KINETIC-STUDIES OF ISOPEPTIDASE-T - MODULATION OF PEPTIDASE ACTIVITY BY UBIQUITIN

We have investigated the specificity of isopeptidase T toward peptide- AMC substrates based on the C-termini of ubiquitin. The substrates inv estigated were Z-Gly-Gly-AMC, Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC , and Z-Arg-Leu-Arg-Gly-Gly-AMC and were hydrolyzed by isopeptidase T with k(c)/K-m values of <0.1, 1, 18, and 95 M(-1) s(-1), respectively. In the course of these experiments, we observed that the hydrolytic a ctivity of isopeptidase T toward these substrates is modulated by ubiq uitin in a biphasic fashion. While submicromolar concentrations of ubi quitin activate isopeptidase T, higher concentrations are inhibitory. In the activation phase, the extent of stimulation of k(c)/K-m, varies with substrate and is 8-, 50-, and 70-fold for Z-Arg-Gly-Gly-AMC, Z-L eu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC, respectively. K-d f or ubiquitin in this phase is, of course, independent of substrate and equals 0.10 +/- 0.03 mu M. At higher concentrations, ubiquitin is inh ibitory and titrates k(c)/K-m with an average Ki value of 3.0 +/- 1.3 mu M for all three substrates. To explain these observations, we propo se a structural model for isopeptidase T that involves two binding sit es for ubiquitin. We propose that the two sites are adjacent to one an other and are the extended active site that binds two ubiquitin moieti es of a polyubiquitin chain for isopeptide bond hydrolysis. We found t hat the ''activation site'' requires ubiquitin to have a free carboxy terminus and propose that this is the site that binds the ubiquitin mo lecule of the polyubiquitin chain that donates Lys(48) to the isopepti de bond. The ''inhibitory site'' is adjacent and binds the ubiquitin m olecule of the polyubiquitin chain that donates the C-terminal Gly(76) to the isopeptide bend. Finally, we propose that these observations m ay have physiological relevance for the regulation of IPaseT activity since high intracellular levels of ubiquitin will inhibit continued hy drolysis of polyubiquitin chains.