title: Substrate positioning by Gln(239) stimulates turnover in factor inhibiting HIF, an αKG-dependent hydroxylase.
abstract: Nonheme Fe(II)/αKG-dependent oxygenases catalyze diverse reactions, typically inserting an O atom from O2 into a C-H bond. Although the key to their catalytic cycle is the fact that binding and positioning of primary substrate precede O2 activation, the means by which substrate binding stimulates turnover is not well understood. Factor Inhibiting HIF (FIH) is a Fe(II)/αKG-dependent oxygenase that acts as a cellular oxygen sensor in humans by hydroxylating the target residue Asn(803), found in the C-terminal transactivation domain (CTAD) of hypoxia inducible factor-1. FIH-Gln(239) makes two hydrogen bonds with CTAD-Asn(803), positioning this target residue over the Fe(II). We hypothesized the positioning of the side chain of CTAD-Asn(803) by FIH-Gln(239) was critical for stimulating O2 activation and subsequent substrate hydroxylation. The steady-state characterization of five FIH-Gln(239) variants (Ala, Asn, Glu, His, and Leu) tested the role of hydrogen bonding potential and sterics near the target residue. Each variant exhibited a 20-1200-fold decrease in kcat and kcat/KM(CTAD), but no change in KM(CTAD), indicating that the step after CTAD binding was affected by point mutation. Uncoupled O2 activation was prominent in these variants, as shown by large coupling ratios (C = [succinate]/[CTAD-OH] = 3-5) for each of the FIH-Gln(239) → X variants. The coupling ratios decreased in D2O, indicating an isotope-sensitive inactivation for variants, not observed in the wild type. The data presented indicate that the proper positioning of CTAD-Asn(803) by FIH-Gln(239) is necessary to suppress uncoupled turnover and to support substrate hydroxylation, suggesting substrate positioning may be crucial for directing O2 reactivity within the broader class of αKG hydroxylases.
authors.affiliation: Department of Chemistry, University of Massachusetts at Amherst , Amherst, Massachusetts 01003, United States.
authors.forename: John A
authors.initials: JA
authors.lastname: Hangasky
authors.forename: Geoffrey T
authors.initials: GT
authors.lastname: Ivison
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Knapp
mesh_terms: D000595:Amino Acid Sequence; D005998:Glycine / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D012097:Repressor Proteins; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; D005998:Glycine