Kinetic Mechanisms of I B-related Kinases (IKK) Inducible IKK and TBK-1 Differ from IKK-1/IKK-2 Heterodimer*

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 15, Issue of April 12, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Kinetic Mechanisms of I B-related Kinases (IKK) Inducible IKK and TBK-1 Differ from IKK-1/IKK-2 Heterodimer* Received for publication, December 3, 2001, and in revised form, January 23, 2002 Published, JBC Papers in Press, January 28, 2002, DOI /jbc.M Q. Khai Huynh, Nandini Kishore, Sumathy Mathialagan, Ann M. Donnelly, and Catherine S. Tripp From the Department of Arthritis and Inflammation Pharmacology, Discovery Research, Pharmacia Corporation, St. Louis, Missouri Nuclear factor- B activation depends on phosphorylation and degradation of its inhibitor protein, I B. The phosphorylation of I B on Ser 32 and Ser 36 is initiated by an I B kinase (IKK) complex that includes a catalytic heterodimer composed of I B kinase 1 (IKK-1) and I B kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF- B essential modulator. Recently, two related I B kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser 36 of I B. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhtbk-1, rhikk-i, and rhikk-1/ rhikk-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an I B peptide for the rhikk-1/rhikk-2 heterodimer ( 0.99), whereas the binding of ATP decreased the affinity of the I B peptide for both rhtbk-1 ( 10.16) and rhikk-i ( 62.28). Furthermore, the dissociation constants of ATP for rhtbk-1 and rhikk-i are between the expected values for kinases, whereas the dissociation constants of the I B peptide for each IKK isoforms is unique with rhtbk-1 being the highest (K I B M), followed by rhikk-i (K I B 5.47 M) and rhikk-1/ rhikk-2 heterodimers (K I B 0.12 M). Thus this family of I B kinases has very unique kinetic properties. NF- B 1 is an inducible, ubiquitous transcription factor that is primarily involved in immune, inflammatory, and stress responses (1 3). NF- B is present in the cytoplasm of resting cells and enters the nucleus upon cellular activation with inflammatory cytokines such as TNF- and interleukin 1 as well as with many other stimuli including LPS, viral infection, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Both authors contributed equally to this work. To whom correspondence should be addressed: Dept. of Arthritis and Inflammation Pharmacology, Pharmacia Corp., Mailzone T3M, 800 North Lindbergh Blvd., St. Louis, MO Tel.: ; Fax: ; quang.k.huynh@pharmacia.com. 1 The abbreviations used are: NF- B, nuclear factor- B; IKK, I B kinase; IKK-2 (S177E,S181E), a variant of IKK-2 in which Ser 177 and Ser 181 were replaced by Glu; TBK-1, TANK-binding kinase-1; IKK-i, inducible IKK; TNF, tumor necrosis factor; LPS, lipopolysaccharide; NEMO, NF- B essential modulator; MAPK, mitogen-activated protein kinase; TRAF, TNF receptor-associated factor; rh, recombinant human; wt, wild type; DTT, dithiothreitol; BSA, bovine albumin serum; I B - PI, I B peptide inhibitor Asp-Ser(PO 3 H 2 )-Gly-Leu-Asp-Ser-Met phorbol esters, and oxidizing agents (1, 2). In unstimulated cells, NF- B resides as an inactive complex with inhibitory proteins called I Bs, the most well characterized being I (1, 2). In the case of I B, stimulation of cells with agents that activate NF- B-dependent gene transcription results in a phosphorylation of I B at Ser 32 and Ser 36. This phosphorylation facilitates the interaction of I B with -TrCP, which triggers the formation of a ubiquitin-ligase complex responsible for adding ubiquitin groups to I B on specific lysine residues. The ubiquitinated form of the I B is then targeted to the 26 S proteosome and degraded (2). This I B degradation leads to the release of NF- B, which can then translocate to the nucleus and promote gene transcription (1). The protein kinase that phosphorylates I B in response to proinflammatory stimuli is the I B kinase (IKK) complex (4 9). The IKK complex purified from HeLa S3 cells has a molecular mass of kda (6, 7, 9). Among the proteins identified in this complex are two kinases, I B kinase 1 (IKK-1, also designated IKK ) and I B kinase 2 (IKK-2, also designated IKK ). IKK-1 and IKK-2 contain similar structural domains consisting of an N-terminal kinase domain as well as leucine zipper and helix-loop-helix domains in their C terminus; they also share significant sequence homology with 65% homology in the kinase domain (4 8). IKK-1 and IKK-2 associate in cells preferentially as a heterodimer (7), although IKK-2 homodimers are also described (10). Both kinases contain a canonical MAPK kinase activation loop motif in the N terminus, and this motif is the substrate for mitogen activated protein kinase (MAP3K) kinases such as NF- B-inducing kinase and MEKK-1 (4 8). Phosphorylation of the MAPK kinase loop of IKKs is necessary for their activation of kinase activity (4 8). A third protein in the complex is NEMO (also called IKK or IKKAP) (10 12). NEMO associates with IKK-2 and is required for the activation of the IKK-1/IKK-2 heterodimer (11, 12). Recombinant hikk-1 and hikk-2 homodimers have been expressed, isolated, and characterized (10, 13 22). Purified rhikk-2 homodimer is more effective at phosphorylating I Bs than rhikk-1 homodimer (10, 13, 14). In addition, it has been reported that the mutation of Ser 177 and Ser 181 to glutamic acid in rhikk-2 homodimer results in an enzyme with dramatically enhanced, constitutive IKK-2 activity (10). The apparent maximal turnover k cat of rhikk-2 (Ser 177 -Glu and Ser 181 -Glu) was 10-fold higher than that of rhikk-2, whereas rhikk-2 displays a 3 4-fold greater k cat value than rhikk-1 (10). Kinetic analysis of rhikk-2 also indicates that in the presence of NF- B, the K m value for I B decreases from 2.2 to 1.4 M, and the V max value increases by a factor of 4, indicating that rhikk-2 phosphorylates I B bound to NF- B more efficiently then it phosphorylates free I B (19). Both the isolated IKK complex from mammalian cells and the rhikks utilize all three isoforms This paper is available on line at

