Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase

The kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase has been investigated employing the heptapeptide Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as substrate. Initial velocity measurements performed over a wide range of ATP and Kemptide concentrations indicated that the reaction follows a sequential mechanistic pathway. In line with this, the results of product and substrate inhibition studies, the patterns of dead end inhibition obtained employing the nonhydrolyzable ATP analogue, AMP X PNP (5'-adenylylimidodiphosphate), and equilibrium binding determinations, taken in conjunction with the patterns of inhibition observed with the inhibitor protein of the cAMP-dependent protein kinase that are reported in the accompanying paper (Whitehouse, S., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3682-3692), are best fit by a steady state Ordered Bi-Bi kinetic mechanism. Although the inhibition patterns obtained employing the synthetic peptide analogue in which the phosphorylatable serine was replaced by alanine were apparently incompatible with this mechanism, these inconsistencies appear to be due to some element of the structure of this latter peptide such that it is not an ideal dead end inhibitor substrate analogue. The data presented both here and in the accompanying paper suggest that both this substrate, analogue and the ATP analogue, AMP X PNP, do not fully mimic the binding of Kemptide and ATP, respectively, in their mechanism of interaction with the protein kinase. It is proposed that, as with some other kinase reactions, the configuration of the terminal anhydride bond of ATP assumes a conformation once the nucleotide is bound to the protein kinase that assists in the binding of either Kemptide or the inhibitor protein but not the alanine-substituted peptide and that AMP X PNP, because of its terminal phosphorylimido bond, cannot assume this conformation which favors protein (or peptide) binding.     

Kinetic mechanism of the type II calmodulin-dependent protein kinase: studies of the forward and reverse reactions and observation of apparent rapid-equilibrium ordered binding

The kinetic reaction mechanism of the type II calmodulin-dependent protein kinase was studied by using its constitutively active kinase domain. Lacking regulatory features, the catalytic domain simplified data collection, analysis, and interpretation. To further facilitate this study, a synthetic peptide was used as the kinase substrate. Initial velocity measurements of the forward reaction were consistent with a sequential mechanism. The patterns of product and dead-end inhibition studies best fit an ordered Bi Bi kinetic mechanism with ATP binding first to the enzyme, followed by binding of the peptide substrate. Initial-rate patterns of the reverse reaction of the kinase suggested a rapid-equilibrium mechanism with obligatory ordered binding of ADP prior to the phosphopeptide substrate; however, this apparent rapid-equilibrium ordered mechanism was contrary to the observed inhibition by the phosphopeptide which is not supposed to bind to the kinase in the absence of ADP. Inspection of product inhibition patterns of the phosphopeptide with both ATP and peptide revealed that an ordered Bi Bi mechanism can show initial-rate patterns of a rapid-equilibrium ordered system when a Michaelis constant for phosphopeptide, Kip, is large relative to the concentration of phosphopeptide used. Thus, the results of this study show an ordered Bi Bi mechanism with nucleotide binding first in both directions of the kinase reaction. All the kinetic constants in the forward and reverse directions and the Keq of the kinase reaction are reported herein. To provide theoretical bases and diagnostic aid for mechanisms that can give rise to typical rapid-equilibrium ordered kinetic patterns, a discussion on various sequential cases is presented in the Appendix.     

Kinetic mechanism of glutathione synthetase from Arabidopsis thaliana

Glutathione synthetase (GS) catalyzes the ATP-dependent formation of the ubiquitous peptide glutathione from gamma-glutamylcysteine and glycine. The bacterial and eukaryotic GS form two distinct families lacking amino acid sequence homology. Moreover, the detailed kinetic mechanism of the bacterial and the eukaryotic GS remains unclear. Here we have overexpressed Arabidopsis thaliana GS (AtGS) in an Escherichia coli expression system and purified the recombinant enzyme for biochemical characterization. AtGS is functional as a homodimeric protein with steady-state kinetic properties similar to those of other eukaryotic GS. The kinetic mechanism of AtGS was investigated using initial velocity methods and product inhibition studies. The best fit of the observed data was to the equation for a random Ter-reactant mechanism in which dependencies between the binding of some substrate pairs were preferred. The binding of either ATP or gamma-glutamylcysteine increased the binding affinity of AtGS for the other substrate by 10-fold. Likewise, the binding of ATP or glycine increased binding affinity for the other ligand by 3.5-fold. In contrast, binding of either glycine or gamma-glutamylcysteine causes a 6.7-fold decrease in binding affinity for the second molecule. Product inhibition studies suggest that ADP is the last product released from the enzyme. Overall, these observations are consistent with a random Ter-reactant mechanism for the eukaryotic GS in which the binding order of certain substrates is kinetically preferred for catalysis.     

