Fig. 1. Schematic of BiBi kinetic mechanisms. The term “sequential” is used to denote systems in which all substrates must bind to the enzyme before any product is released. A random system is sequential even though both substrates bind randomly. An ordered system in which one substrate must bind before the other is also sequential, because no product is released before formation of the ternary complex (E3:IκB:E2ub). Ping-pong systems, in which a product is released between substrate additions, are nonsequential.

Fig. 2. Progress curves for IκBαee ubiquitylation with and without preincubation of ubiquitylation enzymes. E1 (3 pmol), E2 (3 μg), and/or E3 (10 μg) were preincubated for 20 min with ATP and 5 μM¹²⁵I-ubiquitin, and the reaction was then initiated with the substrate IκBαee (5 μM). All preincubation reactions were carried out at 37°. Reactions with no preincubations were initiated with ATP. At the appropriate time, the reactions were stopped with a 25 mM DTT, followed 5 min later with 5.3 M urea, 59 mM iodoacetamide (final) solution, and ubiquitin conjugated IκBαee separated from free ubiquitin with Ni beads as described in “Materials and Methods.”

Fig. 3. Dependence of IκBαee-ubiquitylation on E1 and methylubiquitin. The left panel shows the saturation of IκBαee ubiquitylation with increasing E1 concentration. E1, E2 (2 μg), and E3 (10 μg) were preincubated for 20 min with ATP and methylubiquitin/ubiquitin mixture, and the reaction was initiated with the addition of substrate IκBαee (5 μM). After 60 min, the reaction was stopped with 25 mM DTT followed 5 min later with 5.3 M urea and 59 mM iodoacetamide (final). Ubiquitin-conjugated IκBαee separated from free ubiquitin with Ni beads as described in the “Materials and Methods” section. The final ubiquitin concentration of 4 μM was composed of 200 pmol unlabeled methylubiquitin and 0.075 μCi, 0.0375 pmol labeled ¹²⁵I-ubiquitin. The right side of the figure shows the effect of titrating methylubiquitin on the IκBαee ubiquitylation activity. Methylubiquitin was added in concentrations from 50 nM to 8 μM (2.5–400 pmol/0.05 ml reaction), and ¹²⁵I -labeled ubiquitin was held constant (0.075 μCi, 0.0375 pmol per reaction). The specific activity was calculated assuming methylubiquitin and ubiquitin contributed equally to the formation of IκBαee–ubiquitin conjugates.

Fig. 4. Saturation curves for the substrates E2UBCH7 and IκBαee. The left panel shows saturation curves for E2UBCH7 at fixed concentrations of IκBαee and the right panel saturation curves for IκBαee at fixed E2UBCH7 concentrations. E1 (0.5 μg), E2, and E3 (10 μg) were preincubated for 20 min with ATP and methylubiquitin/ubiquitin mixture and the reaction then initiated with the substrate IκBαee. After 60 min, the reaction was stopped with 25 mM DTT followed 5 min later with 5.3 M urea and 59 mM iodoacetamide (final), and the ubiquitin-conjugated IκBαee was separated from free ubiquitin with Ni beads as described in the “Materials and Methods” section. The final ubiquitin concentration of 4 μM was composed of 200 pmol unlabeled methylubiquitin and 0.075 μCi, 0.0375 pmol labeled ¹²⁵I-ubiquitin.

Fig. 5. Lineweaver–Burk plots of substrate-dependent monoubiquitylation. Replot of the data in Fig. 4.

Kinetic Parameters for E2UBCH7ub Interactions Dependent on IκBαee

Kinetic constants were determined by fitting to a one-site saturation curve using SigmaPlot software program. Vmax is expressed as pmol/h/0.01 mg E3; parentheses indicate the standard error.

Kinetic Parameters for IκBαee Interactions Dependent on E2UBCH7ub

Kinetic constants were determined by fitting to a one-site saturation curve using SigmaPlot software program. Vmax is expressed as pmol/h/0.01 mg E3; parentheses indicate the standard error.