2 Kinetic Mechanisms of Recombinant Human I B Kinases of I Bs,, and, as substrates equally well (22). However, there are differences in the kinetic data reported for the rhikks and IKK complex isolated from HeLa S3 cells (9, 10, 13, 14). First, a wide range of binding constant values for I B and ATP (K I B and K ATP ) has varied in different publications, ranging from 0.12 to 1.4 and from 0.08 to 7.3 M, respectively (9, 13 14). Second, although the two substrates each bind in a cooperative fashion for the IKK complex isolated from HeLa S3 cells ( 0.11 (9)), kinetic analysis using rhikk-1 homodimer and rhikk-2 homodimer suggests that the binding of one substrate has no effect on the affinity of the other substrate ( value 1.0) (14). Thus the native complex, which can contain both IKK proteins, NEMO and various substrate proteins, has different kinetic properties than those reported for the separate purified protein components. The kinetic analysis of the rhikk-1/rhikk-2 has not been described previously. Recently, two other family members of the IKK-related kinases have been reported, namely TBK-1 (also called NAK or T2K) and IKK-i (also called IKK ) (1, 23 27). TBK-1 and IKK-i are 80- and 84-kDa proteins, respectively, that showed 67% identity to each other in their kinase domains but only 50% identity over their entire sequences. When compared with IKK-1 and IKK-2, IKK-i and TBK-1 exhibited 30% identity within the kinase domains but have similar domain structures consisting of an N-terminal kinase domain as well as leucine zipper and helix-loop-helix domains in their C terminus. IKK-i and TBK-1 have unique MAPK kinase activation loops containing only one of the two phosphoacceptor serines compared with the activation loops of IKK-1 and IKK-2. The upstream kinase(s) that activate TBK-1 and IKK-i have not been identified (1). IKK-i was identified in data bases from sequence homology to IKK-2 and was also cloned from a subtractive hybridization screen designed to identify LPS-inducible mrnas (25, 26). Unlike IKK-1 and IKK-2 which are constitutively expressed in all cell types, IKK-i is highly expressed in thymus, spleen, and peripheral blood leukocytes (25, 26) and can also be induced in some cell types by various agonists and cytokines including LPS, TNF-, and interleukin 1 (26). As mentioned, although IKK-1 and IKK-2 phosphorylate both Ser 32 and Ser 36 of I B, IKK-i is only able to phosphorylate I B on Ser 36 and thus alone would not be able to initiate its degradation (25, 26). It has been suggested recently that IKK-i activates NF- B by phosphorylating I-TRAF/TANK, leading to the release of TRAF-2 resulting in activation of downstream targets including IKK-2 (1, 25, 26). The other related kinase, TBK-1, was identified by its interaction with TANK (22). TBK-1 was also identified through degenerate PCR cloning using IKK-1/IKK-2 primers. Unlike IKK-i but similar to IKK-1 and IKK-2, TBK-1 was found to be expressed ubiquitously (23). TBK-1 like IKK-i phosphorylates I B only on Ser 36. However, when TBK-1 is overexpressed in mammalian cells, it is able to phosphorylate IKK-2. Thus the physiological substrate(s) of IKK-i and TBK-1 may be unique but are unclear at this time. Finally, we have demonstrated recently (42) that recombinant proteins of hikk-i and htbk-1 are enzymatically more similar to each other and distinct from rhikk-1, rhikk-2, and rhikk-1/ rhikk-2 heterodimer in terms of K m values for both I B peptides and proteins as well as for ATP; likewise these various IKK isoforms have very different k cat values with respect to the I B peptide as substrate. Although we have cloned, isolated, and characterized rhikk- 1/rhIKK-2 heterodimer (28), we have not yet reported data describing the kinetic mechanism for rhikk-1/rhikk-2 heterodimer. Likewise there is nothing known about the kinetic mechanisms of rhtbk-1 and rhikk-i. In this paper we studied the kinetic mechanisms of the rhtbk-1 and rhikk-i and compared them to those of rhikk-1/rhikk-2 heterodimer as well as to other published IKKs. The results indicate that each of these enzymes exhibit a random sequential kinetic mechanism. However, rhikk-1/rhikk-2 heterodimer exhibits an 1 which suggests that the affinity of ATP and I B for the free enzyme will be the same as the affinities for each of these substrates for the binary complexes. In contrast, rhikk-i or rhtbk-1 exhibits an value 10 which suggests that the affinity of each substrate for the free enzyme is higher than that of the binary complex, indicating a negative co-operativity exists between the two substrates. During the course of characterization of the rhikks, we also found that the dissociation constants of ATP for rhtbk-1 and rhikk-i were between the suggested values for kinases, namely 0.1 to 30 M (29, 30). However the dissociation constants of I B for rhtbk-l and rhikk-i were significantly higher than that for rhikk-1/rhikk-2 heterodimer. Thus these results indicate that rhikk-i and rhtbk-1 are similar to each other but distinct from rhikk-1/rhikk-2 heterodimer not only with respect to their substrate specificity but also with respect to their kinetic properties. EXPERIMENTAL PROCEDURES Materials SAM TM 96 Biotin capture plates were purchased from Promega. Anti-FLAG affinity resin, Nonidet P-40, BSA, ATP, ADP, and dithiothreitol were obtained from Sigma. Nickel-nitrilotriacetic acid resin was purchased from Qiagen. Peptides were purchased from American Peptide Co. Protease inhibitor mixture tablets were from Roche Molecular Biochemicals. Sephacryl S-300 column was from Amersham Biosciences. Centriprep-10 concentrators with a molecular mass cut-off of 10 kda and membranes with molecular mass cut-off of 30 kda were obtained from Amicon. [ - 33 P]ATP (2500 Ci/mmol) was purchased from Amersham Biosciences. The other reagents used were of the highest grade commercially available. Cloning and Expression of rhikks cdnas of human IKKs were cloned and expressed following methods described previously (28). The cells were lysed at a time that maximal expression and rhikk activity were achieved. Cell lysates were stored at 80 C until purification of the recombinant proteins was undertaken as described below. Enzyme Isolation All purification procedures were carried out at 4 C unless otherwise