Peptides Corresponding to the N and C Termini of IκB-α, -β, and -ε as Probes of the Two Catalytic Subunits of IκB Kinase, IKK-1 and IKK-2*

The signal-inducible phosphorylation of serines 32 and 36 of IκB-α is the key step in regulating the subsequent ubiquitination and proteolysis of IκB-α, which then releases NF-κB to promote gene transcription. The multisubunit IκB 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 IκB-α. Values of 313 μm, 15.5 μm, and 1.7 min−1 were obtained forK peptide, K ATP, andk cat, respectively. The value of α, 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 α andK ATP, but values of 1950 μm and 0.016 min−1 were calculated forK 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 IκB-α 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 IκB-α (−7.9 kcal/mol) with the IKK-2. This is consistent with the observation that IKK-2 is able to phosphorylate the IκB-β and IκB-ε proteins, which have consensus phosphorylation sites nearly identical to that of amino acids 31–37 of IκB-α. A peptide corresponding to amino acids 279–303 in the C-terminal domain of IκB-α 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 IκB-α and the amino acid 26–42 peptide with nearly equal efficiency, while the msIKK catalyzes the phosphorylation of the full-length IκB-α 25,000 times more efficiently than the 26–42 peptide. Therefore, the C terminus of IκB-α is important in activating the msIKK through interactions with subunits other than the IKK-2.

The transcriptional activator NF-B normally resides in the cytoplasm in unstimulated cells as an inactive complex with a member of the IB inhibitory protein family. This class of protein includes IB-␣, IB-␤, and IB-⑀, which all contain ankyrin repeats necessary for complexation with NF-B (for a review, see Ref. 1). In the case of IB-␣, the most carefully studied member of this class, stimulation of cells with agents which activate NF-B-dependent gene transcription results in the phosphorylation of IB-␣ at Ser-32 and Ser-36 (2). This is critical for subsequent ubiquitination and proteolysis of IB-␣, which then leaves NF-B free to translocate to the nucleus and promote gene transcription (3)(4)(5). Indeed, a mutant in which both Ser-32 and Ser-36 have been changed to alanine prevents signal-induced activation of NF-B and results in an IB-␣ which is neither phosphorylated, ubiquitinated, nor proteolytically digested (5). Analogous serines have been identified in both IB-␤ and IB-⑀, and phosphorylation at these residues appears to regulate the proteolytic degradation of these proteins by a mechanism similar to that of IB-␣ (6,7). Because the expression of many pro-inflammatory genes such as the cytokines tumor necrosis factor-␣, interleukin-6, interleukin-8, and interleukin-1␤; the adhesion molecules E-selectin and VCAM-1; and the enzyme nitric-oxide synthase is regulated by NF-B (for reviews, see Refs. 8 and 9), the inhibition of this signal-inducible phosphorylation of IB would be an important target for novel anti-inflammatory agents.
Surprisingly, the multisubunit IKK recognizes and is stimulated by elements of the C terminus of IB␣ (19). In this paper we provide a detailed characterization of the IKK-1 and IKK-2 catalytic subunits using peptides corresponding to the N-and C-terminal sequences of IB proteins as probes. Interestingly, amino acids 31-37 of IB-␣ provide for most of the binding free energy to the catalytic subunits, and regions outside of amino acids 21-47 provide for little interactions. Moreover, consider-ably greater binding interactions between the C terminus of IB-␣ and the regulatory subunits of the msIKK appear to be responsible for the activation of the multisubunit complex to accelerate catalysis by many orders of magnitude.

EXPERIMENTAL PROCEDURES
Materials-Glutathione S-transferase (GST)-IB␣ was purchased from Santa Cruz Biotechnology and cleaved with thrombin to remove the GST tag. [ 33 P]ATP (1000 Ci/mmol) was purchased from Amersham Pharmacia Biotech.
