KINETIC-STUDIES ON THE INHIBITION OF ISOPEPTIDASE-T BY UBIQUITIN ALDEHYDE

Isopeptidase T (IPaseT) can hydrolyze isopeptide bonds of polyubiquiti n (polyUb) chains, simple C-terminal derivatives of Ub, and certain pe ptides. We recently reported that IPaseT is regulated by ubiquitin (Ub ); while submicromolar Ub activates, higher concentrations inhibit thi s enzyme [Stein et al. (1995) Biochemistry 34, 12616]. To explain thes e observations, we proposed a model for IPaseT involving two binding s ites 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 o f a polyUb chain. The ''activation site'' binds the Ub that donates Ly s to the isopeptide bond. The ''inhibition site'' is adjacent and bind s the Ub that donates the C-terminal Gly to the isopeptide bond. We no w report that the interaction of IPaseT with the C-terminal aldehyde o f Ub (Ub-H) is also modulated by Ub. In the absence of Ub, Ub-H inhibi ts IPaseT with a K-i of 2.3 nM, while at 0.6 mu M Ub, where the ''acti vation site'' is occupied, K-i is less than 0.1 nM. At high Ub concent rations, where both the ''activation'' and ''inhibition'' sites are oc cupied, IPaseT cannot bind Ub-H. We also determined the kinetics of in hibition 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 f orm 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-acti vated 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, t he 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 w ith our previous results on catalysis, we propose that binding of Ub e ither to catalytic transition states or to tetrahedral inhibition inte rmediates liberates more free energy than binding of Ub to the reactan t state of IPaseT and that IPaseT can utilize this binding energy to s tabilize both of these tetrahedral species. The overall effect is a Ub -induced increase in catalytic efficiency or inhibitory potency.