SUBSTRATE-BINDING AND CATALYSIS BY UBIQUITIN C-TERMINAL HYDROLASES - IDENTIFICATION OF 2 ACTIVE-SITE RESIDUES

Ubiquitin C-terminal hydrolases (UCH's) are a newly-defined class of t hiol proteases implicated in the proteolytic processing of polymeric u biquitin, They are important for the generation of monomeric ubiquitin , the active component of the eukaryotic ubiquitin-dependent proteolyt ic system, There are at least three mammalian isozymes which are tissu e specific and developmentally regulated. To study the structure and f unctional roles of these highly homologous enzymes, we have subcloned and overexpressed two of these isozymes, UCH-L1 and UCH-L3. Here, we r eport their purification, physical characteristics, and the mutagenesi s of UCH-L1. Site-directed mutagenesis of UCH-L1 reveals that C90 and H161 are involved in catalytic rate enhancement. Data from circular di chroic and Raman spectroscopy, as well as secondary structure predicti on algorithms, indicate that both isozymes have a significant amount o f 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 param eters typical of single domain globular proteins. Substrate binding af finity to UCH-L3 was directly measured by equilibrium gel filtration ( K-d = 0.5 mu M), and the results are similar to the kinetically determ ined K-m for ubiquitin ethyl ester (0.6 mu M). The binding is primaril y electrostatic in nature and indicates the existence of a specific an d extensive binding site for ubiquitin on the surface of the enzyme.