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Residues near the active site of UCH enzymes that may contribute to isopeptide binding in ubiquitin‐linked substrates (LB257)
Author(s) -
Chaney Joseph,
Ronau Judith,
Paul Lake,
Das Chitta
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb257
Subject(s) - ubiquitin , deubiquitinating enzyme , active site , alanine , tryptophan , residue (chemistry) , biochemistry , mutant , hydrolase , chemistry , enzyme , moiety , alanine scanning , ubiquitin ligase , stereochemistry , amino acid , mutagenesis , gene
Ubiquitin carboxyl terminal hydrolase (UCH) enzymes release small peptide and protein leaving groups from the C‐terminus of ubiquitin. Although the interaction with ubiquitin is fairly well understood in these enzymes, recognition of the isopeptide moiety of the substrate at the active site has not been well characterized. The crystal structure of TsUCH37 ( T. spiralis) bound to suicide substrate, UbVMe, reveals a conserved tryptophan residue that may play a role in stabilizing the isopeptide linkage in the active site. To study the contribution of this tryptophan residue, the equivalent residue in human UCH37, W55, was mutated to alanine and phenylananine. The phenylalanine mutant retained most of its ubiquitin‐AMC (Ub‐AMC) hydrolysis activity as compared to the wild type enzyme, but the alanine mutant was substantially impaired. The loss of activity with the alanine mutant is not due to any alteration in the three‐dimensional fold of the enzyme. Considering the distance of this tryptophan residue from G76 of ubiquitin (~5 Å), this observation suggests that W55 may be involved in binding to the AMC portion of the Ub‐AMC substrate, which may indicate that it makes contact with the isopeptide moiety in ubiquitin‐linked substrates. This is being further investigated in other members of the UCH family, such as BAP‐1 and UCHL3. Grant Funding Source : Supported by National Institute of Health