title: Kinetic studies of isopeptidase T: modulation of peptidase activity by ubiquitin.
abstract: We have investigated the specificity of isopeptidase T toward peptide-AMC substrates based on the C-termini of ubiquitin. The substrates investigated 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 kc/Km values of < 0.1, 1, 18, and 95 M-1 s-1, respectively. In the course of these experiments, we observed that the hydrolytic activity of isopeptidase T toward these substrates is modulated by ubiquitin in a biphasic fashion. While submicromolar concentrations of ubiquitin activate isopeptidase T, higher concentrations are inhibitory. In the activation phase, the extent of stimulation of kc/Km varies with substrate and is 8-, 50-, and 70-fold for Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC, respectively. Kd for ubiquitin in this phase is, of course, independent of substrate and equals 0.10 +/- 0.03 microM. At higher concentrations, ubiquitin is inhibitory and titrates kc/Km with an average Ki value of 3.0 +/- 1.3 microM for all three substrates. To explain these observations, we propose a structural model for isopeptidase T that involves two binding sites for ubiquitin. We propose that the two sites are adjacent to one another and are the extended active site that binds two ubiquitin moieties of a polyubiquitin chain for isopeptide bond hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: ProScript, Cambridge, Massachusetts 02139, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Stein
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: F
authors.initials: F
authors.lastname: Melandri
mesh_terms: D000595:Amino Acid Sequence; D019759:Carbon-Nitrogen Lyases; D002384:Catalysis; D010450:Endopeptidases / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008190:Lyases / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014452:Ubiquitins / Q000494:pharmacology*
chemical_list: D014452:Ubiquitins; D010450:Endopeptidases; D008190:Lyases; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase