenzyme, but show no such structural change upon binding of Mn(II) and E-F-P-enolpyruvate. Analogous results are observed from CD studies at the aromatic amino acid region (250-350 nm). The stereoselective catalytic activities of P-enolpyruvate carboxykinase with F-P-enolpyruvate analogs can be explained by different interactions of these ligands within the catalytic site of the enzyme.
authors.affiliation: Department of Chemistry, University of Notre Dame, Indiana 46556.
authors.forename: S H
authors.initials: SH
authors.lastname: Hwang
authors.forename: T
authors.initials: T
authors.lastname: Nowak
mesh_terms: D000818:Animals; D002645:Chickens; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D008962:Models, Theoretical; D010728:Phosphoenolpyruvate / Q000031:analogs & derivatives* / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism; D013237:Stereoisomerism; D013816:Thermodynamics
chemical_list: D008024:Ligands; D008345:Manganese; D010728:Phosphoenolpyruvate; C041143:phosphoenol-3-fluoropyruvate; D010729:Phosphoenolpyruvate Carboxykinase (GTP)