noted. The buffers used are as follows: buffer A, 20 mm Tris-HCl, ph 7.6, containing 50 mm NaCl, 20 mm NaF, 20 mm -glycerophosphate, 500 M sodium orthovanadate, 2.5 mm metabisulfite, 5 mm benzamidine, 1 mm EDTA, 0.5 mm EGTA, 10% glycerol, 1 mm DTT, 1 Complete protease inhibitors; buffer B, same as buffer A except 150 mm NaCl; and buffer C, same as buffer A except 500 mm NaCl. Isolation of rhikk-1/rhikk-2 Heterodimer The heterodimer enzyme was produced by co-infection in a baculovirus system (FLAG IKK-2/IKK-1 His; multiplicity of infection 0.1 and time of expression 72 h). Infected cells were centrifuged, and the cell pellet (10.0 g) was suspended in 50 ml of buffer A. The protein suspension was microfluidized and centrifuged at 100,000 g for 45 min. The heterodimer enzyme was further purified to homogeneity using nickel-nitrilotriacetic acid resin and anti-flag M2-agarose affinity resin as described previously (28). Isolation of rhtbk-1 Cells from a 20-liter fermentation from baculovirus of N-terminal FLAG-tagged rhtbk-1 were microfluidized, and the ph was adjusted to 7.6 using NaOH. The sample was centrifuged for 1 h at 26,000 g. Anti-FLAG M2 antibody resin, 50 ml, was added to the pool and allowed to mix overnight. The resin was batch-washed using 4 bed volumes of buffer A per wash. The resin slurry was then poured into a 26/20 column and washed with 15 column volumes of buffer B. The rhtbk-1 protein was eluted using an anti-flag peptide in buffer A. Dithiothreitol was added to the pool to make 5 mm and concentrated in a stirred cell using an Amicon YM30 membrane. BSA was added to the concentrated pool to make 0.1%, and the pool was aliquoted into 1-ml samples. The samples were frozen at 80 C until ready for use. Isolation of rhikk-i Cells from a 20-liter fermentation of baculovi-

3 12552 Kinetic Mechanisms of Recombinant Human I B Kinases rus-expressed rhikk-i with an N-terminal FLAG tag were microfluidized and centrifuged at 26,000 g for 1 h. The supernatant was collected and the ph adjusted to 7.6 using NaOH. Anti-FLAG M2 affinity gel, 25 ml, pre-equilibrated in buffer A was added to the supernatant pool and allowed to mix overnight. The resin was washed in batch using 4 resin volumes of buffer A per wash. The resin slurry was then poured into a 26/20-ml column and washed with 15 resin volumes of buffer B. The FLAG rhikk-i was eluted using a FLAG peptide in buffer A. Dithiothreitol was added to the pool to make 5 mm followed by concentrating in a stirred cell using an Amicon YM30 membrane. BSA was added to the concentrated pool to make 0.1%, and the pool was frozen as 1-ml aliquots at 80 C. SDS-PAGE and isoelectric focusing gel electrophoresis of the isolated enzymes indicated that they are homogeneous proteins (data not shown). When assayed using I as phosphoacceptor peptide, both rhikk-i (395.0 units/mg of protein) and rhtbk-1 (339.0 units/mg of protein) had a dramatically higher specific activity compared with rhikk-1/rhikk-2 heterodimer (8.2 units/mg of protein) (42). However, because I B is a significantly better substrate for rhikk-1/rhikk-2 heterodimer with more than 100-fold lower K m compared with rhikk-i and rhtbk-1, the catalytic efficiency (k cat /K m )is8 12-fold higher for rhikk-1/rhikk-2 heterodimer (81.1 h 1 M 1 ) compared with rhikk-i (9.7 h 1 M 1 ) and rhtbk-1 (6.4 h 1 M 1 ) (42). Recombinant hikks when expressed in a baculovirus system are phosphorylated and require phosphorylation for their kinase activity because it is abolished when these proteins were treated with recombinant phosphatase (42). Data maps the phosphorylation of the rhikks to the MAPK kinase activation loop (10, 42). Phosphopeptide mapping and site-specific mutagenesis indicated that rhikk-i and rhtbk-1 are phosphorylated on Ser 172 (42). All of the wild type rhikks have phosphorylation-dependent kinase activity, whereas the constitutively active mutant rhikk2 (S177E,S181E) and S172E mutant of rhikk-i do not (28, 42). Details of the expression, isolation, characterization, and phosphorylation status of rhikks will be described elsewhere (42). Enzyme Assay Kinase activity was measured using a biotinylated I B peptide (Gly-Leu-Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser 32 -Gly- Leu-Asp-Ser 36 -Met-Lys-Asp-Glu-Glu), 96-well streptavidin Promega plate, and a vacuum system as described previously (28). The standard reaction mixture for heterodimer rhikk-1/rhikk-2 assay contained 5 M biotinylated I B peptide, 1 M [ - 33 P]ATP (about cpm), 1 mm DTT, 50 mm KCl, 2 mm MgCl 2,2mM MnCl 2,10mM NaF, 25 mm Hepes buffer, ph 7.6, and enzyme solution (0.02 to 0.2 g) in a final volume of 50 l. After incubation at 25 C for 30 min, 25 l ofthe reaction mixture was withdrawn and added to a 96-well Promega plate. Each well was then washed successively with 800 lof2mnacl, 1.2 ml of NaCl containing 1% H 3 PO 4, 400 lofh 2 O, and 200 l of 95% ethanol. The plate was allowed to dry in a hood at 25 C for 1 h, and then 25 l of scintillation fluid (Microscint 20) was added to each well. Incorporation of [ - 33 P]ATP was measured using a Top-Count NXT (Packard Instrument Co.). Due to its higher K m for I B peptide and for ATP, rhtbk-1 and rhikk-i were assayed as described above but with 300 M biotinylated I B peptide and 5 M [ - 33 P]ATP ( cpm). Under each assay condition, the degree of phosphorylation of I B peptide substrate was linear with time and concentration of all purified enzymes. An ion exchange resin assay was also employed using [ - 33 P] ATP and I B peptide as the substrates. Each assay system yielded consistent results in regard to K m and specific activities for each of the purified kinase isoforms. One unit of enzyme activity was defined as the amount required to catalyze the transfer of 1 nmol of phosphate from ATP to I B peptide per min. Specific activity is expressed as units per mg of protein. For experiments related to kinetic constants of purified enzymes, various concentrations of ATP or I peptide were used in the assay at either a fixed I B or ATP concentration. Due to their high activity and higher K m for I B peptide, rhtbk-1 or rhikk-i (0.01 to g) was assayed with higher concentrations of I B