title: Increased Turnover at Limiting O2 Concentrations by the Thr(387) → Ala Variant of HIF-Prolyl Hydroxylase PHD2.
abstract: PHD2 is a 2-oxoglutarate, non-heme Fe(2+)-dependent oxygenase that senses O2 levels in human cells by hydroxylating two prolyl residues in the oxygen-dependent degradation domain (ODD) of HIF1α. Identifying the active site contacts that determine the rate of reaction at limiting O2 concentrations is crucial for understanding how this enzyme senses pO2 and may suggest methods for chemically altering hypoxia responses. A hydrogen bonding network extends from the Fe(II) cofactor through ordered waters to the Thr(387) residue in the second coordination sphere. Here we tested the impact of the side chain of Thr(387) on the reactivity of PHD2 toward O2 through a combination of point mutagenesis, steady state kinetic experiments and {FeNO}(7) EPR spectroscopy. The steady state kinetic parameters for Thr(387) → Asn were very similar to those of wild-type (WT) PHD2, but kcat and kcat/KM(O2) for Thr(387) → Ala were increased by roughly 15-fold. X-Band electron paramagnetic resonance spectroscopy of the {FeNO}(7) centers of the (Fe+NO+2OG) enzyme forms showed the presence of a more rhombic line shape in Thr(387) → Ala than in WT PHD2, indicating an altered conformation for bound gas in this variant. Here we show that the side chain of residue Thr(387) plays a significant role in determining the rate of turnover by PHD2 at low O2 concentrations.
authors: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States.
authors: Serap
authors: S
authors: Pektas
authors: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States.
authors: Cornelius Y
authors: CY
authors: Taabazuing
authors: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States.
authors: Michael J
authors: MJ
authors: Knapp
mesh_terms: D000409:Alanine / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D010100:Oxygen / Q000737:chemistry*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013912:Threonine / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D013912:Threonine; C490190:EGLN1 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D000409:Alanine; D010100:Oxygen