Kinetic studies on the inhibition of isopeptidase T by ubiquitin aldehyde

Isopeptidase T (IPaseT) can hydrolyze isopeptide bonds of polyubiquitin (polyUb) chains, simple C-terminal derivatives of Ub, and certain peptides. We recently reported that IPaseT is regulated by ubiquitin (Ub); while submicromolar Ub activates, higher concentrations inhibit this enzyme [Stein et al. (1995) Biochemistry 34, 12616]. To explain these observations, we proposed a model for IPaseT involving two binding sites for Ub. According to the model, the two sites are adjacent to one another and are the extended active site that binds two Ub moieties of a polyUb chain. The ''activation site'' binds the Ub that donates Lys to the isopeptide bond. The ''inhibition site'' is adjacent and binds the Ub that donates the C-terminal Gly to the isopeptide bond. We now report that the interaction of IPaseT with the C-terminal aldehyde of Ub (Ub-H) is also modulated by Ub. In the absence of Ub, Ub-H inhibits IPaseT with a K-i of 2.3 nM, while at 0.6 mu M Ub, where the ''activation site'' is occupied, K-i is less than 0.1 nM. At high Ub concentrations, where both the ''activation'' and ''inhibition'' sites are occupied, IPaseT cannot bind Ub-H. We also determined the kinetics of inhibition of IPaseT by Ub-H. In the absence of Ub, a two-step mechanism is followed. In the first step, Ub-H slowly combines with IPaseT to form a relatively weak complex (K-1 = 260 nM) that slowly isomerizes to the final, stable complex that accumulates in the steady-state (k(2) = 2 x 10(-3) s(-1); k(-2) = 0.02 x 10(-3) s(-1)). In contrast, Ub-activated IPaseT is inhibited by Ub-H through a three-step process. In the first step, Ub-H rapidly combines with IPaseT to form a complex (K-1 = 10 nM) that slowly isomerizes to a second, more stable complex (k(2) = 18 x 10(-3) s(-1) k(-2) = 1.5 x 10(-3) s(-1)). In the third step, the second complex converts to the final complex (k(3) = 1.5 x 10(-3) s(-1) k(-3) < 0.2 x 10(-3) s(-1)). To unify the results of this study with our previous results on catalysis, we propose that binding of Ub either to catalytic transition states or to tetrahedral inhibition intermediates liberates more free energy than binding of Ub to the reactant state of IPaseT and that IPaseT can utilize this binding energy to stabilize both of these tetrahedral species. The overall effect is a Ub-induced increase in catalytic efficiency or inhibitory potency.