Increased Turnover at Limiting O-2 Concentrations by the Thr(387) -> Ala Variant of HIF-Prolyl Hydroxylase PHD2

Pektas S., Taabazuing C. Y., Knapp M. J.

BIOCHEMISTRY, vol.54, no.18, pp.2851-2857, 2015 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 54 Issue: 18
  • Publication Date: 2015
  • Doi Number: 10.1021/bi501540c
  • Journal Name: BIOCHEMISTRY
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.2851-2857
  • Recep Tayyip Erdoğan University Affiliated: Yes


PHD2 is a 2-oxoglutarate, non-heme Fe2+-dependent oxygenase that senses O-2 levels in human cells by hydroxylating two prolyl residues in the oxygen-dependent degradation domain (ODD) of HIF1 alpha. Identifying the active site contacts that determine the rate of reaction at limiting O-2 concentrations is crucial for understanding how this enzyme senses pO(2) 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 O-2 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 k(cat) and k(cat)/K-M(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 O-2 concentrations.