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Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
The second coordination sphere of FIH controls hydroxylation
Evren Saban1, Yuan-Han Chen, John A Hangasky
1Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
Biochemistry
|April 5, 2011
Summary
The factor inhibiting HIF (FIH) enzyme uses second-sphere residues to regulate oxygen sensing and hypoxia-inducible factor (HIF) hydroxylation. Mutations impairing oxidative decarboxylation and substrate positioning affect FIH
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- The factor inhibiting HIF (FIH) acts as a cellular oxygen sensor by hydroxylating the hypoxia-inducible factor (HIF).
- FIH is an iron(II)-dependent dioxygenase utilizing α-ketoglutarate (αKG) and requiring specific coordination spheres for its catalytic activity.
- Second-sphere residues and hydrogen bonding interactions are implicated in modulating the active site chemistry of FIH.
Purpose of the Study:
- To investigate the functional role of specific second-sphere residues in the catalytic mechanism of FIH.
- To elucidate how these residues influence key steps such as Fe(II) priming, oxidative decarboxylation, and substrate positioning.
- To understand the interplay between the primary coordination sphere and the second coordination sphere in FIH's oxygen-sensing function.
Main Methods:
- Site-directed mutagenesis was employed to generate point mutants of FIH, targeting residues in the αKG and HIF-Asn(803) binding sites.
- Steady-state kinetics and autohydroxylation assays were performed to assess catalytic efficiency, priming, and positioning.
- Electronic spectroscopy (UV-Vis) was used to analyze changes in the primary coordination sphere and the electrophilicity of αKG.
Main Results:
- Mutants Asn(205)→Ala and Asn(294)→Ala showed reduced steady-state turnover rates, indicating impaired oxidative decarboxylation.
- The Arg(238)→Met mutant displayed significantly diminished steady-state rates and product yields, suggesting issues with substrate positioning or priming, although O(2) activation was retained.
- The Gln(239)→Asn mutant also exhibited slowed kinetics and reduced product yields, likely due to mispositioning of the HIF-Asn(803) substrate.
Conclusions:
- Second-sphere hydrogen bonds in FIH are crucial for promoting oxidative decarboxylation and priming Fe(II) for O(2) binding.
- Specific residues, such as Arg(238) and Gln(239), play critical roles in substrate positioning, directly impacting FIH's catalytic efficiency.
- This study highlights the importance of the second coordination sphere in fine-tuning the enzymatic activity of FIH, essential for its role as an oxygen sensor.
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