KINETIC AND MECHANISTIC STUDIES ON THE HYDROLYSIS OF UBIQUITIN C-TERMINAL 7-AMIDO-4-METHYLCOUMARIN BY DEUBIQUITINATING ENZYMES

Deubiquitinating enzymes constitute a family of cysteine hydrolases th at specifically cleave ubiquitin-derived substrates of general structu re Ub-X, where X can be any number of leaving groups ranging from smal l thiols and amines to Ub and other proteins (Ub, ubiquitin). We have developed a general assay for deubiquitinating enzymes based on the su bstrate ubiquitin C-terminal 7-amido-4-methylcoumarin (Ub-AMC). Ub-AMC is efficiently hydrolyzed with liberation of highly fluorescent AMC b y two rabbit reticulocyte deubiquitinating enzymes: isopeptidase T (IP aseT), a member of the gene family of ubiquitin-specific processing en zymes, and UCH-L3, a member of the family of ubiquitin C-terminal hydr olases, We used this new assay to probe kinetic and mechanistic aspect s of catalysis by IPaseT and UCH-L3. Results from four series of exper iments are discussed: ii) For UCH-L3, we determined steady-stale kinet ic parameters that suggest a diffusion-limited reaction of UCH-Lj with Ub-AMC. To probe this, we determined the viscosity dependence of k(c) /K-m, as well as k(c). We found complex viscosity dependencies and int erpreted these in the context of a model in which association and acyl ation are viscosity-dependent but deacylation is viscosity-independent . (2) The kinetics of inhibition of UCH-L3 by ubiquitin C-terminal ald ehyde (Ub-H) were determined and reveal a K-i that is less than 10(-14 ) M. Several mechanims are considered to account for the extreme inhib ition. (3) The IPaseT-catalyzed hydrolysis of Ub-AMC is modulated by U b with activation at low [Ub] and inhibition at high [Ub]. (4) Finally , we compare k(c)/K-m values for deubiquitinating enzyme-catalyzed hyd rolysis of Ub-AMC and Z-Leu-Arg-Gly-Gly-AMC. For IPaseT, the ratio of rate constants is 10(4), while for UCM-L3 this ratio is >10(7), These results suggest the following: (i) Deubiquitinating enzymes are able t o utilize the free energy that is released from remote interactions wi th 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 d omain for a Ub ''leaving group''.