peptide ( M) and ATP (5 15 M). In these cases, the reaction mixtures were diluted 20-fold with 25 mm Hepes buffer, ph 7.6, containing 1 mm DTT, 50 mm KCl, 2 mm MgCl 2,2mM MnCl 2,10mM NaF prior to adding 25 l ofthe diluted solution to a 96-well Promega plate. Kinetic Analysis For two-substrate profile analysis, initial velocity studies were performed with varying concentrations of I B at several fixed concentrations of ATP. Lineweaver-Burk double-reciprocal plots were generated SCHEME I. Various models of kinetic mechanisms have been described for enzymes that catalyze two substrates (31 35). Model 1, ordered sequential with ATP binding first; model 2, ordered sequential with I B binding first; model 3, random sequential; and model 4, ping-pong. by linear least square fits of the data (31 35). All enzyme activity data are reported as an average of triplicate determinations. The apparent kinetic constants were determined from the x and y intercepts of the double-reciprocal plots. Secondary plots were generated by replotting the slopes and the y intercepts of the lines as function of 1/[ATP]. Kinetic constants were determined Erithacus Software Grafit as described by Leatherbarrow (36). For the random sequential model, values of K ATP, K I B, and can be determined from the x and y intercepts of the secondary plots. K ATP and K I B are the dissociation constants for ATP and I B, respectively. The constant is the ratio of apparent dissociation constants for binding I B in the presence and absence of ATP (31 35). For inhibition experiments, initial velocity studies were performed with varying ATP concentrations at constant I B concentrations and several fixed inhibitor concentrations. Other Methods Protein concentrations were determined by the method of Bradford (37) or by SDS-PAGE with silver staining (38) using bovine serum albumin as the standard. Purity and molecular weights of the isolated enzyme were confirmed by SDS-PAGE with silver staining (38). RESULTS AND DISCUSSION Various models of kinetic mechanisms have been described for enzymes that catalyze two substrates (31 35). Possible mechanisms for IKK kinases are shown in Scheme I (31 35). Scheme I describes both sequential and ping-pong mechanisms. In sequential reactions, all substrates bind to the enzyme before the first product is released. In ordered sequential mechanisms (Scheme I, models 1 and 2), the substrates and products are bound and released in an obligatory sequence, compare with a random sequential mechanism (Scheme I, model 3) where there is no specific order in which the substrates bind and the products are released. In contrast to a sequential mechanism, a ping-pong mechanism involves one product being released from the enzyme before the second substrate can bind (Scheme I, model 4). The distinctions between these mechanisms are made by analyzing initial velocity patterns when one substrate is varied at several fixed concentrations of the second substrate (31 35). A classical sequential mechanism is indicated when the family of double-reciprocal plots intersects to the left of the y axis and converges at the x axis, because both the slope and the intercept changes as the concentration of the fixed substrate changes. In the case of a ping-pong mechanism, the slope of the series of double-reciprocal plots is unchanged, whereas only the intercept changes as the concentration of the fixed substrate is altered, giving rise to a series of parallel lines. For the twosubstrate profile analysis of the various IKK isoforms, initial

4 Kinetic Mechanisms of Recombinant Human I B Kinases SCHEME II. The equilibria of random sequential mechanism (31 35). velocity studies were performed with varying concentrations of ATP at several fixed concentrations of the I B peptide. Conversely, initial velocity measurements were also made with varying concentrations of the I B peptide at several fixed concentrations of ATP. Lineweaver-Burk, double-reciprocal plots were generated by linear least squares fit of the data (31 36). All enzyme activity data are reported as an average of triplicate determinations as described under Experimental Procedures. Kinetic constants were determined by Erithcus Software Graft as described by Leatherbarrow (36). For a sequential mechanism, the kinetic constants for a two substrate mechanism are shown in Scheme II. K I b and K I b are the dissociation constants for the I B in the absence and presence of ATP in the active site, respectively. Likewise, K ATP and K ATP are the dissociation constants for ATP in the absence and presence of I B in the active site, respectively (31 35). The value of indicates whether the affinity of each substrate for the free enzyme is the same or different from the affinity of each substrate for the binary enzyme complex containing one of the substrates with the enzyme (Equations 1 and 2) (31 35). 1/rate K I B /V max 1 K ATP / ATP 1/ I B 1/rate K ATP /V max 1 K I B / I B 1/ /ATP 1/V max 1 K ATP / ATP]} (Eq. 1) 1/V max 1 K I B / I B ]} (Eq. 2) An value equal to 1 implies that the affinity of ATP and I B for the free enzyme will be the same as the affinities for each of these substrates for the binary complexes, E-I B or E-ATP, respectively. In this instance, the family of 1/rate versus 1/ATP or 1/I B will intersect on the respective x axis of the double-reciprocal plots. If is less than 1, the affinity of ATP or I B to the free enzyme (E) is lower than their respective affinities for the binary complex, E-I B or E-ATP, respectively. In this case, the family of the double-reciprocal plots for the varied substrate (e.g. I B ) intersects at a point above the 1/I B axis and pivots clockwise about this point. Likewise, the family of double-reciprocal plots when ATP is the varied substrate is symmetrical to that described for I B. An value greater than 1 suggests that the affinity of each substrate for the free enzyme is higher than that of the binary complex and suggests a steric hindrance or negative co-operativity exists between the two substrates. In this case, the series of doublereciprocal plots will intersect below the 1/I B and 1/ATP axis, making the plots appear almost parallel. For inhibition experiments, initial velocity studies were performed with varying concentrations of ATP at a constant fixed concentration of I B and several fixed inhibitor concentrations for inhibition at the ATP site, and at varying concentrations of I B at a single fixed concentration of ATP and various inhibitor concentrations at the I B site. Data from inhibition experiments were fitted to either a linear competitive model (Equation 3), a non-competitive model (Equation 4), a mixed inhibition model (Equation 5), or an uncompetitive model (Equation 6) (31 35). 