The -4 phenylalanine is required for substrate ubiquitination catalyzed by HECT ubiquitin ligases.
J Biol Chem
; 279(18): 18935-43, 2004 Apr 30.
Article
in En
| MEDLINE
| ID: mdl-14966115
ABSTRACT
The reaction cycle of HECT domain ubiquitin ligases consists of three steps:
1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the -4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the -4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the -4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine epsilon-amino group of the substrate.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Phenylalanine
/
Ubiquitin
/
Ubiquitin-Protein Ligases
Type of study:
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
J Biol Chem
Year:
2004
Document type:
Article
Affiliation country:
United States