No abstract

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".

We report the preparation and characterization of interleukin-1␤ converting enzyme (ICE) refolded from its p20 and p10 protein fragments. Refolded ICE heterodimer (p20p10) was catalytically active but unstable, and in size exclusion chromatography eluted at an apparent molecular mass of 30 kDa. The mechanisms of the observed instability were pH-dependent dissociation at low enzyme concentrations, and autolytic degradation of the p10 subunit at high concentrations. Binding and subsequent removal of a high affinity peptidic inhibitor increased the apparent molecular mass to 43 kDa (by size exclusion chromatography), and significantly increased its stability and specific activity. Chemical cross-linking and SDS-polyacrylamide gel electrophoresis analysis of the 43-kDa size exclusion chromatography conformer revealed a 60-kDa species, which was absent in the 30-kDa conformer, suggesting that inhibitor binding caused formation of a (p20p10) 2 homodimer. The observation of a reversible equilibrium between ICE (p20p10) and (p20p10) 2 suggests that analogous associations, possibly between ICE and ICE homologs, can occur in vivo, resulting in novel oligomeric protease species.Interleukin-1␤ converting enzyme (ICE) 1 (1, 2) is an intracellular cysteine protease that activates the proinflammatory cytokine interleukin-1␤ (IL-1␤) by cleavage at Asp 116 -Ala 117 (3-5). Several lines of evidence suggest that ICE activity is required for IL-1␤ activation and that this is a crucial step in inflammation. IL-1␤ activation is effectively blocked by CrmA, a cowpox virus serpin that binds and inhibits ICE (6). The tetrapeptide ICE inhibitor acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO) (2) is also effective in blocking IL-1␤ activation (2, 7-9). Mice lacking functional copies of the murine ICE gene (10, 11), and cells derived from those animals, are deficient in IL-1␤ maturation. ICE-deficient mice are also resistant to endotoxic shock (10). These results suggest that inhibition of IL-1␤ activation by ICE is sufficient to block inflamation, and encourage efforts to develop ICE inhibitors as antiinflammatory drugs (12, 13).Several human genes encoding proteins homologous to ICE have been discovered, and elucidation of the biological functions of these proteins is currently an active area of research. These include Ich-1 (14), TX/Ich-2/ICE rel II (15-17), CPP32 (18), ICE rel III (17), Mch2 (19), and Mch3/ICE-LAP3/CMH-1 (20 -22). A clue to the function of ICE homologs, and possibly a second function of ICE itself, is provided by ced-3, a Caenorhabditis elegans protease that is highly homologous to ICE and is required for apoptosis (23,24). The hypothesis that ICE or ICE homologs participate in apoptosis is supported by the antiapoptotic effects of CrmA and the baculovirus protein p35 (25, 26), which is also an inhibitor of ICE and ICE homologs, and by the observation that transient expression of antisense-ICE cDNA blocks Fas-induced apoptosis (27). Overexpression of ICE or many of its homologs in cultured cells causes apoptosis. Biochemical ...

We describe the expression, purification, and characterization of human interleukin-1 beta converting enzyme (ICE) containing an affinity tag and modified to resist autoproteolysis. The point mutation Asp381 to Glu was added to eliminate the major site of autolytic degradation while maintaining catalytic activity, and an N-terminal polyhistidine tag was added in place of the ICE pro-region to facilitate purification. N-His (D381E) ICE was expressed in Escherichia coli and purified by nickel-chelating Sepharose and size-exclusion chromatography (SEC). The enzyme was stabilized greater than 80-fold against autolytic degradation relative to wild-type N-His ICE. SDS-PAGE analysis with silver-staining revealed no impurities, and 85% of the protein was catalytically active as determined by titration with a novel titrant, PD 163594 (3-[2-(2-benzyloxycarbonylamino-3-methylbutyrylamino)prop ionylamino]-4- oxo-5-(2-oxo-2H-chromen-7-yloxypentanoic acid). An oxidized adduct of ICE with glutathione, formed by disulfide rearrangement with oxidized glutathione to inhibit and stabilize the enzyme during purification, was rapidly reduced upon exposure to 5 mM DTT. One mole of glutathione was released per mole of active enzyme. Of the nine cysteines in ICE, eight were present in their reduced form in the glutathione adduct. N-His (D381E) ICE cleaved Ac-YVAD-Amc with the Michaelis-Menten parameters K(M) = 14 microM and Kcat = 0.7 s-1, values essentially identical to those reported for enzyme from natural sources.