Peptide Synthesis-Peptides corresponding to regions of IB-␣, IB-␤, and IB-⑀ were synthesized on Fmoc-Knorr amide resin (N-(9fluorenyl)methoxycarbonyl-Knorr amide-resin; Midwest Biotech, Fishers, IN) with a model 433A synthesizer (Applied Biosystems, Foster City, CA) and the FastMoc chemistry protocol (0.25 mmol scale) supplied with the instrument. Amino acids were double-coupled as their N-␣-Fmoc-derivatives and reactive side chains were protected as follows: Asp and Glu, t-butyl ester; Ser and Thr, t-butyl ether; His, triphenylmethyl; Lys, t-butyloxycarbonyl; Arg, pentamethylchroman-sulfonyl; Cys, triphenylmethyl; and Tyr, t-butyl ether. After the final doublecoupling cycle, the N-terminal Fmoc group was removed by the two-step treatment with piperidine in N-methylpyrrolidone described by the manufacturer. The N-terminal free amines were then treated with 10% acetic anhydride, 5% diisopropylamine in N-methylpyrrolidone to yield the N-acetyl-derivative. The protected peptidyl-resins were simultaneously deprotected and removed from the resin by standard methods, except for peptides IB-␣ 26 -42 and 279 -303, which were extracted as crude products from the resin by a modified procedure described previously (19). The lyophilized peptides were purified on C 18 to apparent homogeneity, as judged by reverse phase-HPLC analysis. Predicted peptide molecular weights were verified by electrospray mass spectrometry.
Expression of Human, Recombinant IKK-1 and IKK-2-The fulllength coding region of IKK-1 and IKK-2 were cloned using reverse transcription-polymerase chain reaction with mRNA from HeLa cells as a template. Identity was confirmed by sequencing. For the protein expression the coding region was cloned into pBMS-1, a derivative of pVL1393 that has the GST coding sequence inserted into the BamHI site of the polylinker followed by a thrombin protease site and by the pVL1393 polylinker with the BamHI site restored. For this purpose, the initiation ATG of the kinases was replaced by GTG preventing potential internal translation initiation. Polymerase chain reaction primers were as follows for IKKA: TCTCATGAATTCCGGTGGAGCGGCCCCCGG and TCATATGCGGCCGCTCATTCTGTTAACCAACTCCAATCAAG, cloned into EcoRI/NotI, and for IKKB: TCTTATTCTAGAGTGAGCTG-GTCACCTTCCCTGACAACG and TCTATAGCGGCCGCGGTCGAGT-CCCCCACATCATGAGG, cloned into the XbaI/NotI site of the pBMS-1 vector.
Spodoptera frugiperda Sf9; ATCC CRL 1711) and Trichoplusia ni (BTI-Tn5B1-4, High Five TM ; Invitrogen Corp.) cells were grown in suspension cultures at 27°C in Sf9 medium (Life Technologies, Inc.). Isolation of recombinant viruses was done as described by Summers and Smith. For the expression of recombinant proteins, High Five TM cells were grown in 1-liter suspensions and infected in the log phase of growth at a cell density of 1.0 ϫ 10 6 to 1.5 ϫ 10 6 cells per ml, using 10 plaque-formung units of virus per cell.
From pellets of High Five cells expressing the recombinant GSTfusion proteins, enzyme isolation was accomplished by first lysing a cell pellet in insect cell lysis buffer (PharMingen) for 45 min at 4°C and then centrifuging the lysate at 40,000 ϫ g for 30 min at 4°C. Glutathione-agarose beads (PharMingen) were added and allowed to incubate with gentle agitation for 5 min at 4°C. The beads were then collected by centrifugation, washed twice with phosphate-buffered saline, and the GST proteins eluted with 5 mM glutathione in 50 mM Tris⅐HCl, pH 8, containing 1 mM dithiothreitol. SDS-polyacryamide gel electrophoresis showed the presence of the desired GST-IKK fusion protein with the only significant contaminant being GST itself. Anti-GST and anti-IKK (Santa Cruz Biotechnology) immunoblots were used to verify the identity and relative concentrations of IKK-1 and IKK-2 in these isolated samples.
Peptides as Substrates-When using peptides as substrates for the IKK-1 or IKK-2, enzymatic assays were performed by adding the enzyme at 30°C to solutions containing peptide and [ 33 P]ATP (1000 Ci/mmol) in 50 mM Tris⅐HCl, 5 mM MgCl 2 at pH 8. After 60 min, the kinase reactions were quenched by addition of EDTA to a concentration of 10 mM. HPLC analysis was performed as described previously (19), and the amount of IKK-catalyzed incorporation of 33 P into each peptide was quantitated by liquid scintillation counting. Under these conditions, the degree of phosphorylation of GST-IB␣ was linear with time and concentration of enzyme.
