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Further characterization of the putative human isopeptidase T catalytic site
Author(s) -
Lacombe Thierry,
Gabriel Jean-Marc
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03586-x
Subject(s) - deubiquitinating enzyme , cleave , ubiquitin , biochemistry , dimer , mutant , cysteine , enzyme , active site , chemistry , hydrolase , residue (chemistry) , serine , proteases , stereochemistry , biology , gene , organic chemistry
The human isopeptidase T (isoT) is a zinc‐binding deubiquitinating enzyme involved in the disassembly of free K48‐linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site‐directed mutagenized. None of the mutants were able to cleave a peptide‐linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48‐linked isopeptide ubiquitin dimer, which is an isoT‐specific substrate that mimics the K48‐linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48‐linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.