The inhibitor of B kinase (IKK) phosphorylates IB twice in a single binding event through a sequential mechanism

Phosphorylation of Inhibitor of & kappa;B (I & kappa;B) proteins by I & kappa;B Kinase & beta; (IKK & beta;) leads to I & kappa;B degradation and subsequent activation of nuclear factor & kappa;B transcription factors. Of particular interest is the IKK & beta;-catalyzed phosphorylation of I & kappa;B & alpha; residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKK & beta;, we performed pre-steady-state kinetic analysis of the phosphor-ylation of I & kappa;B & alpha; protein substrates catalyzed by constitutively active, human IKK & beta;. Phosphorylation of full-length I & kappa;B & alpha; catalyzed by IKK & beta; was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKK & beta;-catalyzed phosphorylation of I & kappa;B & alpha; was 0.32 s-1 and the binding affinity of ATP for the IKK & beta;& BULL;I & kappa;B & alpha; complex (Kd) was 12 & mu;M. Substitution of either Ser32 or Ser36 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 I & kappa;B & alpha; at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the I & kappa;B & alpha;(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT I & kappa;B & alpha; and 4-fold faster than any of the other I & kappa;B & alpha; amino acid substitution mutants, suggesting that phos-phorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experi-ments using GST-I & kappa;B & alpha;(1-54) fusion protein substrates bearing the first 54 residues of I & kappa;B & alpha;. Our data suggest model wherein, IKK & beta; phosphorylates I & kappa;B & alpha; at Ser32 followed by Ser36 within a single binding event.