When using full-length IB-␣ (GST-tagged) as substrate, the assay was allowed to proceed for 10 min before quenching with 2ϫ Laemmli sample buffer and heat treatment at 90°C for 3 min. The samples were then loaded on to 10% Tris-glycine gels (Novex, San Diego, CA). After completion of SDS-polyacryamide gel electrophoresis, gels were dried on a slab gel dryer. The bands were then detected using a 445Si PhosphorImager (Molecular Dynamics), and the radioactivity quantified using the ImageQuant software while employing a mean background correction factor for each 33 P-labeled IB-␣ band. Radioactive standards were run to calculate the absolute (Ci) amount of radioactivity associated with the bands.

IKK-2 and IKK-1 Show Random, Sequential
Binding Kinetics-There are several kinetic mechanisms that have been described for two substrate enzyme systems. Using a peptide corresponding to amino acids 26 -42 of IB-␣ as substrate, Fig.  1 shows a Hanes plot of recombinant IKK-2 activity as a function of the concentration of this peptide substrate at different ATP concentrations. The analysis is best fit to a random, sequential binding mechanism where the enzyme binds both substrates prior to product release. This is in contrast to a ping-pong mechanism, where one product is released before the second substrate binds and would give a Hanes plot with lines intersecting at the y axis (20). Verification of a random versus ordered binding mechanism will be shown later.
The sequential binding mechanism of human, recombinant IKK-2 is defined in Scheme 1, where K IB␣ and ␣K IB␣ are the dissociation constants for IB-␣ in the absence and presence, respectively, of ATP bound to the active site; and K ATP and ␣K ATP are the dissociation constants for ATP in the absence a Multisubunit IB kinase isolated from HeLa S3 cells (19 and presence, respectively, of IB-␣ bound to the active site. Using a non-linear regression analysis of the data from Fig. 1 (21), values for K IB␣ , K ATP , and ␣ were obtained and are shown in Table I. A value of ␣ Ͻ 1 demonstrates that the binding of one substrate increases the affinity for the second substrate (20).
Using recombinant IKK-1 as the enzyme, kinetics consistent with random, sequential binding were also obtained (see Fig.  2). However, the N-terminal peptide substrate bound 6 times less tightly to IKK-1 as compared with IKK-2. Moreover, the k cat with IKK-1 was 2 orders of magnitude smaller than that measured with IKK-2. Interestingly, the dissociation constants measured with IKK-2 agree well with the values measured previously with the multisubunit complex (msIKK) isolated from HeLa S3 cells (see Table I).
N-terminal Peptides as Active Site Probes-In order to determine the effect on active site binding and catalysis of amino acids around the region of Ser-32 and Ser-36 of IB-␣, peptides of various length were prepared and tested as substrates for IKK-2. As shown in Table II, the peptide corresponding to amino acids 26 -42 of IB-␣ bound with a dissociation constant of 23 M, which corresponds to an apparent binding free energy of Ϫ6.4 kcal/mol. This apparent binding free energy may underestimate the intrinsic binding free energy since some of the binding energy may be utilized to accelerate catalysis (22). However, the apparent binding free energies calculated in Table II do provide for a reasonable comparison between peptides since the turnover number (k cat ) is relatively constant between peptides. Accordingly, because a peptide corresponding to amino acids 21-42 also bound with an apparent binding free energy of Ϫ6.4 kcal/mol, it would suggest that amino acids 21-25 contributes a negligible amount of binding interactions. Amino acids 43-47 provide for a small amount of additional binding interactions (ϳ-0.9 kcal/mol) as determined by peptide 26 -47.
The use of a peptide corresponding to amino acids 26 -39 demonstrated that nearly all of the binding free energy (ϳϪ6.2 kcal/mol) with the longer peptides is provided by amino acids 26 -39, with little contribution from amino acids 40 -42. Indeed, the use of a peptide corresponding to amino acids 31-37 demonstrated that this region provided over two-thirds of the binding free energy of the longer peptides.
Peptides corresponding to amino acids 26 -42 were synthesized with either Ser-32 or Ser-36 mutated to aspartate in an effort to probe the relative contributions of serines 32 and 36 to substrate binding interactions. As shown in Table II, these peptides bound to the active site with an avidity roughly equivalent to that of the "wild-type" peptide, demonstrating that having at least one of the serines present allowed for comparable binding interactions. Alternatively, the aspartate at either position may mimic phosphorylation at this site and provide for additional binding interactions lost from eliminating the serine. The turnover number (k cat ) for either of the mutant peptides was approximately half that of the wild-type peptide, probably owing to there being only one phosphorylation site available.