Kinetic mechanism of AKT/PKB enzyme family

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.     

Kinetic mechanisms of Ca++/calmodulin dependent protein kinases

Many of the cellular responses to Ca⁺⁺ signaling are modulated by a family of multifunctional Ca⁺⁺/calmodulin dependent protein kinases (CaMKs): CaMK I, CaMK II and CaMK IV. In order to further understand the role of CaMKs, we investigated the kinetic mechanism of CaMK II isozymes in comparison with those of CaMK I and CaMK IV by analyzing their steady state kinetics using phospholamban as a phosphoacceptor. The results indicated that (a) the CaMK family’s reaction mechanisms were of the sequential type in which all substrates must bind to enzyme before any product is released; (b) CaMK I and CaMK IV exhibited random sequential mechanism where either phospholamban or ATP can bind to the free enzyme; (c) the data of product inhibition for CaMK IIs best fit with an Ordered Bi Bi mechanism in which phospholamban is the first substrate to bind and ADP is the last product to be released; and (d) the constant α (ratio of apparent dissociation constants for binding peptide in the presence and absence of the second ligand) of all isozymes for ATP and peptide was higher than 1 indicating that the binding of phospholamban to CaMK decreased the enzyme’s affinity toward ATP.     

Kinetic mechanism for human Rho-Kinase II (ROCK-II)

Rho-Kinase is a serine/threonine kinase that is involved in the regulation of smooth muscle contraction and cytoskeletal reorganization of nonmuscle cells. While the signal transduction pathway in which Rho-Kinase participates has been and continues to be extensively studied, the kinetic mechanism of Rho-Kinase-catalyzed phosphorylation has not been investigated. We report here elucidation of the kinetic mechanism for Rho-Kinase by using steady-state kinetic studies. These studies used the kinase domain of human Rho-Kinase II (ROCK-II 1-534) with S6 peptide (biotin-AKRRRLSSLRA-NH(2)) as the phosphorylatable substrate. Double-reciprocal plots for two-substrate kinetic data yielded intersecting line patterns with either ATP or S6 peptide as the varied substrate, indicating that Rho-Kinase utilized a ternary complex (sequential) kinetic mechanism. Dead-end inhibition studies were used to investigate the order of binding for ATP and the peptide substrate. The ATP-competitive inhibitors AMP-PCP and Y-27632 were noncompetitive inhibitors versus S6 peptide, and the S6 peptide analogue S6-AA (acetyl-AKRRRLAALRA-NH(2)) was a competitive inhibitor versus S6 peptide and a noncompetitive inhibitor versus ATP. These results indicated a random order of binding for ATP and S6 peptide.     

Kinetic studies of Cdk5/p25 kinase: phosphorylation of tau and complex inhibition by two prototype inhibitors

Cdk5/p25 is a member of the family of cyclin-dependent, Ser/Thr kinases and is thought to play a causal role in Alzheimer's disease (AD) due to its ability to phosphorylate the protein tau, and thus promote the latter's aggregation into intraneuronal tangles. Given this, we and others are seeking inhibitors of cdk5/p25 as possible disease-modifying therapeutics for AD. In this paper, we first report the kinetic mechanism for the cdk5/p25-catalyzed phosphorylation of tau and histone H-1-derived peptide (H1P). These studies served as a necessary kinetic backdrop for investigations of the mechanism of inhibition by prototype inhibitors N4-(6-aminopyrimidin-4-yl)-sulfanilamide (APS) and 1-(5-cyclobutyl-thiazol-2-yl)-3-isoquinolin-5-yl-urea (CTIU). We found that the cdk5/p25-catalyzed phosphorylation of tau follows a rapid equilibrium, random kinetic mechanism, as evidenced by initial velocity analysis indicating sequential addition of tau and ATP, and studies of the mechanism of inhibition by substrate analogue AMP, product ADP, and analogues of peptide substrate H1P. Identical mechanistic conclusions were drawn when H1P was the phosphoryl acceptor. Subsequent studies of inhibition by APS and CTIU revealed that both compounds can bind to all four steady-state forms of the enzyme, to form the complexes E:I, E:I:tau, E:I:ATP, and E:I:tau:ATP. These results contrast with reported claims that APS and CTIU are competitive inhibitors of the binding of ATP.     