1/rate K m /V max 1 I /K i 1/ S 1/V max (Eq. 3) FIG. 1. Effects of Asp-Ser(PO 3 H 2 )-Gly-Leu-Asp-Ser-Met on activity of rhikk-1/rhikk-2 heterodimer, IKK-I, and TBK-1. Different concentrations of Asp-Ser(PO 3 H 2 )-Gly-Leu-Asp-Ser-Met were incubated with rhikk-1/rhikk-2 (E), rhtbk-1 ( ), and rhikk-i ( ), and kinase activities were performed as described under Experimental Procedures. 1/rate K m /V max 1 I /K i 1/ S 1/V max 1 I /K i (Eq. 4) 1/rate K m /V max 1 I /K i 1/ S 1/V max 1 I / K i (Eq. 5) 1/rate I / V max K i 1/V max 1 K m / S (Eq. 6) All IKK family members were cloned, expressed in a baculovirus system, and purified to homogeneity for enzymatic characterization (28, 42). To aid in the study of the kinetic mechanisms of rhikk-1/rhikk-2 heterodimer, rhtbk-1 and rhikk-i, the effects of their products both ADP and phosphorylated I B peptide Gly-Leu-Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His- Asp-Ser 32 (PO 3 H 2 )-Gly-Leu-Asp-Ser 36 (PO 3 H 2 )-Met-Lys-Asp- Glu-Glu were examined on the enzyme activities. Our objective was to identify a suitable product or dead-end inhibitor for both the ATP and I B sites, respectively, for each of the IKK enzymes. We reported recently (42) that ADP is a competitive inhibitor at the ATP site for each of the IKK isoforms. However, each IKK isoform had a distinct IC 50 value of ADP inhibition, with rhikk1/rhikk2 heterodimer 1.7 M, rhikk-i 19.6 M, and TBK M. In contrast, the other product of the kinase reaction, a phosphorylated I B peptide (Gly-Leu-Lys- Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser 32 (PO 3 H 2 )- Gly-Leu-Asp-Ser 36 (PO 3 H 2 )-Met-Lys-Asp-Glu-Glu), was not an effective inhibitor up to 1 mm concentration for any of these IKK isoforms (data not shown). Because a peptide inhibitor for the I B site was necessary for kinetic studies, a series of truncated and phosphorylated peptides derived from the I B peptide sequence were evaluated as inhibitors against these three IKK family members (data not shown). This analysis yielded one truncated peptide, Asp-Ser 32 (PO 3 H 2 )-Gly-Leu-Asp- Ser 36 -Met, that could inhibit all three enzymes, again with varying IC 50 values of inhibition, namely rhikk-1/rhikk-2 heterodimer M, rhtbk M, and rhikk-i M (Fig. 1). We have shown previously (42) that this I B peptide analog was not a substrate for any of these IKK isoforms and therefore constitutes a dead-end inhibitor rather than an alternate substrate. Thus, ADP and the I B peptide inhibitor, Asp-Ser(PO 3 H 2 )-Gly-Leu-Asp-Ser-Met, designated I B -PI, were used to characterize the kinetic mechanism of each IKK isoform. As discussed above, several kinetic mechanisms have been described for enzymes that, like the IKKs, catalyze two substrates. Fig. 2A shows the double-reciprocal plot of rhikk-1/ rhikk-2 heterodimer when initial velocity is measured at varying concentrations of I B peptide and at various fixed concentrations of ATP. A similar family of plots results when the initial velocities are measured at varying concentrations of ATP at several fixed concentrations of I B peptide (Fig. 2B). These data best fit a classical sequential mechanism as de-

5 12554 Kinetic Mechanisms of Recombinant Human I B Kinases FIG. 2. Kinetic patterns for rhikk- 1/rhIKK-2 heterodimer with various concentrations of I B peptide and ATP. A, double-reciprocal plot of 1/rate versus 1/(I B ) were generated at five fixed ATP concentrations of 0.1 ( ), 0.2 ( ), 0.5 (f), and 1.0 M ( ). B, doublereciprocal plot of 1/rate versus 1/(ATP) were generated at five fixed I B peptide concentrations of 0.1 (E), 0.2 ( ), 0.5 ( ), 1.0 (f), and 2.5 M ( ). Assays were performed as described under Experimental Procedures. scribed in Scheme I because the family of curves intersect on the x axis. Recall in a sequential mechanism, the enzyme binds both substrates forming a ternary complex before the first product is released (31 35). Whereas these initial velocity plots suggest a sequential mechanism, it gives no indication of the order of the substrate binding or the order of the product release. As shown in Scheme I, either ATP can bind first (model 1) ori B can bind first (model 2). Alternatively, either ATP or I B bind in a random manner (model 3). Any of these models could result in these initial velocity plots shown in Fig. 2. Therefore, verification of an ordered versus a random mechanism was addressed with inhibition studies. ADP and the I B -PI were used to assess the inhibition patterns. The effect of I B -PI on the activity of rhikk-1/rhikk-2 heterodimer is shown in Fig. 3. Double-reciprocal plots of 1/rate versus 1/I B peptide substrate intersect on the y axis, indicating that the inhibition is competitive with respect to the I B peptide (Fig. 3A). In contrast, increasing concentrations of ATP did not reduce the inhibition of rhikk- 1/rhIKK-2 heterodimer activity by the I B -PI (Fig. 3B). Thus these data indicate that the I B -PI is non-competitive with ATP. This inhibition pattern eliminates an ordered sequential mechanism with ATP binding first because the inhibition pattern with respect to ATP would have been uncompetitive. Additionally, we have shown previously (28) that ADP competitively inhibited rhikk-1/rhikk-2 heterodimer with respect to ATP but was non-competitive with respect to the I B peptide substrate (28). These data again suggest a random sequential mechanism and rule out an ordered sequential mechanism with I B peptide binding first, because in that case, ADP would have been an uncompetitive inhibitor with respect to the I B peptide. Fig. 3C summarizes the inhibition pattern of ADP and I B -PI versus the ATP and I B