期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 295, 期 23, 页码 7826-7838出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.012202
关键词
epigenetics; enzyme kinetics; hypoxia; mass spectrometry (MS); hypoxia-inducible factor (HIF); 2-oxoglutarate; alpha-ketoglutarate; 2OG-dependent dioxygenase; aspartate; asparagine?beta-hydroxylase; AspH; BAH; HAAH; enzyme assays; epidermal growth factor?like domain; epidermal growth factor?like protein 7; EGFL7
资金
- Wellcome Trust [106244/Z/14/Z]
- Cancer Research UK [C8717/A18245]
- Biotechnology and Biological Sciences Research Council [BB/J003018/1]
- Deutsche Forschungsgemeinschaft [BR 5486/2-1]
- BBSRC [BB/J003018/1, BB/L009846/1, BB/L000121/1] Funding Source: UKRI
Human aspartate/asparagine??-hydroxylase (AspH) is a 2-oxoglutarate (2OG)?dependent oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor?like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity (i.e. the Cys 1?2, 3?4, 5?6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo, which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG oxygenases. Of note, a relatively high K-m for O-2 suggested that O-2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG oxygenases.
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