In order to further probe the contribution of the two serines to active site binding interactions, peptides corresponding to amino acids 26 -42 were prepared with both serines replaced with either aspartates (S32D/S36D) or alanines (S32A/S36A). Since these "double mutants" could no longer be used as substrates, their active site affinities were determined by using them as inhibitors of the phosphorylation of the wild-type peptide (amino acids 26 -42). As shown in Fig. 3, the S32D/S36D mutant showed dose-dependent inhibition of IKK-2, which gave a linear Dixon plot. By comparing the slope of this correlation to that shown by a C-terminal peptide inhibitor, which has a dissociation constant (K I app ) of 1.5 M (see below), the dissociation constant for this double mutant was calculated to be 102 M. The S32A/S36A mutant gave similar results (not shown). This corresponds to an apparent binding free energy of Ϫ5.5 kcal/mol, which indicates that the serines themselves only provide Ϫ0.9 kcal/mol of binding free energy for the 26 -42 peptide (Ϫ6.4 versus Ϫ5.5 kcal/mol). This, of course, explains why the S32D or S32A "single mutant" peptides bind well to the enzyme.
Consistent with amino acids 31-37 of IB-␣ providing most of the binding interactions of amino acids 21-47 with the enzyme, peptides corresponding to regions around the analogous serines of IB-␤ and IB-⑀ were also effective substrates as shown in Table III. Indeed, all three of these peptides have a highly conserved phosphorylation site (DSGX 1 X 2 S, where X 1 is either leucine or isoleucine) corresponding to amino acids 31-36 of IB-␣ and bind to the enzyme with roughly equivalent affinity. The three peptides also had dissociation constants roughly equal to each other when using IKK-1 as the enzyme source (results not shown).
Full-length IB-␣ Versus N-terminal Peptide as Substrate for IKK-2-When comparing the N-terminal peptide to the fulllength IB-␣ as a substrate for IKK-2, the results shown in Table IV demonstrate that the dissociation constants and k cat values are similar to those of the 26 -42 peptide. The dissociation constant of 2 M for IB-␣ corresponds to an apparent free energy of binding of Ϫ7.9 kcal/mol. Therefore, the Ϫ4.7 kcal/ mol of binding free energy from amino acids 31-37 (see above) represents most of the binding interactions of the full-length IB-␣ with IKK-2. Interestingly, the GST-IB-␣ fusion protein showed considerably different kinetic constants. As shown in Table IV, the presence of the GST tag greatly diminished the turnover number with a mild effect on the dissociation constant.
C-terminal Peptide as a Substrate and Inhibitor of IKK-2-We have previously reported that a C-terminal peptide corresponding to amino acids 279 -303 of IB-␣ was able to activate the msIKK isolated from HeLa S3 cells to phosphorylate an N-terminal peptide (19). In fact, the C-terminal peptide itself was a reasonable substrate for the msIKK.
When using the human, recombinant IKK-2 or IKK-1 as the enzyme source, however, phosphorylation of the C-terminal peptide was not observed (results not shown). Moreover, the C-terminal peptide failed to potentiate the IKK-2-or IKK-1catalyzed phosphorylation of the N-terminal peptide. As shown in Fig. 4, this C-terminal peptide instead showed pure competitive inhibition with respect to an N-terminal (amino acids 26 -42) peptide substrate. 2 From a nonlinear regression analysis of the data represented in Fig. 4, a K I app value of 1.5 Ϯ 0.3 M was obtained. Thus, the C-terminal peptide binds well to the active site, but does not act as either a substrate or activator of the IKK-2.
