Title |
Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen.
|
---|---|
Published in |
Biochemical Journal, October 2014
|
DOI | 10.1042/bj20140779 |
Pubmed ID | |
Authors |
Hanna Tarhonskaya, Rasheduzzaman Chowdhury, Ivanhoe K H Leung, Nikita D Loik, James S O McCullagh, Timothy D W Claridge, Christopher J Schofield, Emily Flashman |
Abstract |
Prolyl hydroxylase domain 2 (PHD2) catalyses the post-translational hydroxylation of the Hypoxia Inducible Factor (HIF), a modification that regulates the hypoxic response in humans. PHD2 is an FeII/2-oxoglutarate (2OG) oxygenase; its catalysis is proposed to provide a link between cellular HIF levels and changes in O2 availability. Transient kinetic studies have shown that PHD2 reacts slowly with O2 compared to some other studied 2OG oxygenases, a property which may be related to its hypoxia sensing role. PHD2 forms a stable complex with FeII and 2OG; crystallographic and kinetic analyses indicate that an FeII-coordinated water molecule, which must be displaced prior to O2 binding, is relatively stable in the active site of PHD2. We used active site substitutions to investigate whether these properties are related to the slow reaction of PHD2 with O2. Whilst disruption of 2OG binding in a R383K variant did not accelerate O2 activation, we found that substitution of the FeII binding Asp with Glu (D315E) manifested significantly reduced FeII binding yet maintained catalytic activity with a 5-fold faster reaction with O2. The results inform on how the precise active site environment of oxygenases can affect rates of O2 activation and provide insights into limiting steps in PHD catalysis. |
X Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 1 | 100% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
United Kingdom | 2 | 4% |
Unknown | 43 | 96% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 12 | 27% |
Researcher | 6 | 13% |
Student > Doctoral Student | 4 | 9% |
Student > Bachelor | 3 | 7% |
Professor | 3 | 7% |
Other | 6 | 13% |
Unknown | 11 | 24% |
Readers by discipline | Count | As % |
---|---|---|
Chemistry | 15 | 33% |
Biochemistry, Genetics and Molecular Biology | 11 | 24% |
Agricultural and Biological Sciences | 4 | 9% |
Arts and Humanities | 1 | 2% |
Pharmacology, Toxicology and Pharmaceutical Science | 1 | 2% |
Other | 2 | 4% |
Unknown | 11 | 24% |