sites and reports the apparent K i values of inhibition. Note that the K i values for ADP and I B -PI, although lower than the respective IC 50 values, are similar regardless of whether ATP or I B peptide is used as the variable substrate. Taken together, these inhibition studies clearly eliminate an ordered sequential mechanism with either ATP binding first or I B peptide binding first. Furthermore, these data show that the rhikk-1/rhikk-2 heterodimer exhibits a random sequential mechanism where either ATP or I B peptide can bind to the free enzyme. It is also clear from Fig. 3C that the value derived from global fitting of the data in Fig. 2 is close to 1 (0.99) with the K ATP 0.18 M and the K I B 0.12 M. This suggests that the affinity of each substrate for the free enzyme is comparable with their affinity for the binary complex. Thus, the binding of one substrate to the enzyme does not affect the affinity of the other. Our results with the rhikk-1/rhikk-2 heterodimer are similar to the mechanism reported previously (9, 14) for both rhikk-1 homodimer and rhikk-2 homodimer which are summarized for convenience in Table I. However, these results differ from those reported for a native IKK complex partially isolated from HeLa FIG. 3. Inhibition of rhikk-1/rhikk-2 heterodimer by Asp- Ser(PO 3 H 2 )-Gly-Leu-Asp-Ser-Met. Double-reciprocal plots of 1/rate versus 1/(I B ) (A) or 1/(ATP) (B) were generated at various I B peptide inhibitor (Asp-Ser(PO 3 H 2 )-Gly-Leu-Asp-Ser-Met) concentrations as follows: A, 0(E), 10 ( ), 20 ( ), and 40 M (f); B, 0(E), 10 ( ), and 20 M ( ). Assays were performed as described under Experimental Procedures. C shows the kinetic constants and their inhibition patterns by ADP and a peptide inhibitor (Asp-Ser(PO 3 H 2 )-Gly-Leu-Asp- Ser-Met). S3 cells, also depicted in Table I. The IKK complex shows an value of 0.11, indicative of a positive co-operative interaction, where the binding of one substrate to the enzyme significantly enhances the affinity for the other substrate. Also note from Table I that the dissociation constant for K ATP 7.3 M is higher than our finding with respect to the rhikk-1/ikk-2 heterodimer (K ATP 0.18 M) or those observed for either of the homodimers, rhikk-1 and rhikk-2 (K ATP 0.08 M and K ATP 0.13 M, respectively, Table I). Such differences between values for and K ATP for the isolated IKK complex compared with the rhikks suggest that other component(s) in the isolated IKK complex fraction may influence the substrate binding as well as the enzyme mechanism. The difference in values may also reflect differences in assay conditions (Table I) as well as differences in the state of kinase activation by phosphorylation obtained from expression conditions (14) or from TNF- -stimulated HeLa S3 cells (9). Recently, two IKK-related kinases, TBK-1 and IKK-i, have been reported (23 27). TBK-1 and IKK-i are 67% identical to each other in the kinase domain but exhibit only 30% homology to the kinase domains of IKK-1 and IKK-2 (23 27). The recombinant proteins, rhtbk-1 and rhikk-i, are enzymatically distinct from rhikk-2 and rhikk-1 (42). Unlike IKK-1 and IKK-2, TBK-1 and IKK-i specifically phosphorylate Ser 36 but not Ser 32 of the I B peptide (23 27, 42). These two kinases reside in

6 Kinetic Mechanisms of Recombinant Human I B Kinases TABLE I Kinetic constants of rhikk-1, rhikk-2, or IKK complex from HeLa S3 cells and their inhibition patterns by ADP and peptide inhibitors from published work (9, 14) The abbreviations used are: C, competitive; NC, noncompetitive. Kinase Kinetic constants value K ATP K I B Inhibitor M Inhibition patterns Varied substrate Inhibition pattern rhikk-1 a ADP ATP C 0.16 ADP I B NC 0.42 Peptide I B C Peptide ATP NC rhikk-2 a ADP ATP C 0.15 ADP I B NC 0.35 Peptide I B C Peptide ATP NC IKK complex b a From Peet and Li (14). Peptide inhibitor peptide corresponding to amino acids of I B (Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg- His-Asp-Ser 32 -Gly-Leu-Asp-Ser 36 -Met-Lys-Asp-Glu-Glu), substrate His 6 -tagged thioredoxin-i B -(1 54) fusion protein. Assayed samples were analyzed by acid precipitation on a 96-well microtiter plate followed by liquid scintillation counting (13). b From Burke et al. (9). Peptide inhibitors peptide corresponding to amino acids of I B (Leu-Asp-Asp-Arg-His-Asp-Ser 32 -Gly-Leu- Asp-Ser 36 -Met-Lys-Asp-Glu-Glu-Lys), and peptide corresponding to amino acids of I B (MLPESEDEESYDTESEFTEFTEDEL), substrate GST-I B -(1 317) fusion protein. Assayed samples were analyzed by SDS-PAGE. The bands were detected by PhosphorImager and the radioactivities were determined using the ImageQuant software (9). K i M unique protein complexes distinct from the IKK signalosome within the cell as demonstrated by immunoprecipitation studies (23 27). However, like IKK-1 and IKK-2, both kinases are thought to play a role in the activation of NF- B (reviewed in Refs. 1 and 23 27). We characterized each of these recombinant IKK isoforms with respect to their kinetic mechanism. Fig. 4 shows the two substrate profile analyses of rhikk-i and rhtbk-1 using either the I B peptide (Fig. 4, A and C)orATP (Fig. 4, B and D) as the variable substrate. Unlike the pattern seen with the rhikk-1/rhikk-2 heterodimer, the family of double-reciprocal plots for IKK-i and TBK-1 appears to be parallel. As in the case of rhikk-i, the family of double-reciprocal plots intersects far to the left of the 1/rate axis and far below the 1/I B or 1/ATP axis. The seemingly parallel lines, both in the case of rhikk-i and rhtbk-1, deviate from a typical sequential mechanism and resemble the parallel plots expected of a pingpong mechanism (Scheme I, model 4). If indeed the kinetic mechanism for IKK-i and TBK-1 was ping-pong, product inhibition studies would reveal that ADP, the first product to be released, would be competitive with respect to I B (because ADP and I B would have access to the same form of the enzyme). Additionally, ADP would show noncompetitive or mixed type inhibition with respect to ATP. However, as seen in Fig. 5 for rhikk-i and for rhtbk-1, the inhibition of pattern of ADP was the exact opposite. Thus, ADP was competitive with respect to ATP