The signal-inducible phosphorylation of serines 32 and 36 of IkappaB-alpha is the key step in regulating the subsequent ubiquitination and proteolysis of IkappaB-alpha, which then releases NF-kappaB to promote gene transcription. The multisubunit IkappaB kinase (msIKK) responsible for this phosphorylation contains two catalytic subunits, termed IKK-1 and IKK-2. Using recombinant IKK-2, a kinetic pattern consistent with a random, sequential binding mechanism was observed with the use of a peptide corresponding to amino acids 26-42 of IkappaB-alpha. Values of 313 microM, 15.5 microM, and 1.7 min(-1) were obtained for K(peptide), K(ATP), and k(cat), respectively. The value of alpha, a factor by which binding of one substrate changes the dissociation constant for the other substrate, was determined to be 0.2. Interestingly, the recombinant IKK-1 subunit gave similar values for alpha and K(ATP), but values of 1950 microM and 0.016 min(-1) were calculated for K(peptide) and k(cat), respectively. This suggests that the IKK-2 catalytic subunit provides nearly all of the catalytic activity of the msIKK complex with the IKK-1 subunit providing little contribution to catalysis. Using peptides corresponding to different regions of IkappaB-alpha within amino acids 21-47, it was shown that amino acids 31-37 provide most binding interactions (-4.7 kcal/mol of binding free energy) of the full-length IkappaB-alpha (-7.9 kcal/mol) with the IKK-2. This is consistent with the observation that IKK-2 is able to phosphorylate the IkappaB-beta and IkappaB-epsilon proteins, which have consensus phosphorylation sites nearly identical to that of amino acids 31-37 of IkappaB-alpha. A peptide corresponding to amino acids 279-303 in the C-terminal domain of IkappaB-alpha was unable to activate IKK-2 to phosphorylate an N-terminal peptide, which is in contrast to the results observed with the msIKK. Moreover, the IKK-2 catalyzes the phosphorylation of the full-length IkappaB-alpha and the amino acid 26-42 peptide with nearly equal efficiency, while the msIKK catalyzes the phosphorylation of the full-length IkappaB-alpha 25,000 times more efficiently than the 26-42 peptide. Therefore, the C terminus of IkappaB-alpha is important in activating the msIKK through interactions with subunits other than the IKK-2.