14-855 Sigma-AldrichUbiquitin C-terminal hydrolase L1 (UCH-L1), 50 µg

The assumption that each enzyme expresses a single enzymatic activity in vivo is challenged by the linkage of the neuronal enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) to Parkinson's disease (PD). UCH-L1, especially those variants linked to higher susceptibility to PD, causes the accumulation of alpha-synuclein in cultured cells, an effect that cannot be explained by its recognized hydrolase activity. UCH-L1 is shown here to exhibit a second, dimerization-dependent, ubiquityl ligase activity. A polymorphic variant of UCH-L1 that is associated with decreased PD risk (S18Y) has reduced ligase activity but comparable hydrolase activity as the wild-type enzyme. Thus, the ligase activity as well as the hydrolase activity of UCH-L1 may play a role in proteasomal protein degradation, a critical process for neuronal health.

Deubiquitinating enzymes constitute a family of cysteine hydrolases that specifically cleave ubiquitin-derived substrates of general structure Ub-X, where X can be any number of leaving groups ranging from small thiols and amines to Ub and other proteins (Ub, ubiquitin). We have developed a general assay for deubiquitinating enzymes based on the substrate ubiquitin C-terminal 7-amido-4-methylcoumarin (Ub-AMC). Ub-AMC is efficiently hydrolyzed with liberation of highly fluorescent AMC by two rabbit reticulocyte deubiquitinating enzymes: isopeptidase T (IPaseT), a member of the gene family of ubiquitin-specific processing enzymes, and UCH-L3, a member of the family of ubiquitin C-terminal hydrolases. We used this new assay to probe kinetic and mechanistic aspects of catalysis by IPaseT and UCH-L3. Results from four series of experiments are discussed: (1) For UCH-L3, we determined steady-state kinetic parameters that suggest a diffusion-limited reaction of UCH-L3 with Ub-AMC. To probe this, we determined the viscosity dependence of kc/Km, as well as kc. We found complex viscosity dependencies and interpreted these in the context of a model in which association and acylation are viscosity-dependent but deacylation is viscosity-independent. (2) The kinetics of inhibition of UCH-L3 by ubiquitin C-terminal aldehyde (Ub-H) were determined and reveal a Ki that is less than 10(-14) M. Several mechanisms are considered to account for the extreme inhibition. (3) The IPaseT-catalyzed hydrolysis of Ub-AMC is modulated by Ub with activation at low [Ub] and inhibition at high [Ub]. (4) Finally, we compare kc/Km values for deubiquitinating enzyme-catalyzed hydrolysis of Ub-AMC and Z-Leu-Arg-Gly-Gly-AMC. For IPaseT, the ratio of rate constants is 10(4), while for UCH-L3 this ratio is > 10(7). These results suggest the following: (i) Deubiquitinating enzymes are able to utilize the free energy that is released from remote interactions with Ub-containing substrates for stabilization of catalytic transition states, and (ii) UCHs are more efficient at utilizing the energy from these interactions, presumably because they do not possess a binding domain for a Ub "leaving group".

Database search using a bovine thymus ubiquitin C-terminal hydrolase sequence indicated 54% sequence identity with the abundant human neuron-specific protein gene product 9.5 (PGP9.5), which was then shown to possess the same activity [Wilkinson, Lee, Deshpande, Duerksen-Hughes, Boss & Pohl (1989) Science 246, 670-673]. A yeast counterpart of the enzyme is also known. The human PGP9.5 gene, described here, spans 10 kb, contains nine exons and displays 5' features some common to many genes and some common with neurofilament neuron-specific enolase and Thy-1-antigen gene 5' regions.