both in the case of rhikk-i (Fig. 5A) and rhtbk-1 (Fig. 5C) and non-competitive with respect to the I B site for both rhikk-i (Fig. 5B) and rhtbk-1 (Fig. 5D). This would only be possible when a ternary complex consisting of ATP, I B peptide, and the enzyme was formed, which therefore suggests a sequential mechanism. The fact that ADP shows non-competitive inhibition with respect to the I B peptide also eliminates an ordered sequential mechanism with the I B peptide binding first. If this were the case, ADP would have been uncompetitive with respect to I B peptide. This inhibition pattern does not, however, distinguish if the mechanism is random sequential or ordered sequential with ATP binding first. To further distinguish between these possibilities, we next examined the inhibition pattern with the I B -PI at the I B site. The double-reciprocal plots of 1/rate versus 1/I B peptide at various fixed concentrations of the I B -PI yielded straight lines that intersected on the y axis, confirming its being a competitive inhibitor with respect to the I B peptide both in the case of rhikk-i (Fig. 6A) and rhtbk-1 (Fig. 6C). When ATP was the variable substrate, the series of double-reciprocal plots yielded straight lines that intersected far to the left of the 1/rate axis either above the 1/ATP axis indicating a mixed type of non-competitive inhibition, as in the case of rhikk-i (Fig. 6B), or at the 1/ATP axis indicating non-competitive inhibition, as in the case of rhtbk-1 (Fig. 6D). This inhibition pattern confirms a random sequential mechanism for rhikk-i and rhtbk-1 and eliminates an ordered sequential mechanism with ATP binding first, because, in that case, the I B -PI would have been uncompetitive with respect to ATP. The results of inhibitor studies with rhikk-i and rhtbk-1 are summarized in Table II. Although the binding constant K ATP values of all kinases are in the range for published data (from 0.1 to 30 M), the K I B values for rhtbk-1 and rhikk-i are higher than those reported for rhikk-2 homodimer and rhikk-1/rhikk-2 heterodimer (10, 13, 14, 28; Table I and Fig. 3C). These observations were confirmed by the fact that K m values of I B peptide for rhtbk-1 and rhikk-i are over 200- fold higher than those reported for rhikk-2 homodimer and rhikk-1/rhikk-2 heterodimer (10, 13, 14, 28, 42). These data suggest that rhtbk-1 and rhikk-i have less affinity toward I B peptide than rhikk-1/rhikk-2 heterodimer. Because it has been reported that endogenous IKK-i is associated with another kinase in a high molecular weight complex and the complex could be activated to phosphorylate both Ser 32 and Ser 36 of I B peptide when the cells were stimulated with phorbol ester (26), it is possible that Ser 32 of I B has to be phosphorylated first by the other kinase, before it can be an efficient substrate for IKK-i. To test this hypothesis, we examined the activity of rhtbk-1 and rhikk-i using phosphorylated I B peptide at position Ser 32 as a substrate (Gly-Leu-Lys- Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser 32 (PO 3 H 2 )- Gly-Leu-Asp-Ser 36 -Met-Lys-Asp-Glu-Glu). However, no significant change for K m and k cat values for rhtbk-1 and rhikk-i could be demonstrated with this I B peptide phosphorylated at Ser 32 (42). The possibility still remains that IKK-i and TBK-1 may have better affinity toward full-length I B in the presence of NF- B. Because it has been reported that overexpressed TBK-1 can activate the IKK complex in mammalian cells by phosphorylating IKK-2 in the activation loop (24), we evaluated a 17 amino acid peptide (Ala 170 -Lys-Glu-Leu-Asp-Gln-Gly-Ser-Leu- Cys-Thr-Ser-Phe-Val-Gly-Thr-Leu-Gln 187 ) derived from the activation loop of IKK-2. We have shown that this IKK-2 activation loop peptide was a more efficient substrate for both rhikk-i and rhtbk-1 with a 6 8-fold higher catalytic efficiency and a greater than 20-fold lower K m value compared with the I B peptide (42). The k cat /K m values obtained for the IKK-2 loop peptide were 34.12, 41.95, and h 1 M 1 for rhikk-i, rhtbk-1, and rhikk-2, respectively (42). By using this IKK-2 loop peptide, we also examined the kinetic mechanisms of rhtbk-1 and rhikk-i. We measured initial velocity patterns of rhikk-i and rhtbk-1 using different fixed concentrations of this IKK-2 loop peptide substrate to determine whether the affinity of ATP to the enzyme-peptide binary complex was reduced as in the case of I B peptide substrate. The initial velocity plots for rhikk-i intersected below the x axis but with an value of 4 compared with greater than 50 when I B peptide was used as the

7 12556 Kinetic Mechanisms of Recombinant Human I B Kinases FIG. 4.Kinetic patterns for rhikk-i (A and B) and rhtbk-1 (C and D) with various concentrations of I B peptide and ATP. A, double-reciprocal plots of 1/rate versus 1/(I B ) were generated at five fixed ATP concentrations of 1.25 ( ), 2.5 ( ), 5.0 (f), and 10 M ( ). B, double-reciprocal plots of 1/rate versus 1/(ATP) were generated at four fixed I B peptide concentrations of 50 (E), 100 ( ), 200 ( ), 500 (f), and 1000 M ( ). C, double-reciprocal plots of 1/rate versus 1/(I B ) were generated at five fixed ATP concentrations of 0.62 ( ), 1.25 ( ), 2.5 (f), and 5.0 M ( ). D, double-reciprocal plot of 1/rate versus 1/(ATP) were generated at four fixed I B peptide concentrations of 31.2 (E), 62.5 ( ), 125 ( ), 250 (f), and 500 M ( ). Assays were performed as described under Experimental Procedures. FIG. 5.Inhibition of rhikk-i (A and B) and rhtbk-1 (C and D) by ADP. Double-reciprocal plots of 1/rate versus 1/(ATP) and 1/(I B ) at fixed ADP concentrations as indicated. Concentrations of ADP used are as follows: A and B,0(E), 6.25 ( ), 12.5 ( ), and 25 M (f); C and D, 0(E), 50 ( ), 100 ( ), and 250 M.