Abstract/Details

The active site chemistry of factor inhibiting HIF-1, coordination, bonding, and reaction


2009 2009

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Abstract (summary)

Oxygen is vital for aerobic life. A shortage of oxygen can produce disastrous outcomes to cellular functions. To avoid this, eukaryotic cells developed an oxygen sensing machinery of which the key component is hypoxia inducible factor (HIF). The function of HIF is controlled by two α-ketoglutarate-dependent non-heme iron dioxygenases, HIF-1 prolyl hydroxylase (PHD) and HIF-1 asparaginyl hydroxylase (FIH-1). FIH-1 is the focus of this study. When oxygen is in short supply, FIH-1 cannot hydroxylate HIF. HIF then binds on DNA and turns on genes which control energy production, oxygen uptake, angiogenesis and cell growth. When the concentration of oxygen is optimal, FIH-1 hydroxylates the C-terminal activation domain (CTAD) of HIFα and turns-off the transcriptional machinery. Studying FIH-1 will not only help to provide a clear image about the reaction mechanism of FIH-1, but also guide the way in how to modulate the hypoxic responses. The three topics of concentration for FIH-1 are active site coordination, functionality, and hydrogen bond networking.

The iron center in the FIH-1 active site is octahedral in the resting state, coordinated by a His2Asp facial triad, bidentate α-ketoglutarate, and an axial water ligand. In the consensus mechanism, the water molecule leaves the active site when primary substrate, HIFα, binds with FIH-1 and generates a five coordinated iron center. The results shown in this thesis indicate that the five coordinated iron center occurs even without HIFα binding. This leads to an uncoupled and suicide reaction termed auto-hydroxylation.

The auto-hydroxylation reaction is studied in the functionality assays, which occurs when oxygen is sufficient and in the absence of HIFα. In this aberrant reaction, FIH-1 will use one oxygen atom to cleave α-ketoglutarate and use the other oxygen atom to hydroxylate Trp296. This auto-hydroxylation reaction leads to chromophore formation, that is shown to be iron and α-ketoglutarate dependent, and sensitive to reductant and preservative. Since the reaction condition for FIH autohydroxylation, planty of oxygen but absent of HIF-1α, does exist in normoxic environment, it is important to understand this reaction. The detail of this reaction provides insights not only into how FIH-1 controls O2 activation, but also how cells deal with reactive oxygen species.

The active site of FIH-1 has a hydrogen bond network, which involves Asn803 on HIFα and four residues from FIH-1: Gln205, Arg238, Gln239, and Gln294. FIH-1 residues involved in hydrogen bonding are mutated into alanine one by one, and the impact of these mutations are analyzed. The results suggest that Gln205 and Gln294 act as a decelerator for O2 activation, and Arg238 and Gln239 act as an accelerator to prime the Fe(II) for reaction with O2 following HIF binding.

FIH-1 is crucial for oxygen regulation in human cells. The results of these experiments provide detailed knowledge about FIH-1, which provides broader insight into the control of oxidation chemistry, and may aid in the therapeutic targeting in multiple diseases that are associated with hypoxic pathways.

Indexing (details)


Subject
Biochemistry
Classification
0487: Biochemistry
Identifier / keyword
Pure sciences; Autohydroxylation; Factor inhibiting HIF; Iron center coordination
Title
The active site chemistry of factor inhibiting HIF-1, coordination, bonding, and reaction
Author
Chen, Yuan-Han
Number of pages
153
Publication year
2009
Degree date
2009
School code
0118
Source
DAI-B 70/09, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9781109352009
Advisor
Knapp, Michael J.
Committee member
Eyles, Stephen J.; Martin, Craig T.; Weis, Robert M.
University/institution
University of Massachusetts Amherst
Department
Molecular & Cellular Biology
University location
United States -- Massachusetts
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3372258
ProQuest document ID
304920552
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/304920552
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