Substrate binding and catalysis by ubiquitin C-terminal hydrolases: identification of two active site residues.
Ubiquitin C-terminal hydrolases (UCH's) are a newly-defined class of thiol proteases implicated in the proteolytic processing of polymeric ubiquitin. They are important for the generation of monomeric ubiquitin, the active component of the eukaryotic ubiquitin-dependent proteolytic system. There are at least three mammalian isozymes which are tissue specific and developmentally regulated. To study the structure and functional roles of these highly homologous enzymes, we have subcloned and overexpressed two of these isozymes, UCH-L1 and UCH-L3. Here, we report their purification, physical characteristics, and the mutagenesis of UCH-L1. Site-directed mutagenesis of UCH-L1 reveals that C90 and H161 are involved in catalytic rate enhancement. Data from circular dichroic and Raman spectroscopy, as well as secondary structure prediction algorithms, indicate that both isozymes have a significant amount of alpha-helix (> 35%), and contain no disulfide bonds. Both enzymes are reasonably stable, undergoing a reversible thermal denaturation at 52 degrees C. These transitions are characterized by thermodynamic parameters typical of single domain globular proteins. Substrate binding affinity to UCH-L3 was directly measured by equilibrium gel filtration (Kd = 0.5 microM), and the results are similar to the kinetically determined Km for ubiquitin ethyl ester (o.6 microM). The binding is primarily electrostatic in nature and indicates the existence of a specific and extensive binding site for ubiquitin on the surface of the enzyme.
Larsen
C N
CN
Department of Biochemistry, Emory University, Atlanta, Georgia 30322, USA.
Price
J S
JS
Wilkinson
K D
KD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine; D004330:Drosophila; D004926:Escherichia coli; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase
D007527:Isoenzymes; D011994:Recombinant Proteins; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D003545:Cysteine