That the C-terminal peptide showed competitive inhibition with respect to the N-terminal peptide substrate is expected from a mechanism detailed in Scheme 2, where the binding of the inhibitor (i.e. the C-terminal peptide) competes with the binding of IB-␣, but not ATP. Also consistent with this mechanism, the inhibition observed with the C-terminal peptide while keeping the concentration of peptide substrate fixed and varying the concentration of ATP showed mixed-type (noncompetitive) inhibition as shown in Fig. 5. As expected from Scheme 2, infinitely large concentrations of ATP are unable to completely overcome the inhibition produced by the C-terminal peptide. However, this analysis does not rule out an ordered binding mechanism, which would have shown a similar inhibition pattern if the peptide substrate binds before ATP (23). Verification of a random sequential binding mechanism comes from the use of staurosporine as an inhibitor. Staurosporine, which is known to bind to the ATP binding site of kinases (24,25), shows competitive inhibition with respect to ATP (Fig. 6) and mixed-type noncompetitive inhibition with respect to peptide substrate (see Fig. 7). This unequivocally demonstrates a random sequential binding mechanism since an ordered binding mechanism would have shown uncompetitive inhibition with respect to peptide substrate (23).
Scheme 2 shows equilibria in a random sequential mechanism showing an inhibitor (I), which competes with IB-␣ (or peptide substrate) but allows ATP to bind (20). Here, K I and ␤K I represent the dissociation constants of the inhibitor in the absence and presence, respectively, of ATP; and K ATP and ␤K ATP represent the dissociation constants of ATP in the absence and presence, respectively, of inhibitor. The other constants are defined elsewhere.

DISCUSSION
Random Sequential Kinetics-The use of peptide substrates and inhibitors in the present work has provided valuable insights into the interactions between IB and IKK. In the case of IKK-2, a random sequential mechanism was unequivocally demonstrated by the use of these peptides. This is in agreement with the mechanism determined for the msIKK isolated from HeLa S3 cells (19). A sequential binding mechanism was also recently determined by Li and co-workers (16) with recombinant IKK subunits using full-length IB-␣. However, Li et al.  measured an apparent dissociation constant of 0.13 M for ATP and failed to show a cooperative binding between ATP and IB-␣ (i.e. ␣ ϭ 1). This is in contrast to the present work, where values of 0.2 and 2 M were determined for ␣ and K ATP app , respectively. While the basis of this difference is not clear, we have found that the use of NaCl in the kinase assay (Li and coworkers used 100 mM NaCl) quite often failed to give linear reaction rates for more than a few minutes, especially when using the full-length IB-␣ as substrate. In contrast, the use of the peptide substrates in the present research gave reaction rates that were linear for Ͼ2 h. It is interesting to note that the K ATP app values determined here agree well with the value determined with the msIKK (19) and are more in line with the dissociation constants determined with other protein kinases such as mitogen-activated protein kinase (26) and cAMP-dependent protein kinase (27), which have ATP dissociation constants in the 5-10 M range.
Differences between IKK-1 and IKK-2 Catalytic Subunits-While the kinetics of the IKK-1 subunit are also consistent with a random sequential binding mechanism, this catalytic subunit proved to be a much less effective catalyst for the phosphorylation of IB-␣ peptides than the IKK-2 catalytic subunit as others have also noted (16,41). This resulted from a 6-fold increase in the peptide substrate dissociation constant as well as a 100-fold decrease in the turnover number when compared with IKK-2. This translates into an apparent second order rate constant (k cat /K m ) for IKK-1 that is 650 times smaller than with IKK-2.
This observation suggests that the IKK-2 catalytic subunit of the msIKK provides most if not all of the catalytic activity, with the IKK-1 catalyzing very little of the phosphorylation. Indeed, the dissociation constant of peptide substrate measured with the msIKK is very similar to the value measured with the IKK-2 and not the IKK-1 (see Table I). This conclusion is also consistent with a number of recent reports, which indicated that IKK-2 plays a larger role than IKK-1 in the phosphorylation of IB proteins and subsequent NF-B activation in cells.   For instance, the translation of a catalytically inactive mutant of IKK-1 resulted in a multisubunit complex that was only slightly less active than with wild-type IKK-1, while the translation of a catalytically inactive mutant of IKK-2 obliterated the activity of the multisubunit complex (13). Moreover, the use of these catalytically inactive, dominant-negative mutants of the two catalytic subunits expressed in cells showed that the IKK-2 played a larger role in the tumor necrosis factor-␣stimulated NF-B translocation in HeLa cells and lipopolysaccharide-induced NF-B-dependent transcription in monocytic cells (14,28). However, the IKK-1 and IKK-2 subunits appear to contribute equally in CD28-dependent and HTLV-1 taxmediated activation of NF-B in Jurkat cells (29,30).