(f). Assays were performed as described under Experimental Procedures. substrate (data not shown). Similar results were noted for rhtbk-1, where the value using the IKK-2 loop peptide was 2 compared with greater than 10 for the I B peptide substrate (data not shown). These data suggest that the affinity of ATP for the enzyme is not decreased to the same extent with the IKK-2 loop peptide as with the I B peptide. This is consistent with the observation that the K m(app) for ATP in the presence of saturating concentrations of the IKK-2 loop peptide is fold lower than in the presence of saturating concentrations of I B peptide (data not shown). Thus, the kinetic mechanism of rhikk-1/rhikk-2 heterodimer, rhikk-i, and rhtbk-1 remain the same with different peptide substrates as phospho-acceptors, although the kinetic constants did vary. The K i values for ADP of and 8.30 M for rhtbk-1 and rhikk-i were obtained, respectively (Table II). Note that the K i of ADP versus the ATP site is 5 6-fold lower than the K i for ADP versus the I B site, both with respect to rhikk-i and rhtbk-1. Likewise, the K i of the I B -PI for the I B site is 5 6-fold lower than the K i with respect to the ATP site, again with respect to rhikk-i and rhtbk-1. These findings, combined with the typical initial velocity plots are consistent with the observation that the value is 10 for both rhikk-i and rhtbk-1 (Table II). As described in Scheme II, an value greater than 10 implies that the affinity of either ATP or I B peptide for the free enzyme is higher than their affinity to the binary complex of either the enzyme and I B or the enzyme and ATP, respectively. Thus whereas the binding of one substrate has no effect on the affinity of the other for rhikk-1/ rhikk-2 heterodimer, the binding of ATP significantly decreased the affinity of I B for rhikk-i and rhtbk-1. Parallel lines obtained in the initial velocity studies have been observed for other protein kinases that exhibit sequential mechanisms when inhibition studies are conducted (39). Thus Gold and Segel (39) showed that a protein kinase isolated from

8 Kinetic Mechanisms of Recombinant Human I B Kinases FIG. 6.Inhibition of rhikk-i (A and B) and rhtbk-1 (C and D) by Asp- Ser(PO 3 H 2 )-Gly-Leu-Asp-Ser-Met. Double-reciprocal plots of 1/rate versus 1/(ATP) and 1/(I B ) at fixed peptide inhibitor (Asp-Ser(PO 3 H 2 )-Gly-Leu-Asp- Ser-Met) concentrations as indicated. Concentrations of peptide inhibitor used are as follows: A and B,0(E), 100 ( ), 200 ( ), and 500 M (f); C and D, 0 (E), 50 ( ), 100 ( ), and 250 M (f). Assays were performed as described under Experimental Procedures. TABLE II Kinetic constants of rhikk-i or rhtbk-1 and their inhibition patterns by ADP and a peptide inhibitor The abbreviations used are: C, competitive; NC, noncompetitive; M, mixed type. Kinase Kinetic constants value K ATP K I B Inhibitor a substrate Varied Inhibition patterns Inhibition pattern rhikk-i ADP ATP C 8.30 ADP I B NC Peptide I B C Peptide ATP M (K i ) rhtbk ADP ATP C ADP I B NC Peptide I B C Peptide ATP NC a Peptide inhibitor Asp-Ser(PO 3 H 2 )-Gly-Leu-Asp-Ser-Met; I B substrate peptide corresponding to amino acids of I B (Gly- Leu-Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser 32 -Gly-Leu- Asp-Ser 36 -Met-Lys-Asp-Glu-Glu). Assays were performed as described under Experimental Procedures. K i M TABLE III Kinetic mechanism of IKKs compared with other published kinases (9, 14, 39 49) Kinases Suggested mechanism Refs. rhikk-i Random sequential This work rhtbk-1 Random sequential This work rhikk-1/rhikk-2 heterodimer Random sequential This work IKK complex from HeLa S3 cells Random sequential 9 rhikk-1 homodimer Random sequential 14 rhikk-2 homodimer Random sequential 14 Protein kinase Random sequential 39 Phosphofructose kinase Random sequential p38 MAPK Ordered sequential 36, 43 camp-dependent protein kinase Random sequential Ordered sequential C-terminal Src kinase Random sequential 46 pp60 c-src Random sequential 47 Epidermal growth factor receptor Random sequential Ordered sequential Neurospora crassa demonstrated a random sequential mechanism despite the parallel initial velocity patterns. Here, the was significantly greater than 1, suggesting that the binding of one substrate greatly decreased the affinity of the enzyme for the second substrate. Direct equilibrium binding studies would confirm or negate the possibility of steric hindrance of one substrate affecting the binding of the other. Thus rhikk-i and rhtbk-1 exhibit random sequential mechanisms similar to rhikk-1/rhikk-2 heterodimer. These results were not surprising because it has been suggested that parallel initial velocity patterns can occur in rapidly equilibrating random systems when the binding of one substrate strongly inhibits the binding of the other (i.e. 1) (35). A product will be competitive with whichever substrate occupies the same binding site and a non-competitive or mixed type inhibition with whichever substrate it forms a dead-end complex. The noncompetitive or mixed type patterns will appear uncompetitive if the binding of the product does not strongly inhibit the binding of the co-substrate and vice versa (35). For example, the phosphofructokinase of Dictyostelium discoideum exhibited parallel reciprocal plots when either ATP or fructose 6-phosphate is varied (40, 41). Furthermore, a non-rapid equilibrium random system will also yield seemingly parallel plots if the rate constants for the release of substrates are lower than V max (41). Table III summarizes the published kinetic mechanisms of several known kinases (9, 14, 39 49). Similar to IKKs, several tyrosine kinase including C-terminal Src kinase and pp60 c-src showed random sequential mechanisms (44, 45). Differing kinetic data generated with various substrates is not surprising because other investigators have shown that the phospho-acceptor substrate could affect initial velocity plots as well as the enzyme mechanism (44 49). This is illustrated in the case of the kinetic mechanism of p38 kinase reported by two different laboratories using different phospho-acceptor substrates. Whereas LoGrasso et al. (30) obtained initial velocity plots intersecting well below the x axis suggesting an value of greater than 1 using glutathione S-transferase-activating transcription factor 2 as the substrate, Chen et al. (43) used a 16-amino acid synthetic peptide derived from the epidermal growth factor receptor phosphorylation site (Thr 669 )asasubstrate and showed that the initial velocity plots intersected on the x axis which suggested an value close to 1. In the former