Lacombe Thierry, Gabriel Jean Marc
Department of Medical Biochemistry, University of Geneva, CMU, 1 rue Michel Servet, CH-1211 4, Genève, Switzerland.
FEBS Lett. 2002 Nov 20;531(3):469-74. doi: 10.1016/s0014-5793(02)03586-x.
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.
人异肽酶T（isoT）是一种锌结合去泛素化酶，参与将游离的K48连接的多聚泛素链分解为泛素单体。该酶的催化位点被认为由Cys335、Asp435、His786和His795组成。对这四个残基进行了定点诱变。没有一个突变体能够切割肽连接的泛素二聚体。同样，C335S、D435N和H795N突变体对K48连接的异肽泛素二聚体几乎没有活性，K48连接的异肽泛素二聚体是一种isoT特异性底物，模拟K48连接的多聚泛素链。另一方面，H786N突变体对K48连接的底物保留了部分活性，这表明His786残基可能不是催化位点的一部分。没有一个突变显著影响isoT结合泛素和锌的能力。因此，泛素特异性蛋白酶（UBPs）的催化位点可能类似于其他半胱氨酸蛋白酶的催化位点，后者包含一个Cys、一个Asp和一个His。