While it is possible that a heterodimer of IKK-1 and IKK-2 may show different characteristics than the homodimers of each catalytic subunit used in the present study, this is unlikely since it has been shown that the two catalytic subunits are not affected by each other when present as heterodimers (16).
Binding Free Energies-Using peptides corresponding to amino acids 21-47 of IB-␣, the present research indicates that most of the binding interactions come from amino acids 31-37, the region encompassing the serines (32 and 36) that are phosphorylated by the IKK (see Table II). This explains the ability of IKK to phosphorylate IB-␤ and IB-⑀ as well, since these proteins have highly conserved phosphorylation sites as shown in Table III (31). It has previously been shown that a peptide corresponding to amino acids 279 -303 (the "C-terminal peptide") activates the multisubunit IKK to catalyze the phosphorylation of an Nterminal peptide containing serines 32 and 36 (19). The present research shows that the isolated IKK-1 and IKK-2 was not activated by this C-terminal peptide. This indicates that only the msIKK, and not the catalytic subunits, is able to bind to and be activated by the C terminus of IB-␣. Consistent with this conclusion is the observation that the dissociation constants and V max for the 26 -42 peptide substrate and the fulllength IB-␣ are similar when using the recombinant IKK-2 as enzyme. This is in contrast to the msIKK, which showed a 10-fold increase in V max and a 2500-fold decrease in the K m when comparing the peptide to the full-length length IB-␣ (19). This corresponds to a 25,000-fold greater apparent second order rate constant (k cat /V max ) for the full-length IB-␣ as compared with the peptide substrate.
Therefore, the effects observed with the msIKK must be due to either C-terminal peptide interactions with other subunits within the msIKK, or that the presence of other regulatory subunits within the complex affects the conformation of the catalytic subunits so that activation by the C-terminal peptide can occur. Indeed, two other subunits of the msIKK, termed IKAP and IKK-␥, have recently been identified, and the IKK-␥ in particular has been shown to interact with and regulate the activity of the catalytic subunits (32,33). Interestingly, the IKK-␥ appears to interact preferentially with IKK-2. We are currently investigating how IKK-␥ affects the kinetics of IKK-2-catalyzed phosphorylation of IB. The conclusion that the C terminus of IB-␣ activates the msIKK is also consistent with reports that the presence and phosphorylation state of the C terminus of IB-␣ play a role in the signal-induced degradation of IB-␣ (34 -36). An analogous C-terminal region rich in proline, glutamate, aspartate, serine, and threonine residues (termed the PEST domain) has also been identified in IB-␤ (37,38). Interestingly, a recent report has indicated that recombinantly transferring the C terminus (along with the N terminus) of IB-␣ to an unrelated protein such as GST enables GST to be phosphorylated and degraded in a signal-responsive way (39), although this may result from a recognition of the C terminus by the proteasome (40). Transferring only the N terminus of IB-␣ to GST did not result in a recombinant protein that underwent signal-induced proteolysis.
Also related to the conclusion that regions of IB-␣ outside of amino acids 21-47 interact with the msIKK to affect catalysis, it has been recently reported that IB-␣ complexed with NF-B is a more efficient substrate (lower K m and greater V max ) than IB-␣ alone (41). This may result from a conformational change in IB-␣, which may further maximize binding interactions that are utilized for rate acceleration. It should be noted, however, that the GST fusion protein of IB-␣ was used in those studies. The present research indicates that the GST tag reduced the effectiveness of the IB-␣ as a substrate. Since untagged IB-␣ is a better substrate (lower K m and greater V max ), the effects with the GST-tagged protein as substrate when complexed with NF-B may simply reflect a change in the conformation of GST-IB-␣ that removes the detrimental affects of the the GST tag. We are currently investigating these possibilities.
In summary, the present work demonstrates that both the IKK-2 and IKK-1 catalytic subunits follow a random, sequential binding mechanism as was observed with the msIKK. The much greater catalytic efficiency observed with IKK-2 as compared with IKK-1 suggests that the IKK-2 subunit contributes nearly all of the catalytic activity of the msIKK complex with the IKK-1 subunit providing little contribution to catalysis. In addition, amino acids 31-37 of IB-␣, which contains the consensus phosphorylation site, appear to contribute most of the binding interactions of the full-length IB-␣ with the IKK-2. However, areas in the C terminus of IB-␣ contribute greatly to binding interactions with the msIKK, which provides for extremely large rate accelerations.