The inhibitor of κB kinase β (IKKβ) phosphorylates IκBα twice in a single binding event through a sequential mechanism.

Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser and Ser within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre-steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (k) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s and the binding affinity of ATP for the IKKβ•IκBα complex (K) was 12 μM. Substitution of either Ser or Ser with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser and Ser, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser increases the phosphorylation rate of Ser. These conclusions were supported by parallel experiments using GST-IκBα(1-54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser followed by Ser within a single binding event.