Divalent metal ions influence catalysis and active-site accessibility in the cAMP-dependent protein kinase.

Phosphorylation of the peptide LRRASLG by the catalytic subunit of cAMP-dependent protein kinase was measured in the presence of various divalent metals to establish the role of electrophiles in the kinetic mechanism. Under conditions of low or high metal concentrations, the apparent second-order rate constant, kcat/Kpeptide, and the maximal rate constant, kcat, followed the trend Mg2+ > Co2+ > Mn2+. Competitive inhibition studies indicate that the former effect is not due to destabilization of the substrate complex, E.ATP.S. The effects of solvent viscosity on the steady-state kinetic parameters were interpreted according to a simple mechanism involving substrate binding, phosphotransfer, and product release steps and two metal chelation sites in the nucleotide pocket. Decreases in kcat and kcat/Kpeptide result mostly from attenuations in the dissociation rate constant for ADP and the association rate constant for the substrate, respectively. Decreases in the phosphoryl transfer rate constant have only negligible to moderate effects on these parameters. The low observed values for the association rate constant of the substrate indicate that the metals control the concentration of the productive binary form, Ea.ATP, and indirectly the accessibility of the active site. By comparison, Mg2+ is the best divalent metal catalyst because it uniformly lowers the transition state energies for all steps in the kinetic mechanism, permitting maximum flux of substrate to product. The data suggest that cAMP-dependent protein kinase uses metal ions to serve multiple roles in facilitating phosphotransfer and accelerating substrate association and product dissociation.     

Kinetic mechanism of fully activated S6K1 protein kinase

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His(6)-S6K1alphaII(DeltaAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (K(d)(ATP)=5-6 microM) was followed by low affinity binding of Tide (K(d)(Tide)=180 microM), and values of K(m)(ATP)=5-6 microM and K(m)(Tide)=4-5 microM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.     

Kinetic analysis of protein kinase C: product and dead-end inhibition studies using ADP, poly(L-lysine), nonhydrolyzable ATP analogues, and diadenosine oligophosphates

The kinetic mechanism of protein kinase C (PKC) was analyzed via inhibition studies using the product MgADP, the nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-imidotriphosphate) (MgAMPPNP), the peptide antagonist poly(L-lysine), and several naturally occurring ATP analogues that are produced in rapidly growing cells, i.e., the diadenosine oligophosphates (general structure: ApnA; n = 2-5). By use of histone as the phosphate acceptor, the inhibition of PKC by MgAMPPNP and MgADP was found to be competitive vs MgATP (suggesting that these compounds bind to the same enzyme form), whereas their inhibition vs histone was observed to be noncompetitive. In contrast, the inhibition by poly(L-lysine) appeared competitive vs histone but uncompetitive vs MgATP, which is consistent with a model wherein MgATP binding promotes the binding of poly(L-lysine) or histone. With the diadenosine oligophosphates, the degree of PKC inhibition was found to increase according to the number of intervening phosphates. The diadenosine oligophosphates Ap4A and Ap5A were the most effective antagonists of PKC, with Ap5A being approximately as potent as MgADP and MgAMPPNP. However, as opposed to MgADP and MgAMPPNP, Ap4A and Ap5A appear to act as noncompetitive inhibitors vs both MgATP and histone, suggesting that they can interact at several points in the reaction pathway. These studies support the concept of a steady-state mechanism where MgATP binding preferentially precedes that of histone, followed by the release of phosphorylated substrate and MgADP. Furthermore, these results indicate a differential interaction of the diadenosine oligophosphates with PKC, when compared to other adenosine nucleotides.     

Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in k_cat for PKN1 and PKN2, and a decrease in peptide K_M for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (K_i<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.     

Kinetic characterization of the peptidase activity of Escherichia coli Lon reveals the mechanistic similarities in ATP-dependent hydrolysis of peptide and protein substrates

Lon is an ATP-dependent protease that degrades unstructured proteins. In this study, we have examined the ATP dependency of Escherichia coli Lon catalyzing the hydrolysis of a defined fluorogenic peptide known as S3. Steady-state velocity analyses of S3 degradation in the presence of ATP, or the nonhydrolyzable ATP analogue AMPPNP, indicate a sequential mechanism, and the k(cat) of the reaction was 7-fold higher in the presence of ATP. Comparing the pre-steady-state time courses of the ATP- versus AMPPNP-mediated S3 hydrolysis reveals that ATP hydrolysis accelerates a slow step before the chemical cleavage of peptide. Product inhibition studies indicate that ADP is competitive versus ATP but noncompetitive versus the S3 substrate. In the absence of S3, Lon exhibits a 10-20-fold higher affinity for ADP than ATP. However the S3 substrate weakens the affinity of Lon for ADP by 7-19-fold, indicating that this peptide also promotes ADP/ATP exchange in Lon similar to that observed with protein substrates. The hydrolyzed peptide product, Pd1, exhibited noncompetitive inhibition versus both ATP and S3 substrates. Together with the small change in the K(i) of Pd1 at increasing S3 concentrations, the Pd1 inhibition data support the existence of an isomechanism in Lon catalyzing the hydrolysis of S3 in the presence of ATP or AMPPNP. Upon the basis of the collected data, an extended kinetic mechanism is proposed for the ATP-dependent peptidase mechanism of Lon.     

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases. Kinetic and equilibrium binding measurements have been used to evaluate the proposed mechanism. Equilibrium binding studies show that APS, PAPS, ADP, and the ATP analog AMPPNP each bind at a single site per subunit; thus, substrates can bind in either order. When ATPgammaS replaces ATP as substrate the V(max) is reduced 535-fold, the kinetic mechanism is sequential at each APS concentration, and substrate inhibition is not observed. The results indicate that substrate inhibition arises from a kinetic phenomenon in which product formation from ATP binding to the E. APS complex is much slower than paths in which product formation results from APS binding either to the E. ATP complex or to E-P. APS kinase requires divalent cations such as Mg(2+) or Mn(2+) for activity. APS kinase binds one Mn(2+) ion per subunit in the absence of substrates, consistent with the requirement for a divalent cation in the phosphorylation of APS by E-P. The affinity for Mn(2+) increases 23-fold when the enzyme is phosphorylated. Two Mn(2+) ions bind per subunit when both APS and the ATP analog AMPPNP are present, indicating a potential dual metal ion catalytic mechanism.     

Biochemical characterization of human SET and MYND domain-containing protein 2 methyltransferase

SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s(-1), with K(M)s for AdoMet and the p53 peptide of 0.031 ± 0.01 μM and 0.68 ± 0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.     

Kinetic mechanism of the beta-lactam synthetase of Streptomyces clavuligerus

Streptomyces clavuligerus beta-lactam synthetase (beta-LS) was recently demonstrated to catalyze an early step in clavulanic acid biosynthesis, the ATP/Mg(2+)-dependent intramolecular closure of the beta-amino acid N(2)-(carboxyethyl)-L-arginine (CEA) to the monocyclic beta-lactam deoxyguanidinoproclavaminic acid (DGPC). Here we investigate the steady-state kinetic mechanism of the beta-LS-catalyzed reaction to better understand this unprecedented secondary metabolic enzyme. Initial velocity patterns were consistent with a sequential ordered bi-ter kinetic mechanism. Product inhibition studies with PP(i) and DGPC demonstrated competitive inhibition versus their cognate substrates ATP and CEA, respectively, and noncompetitive inhibition against their noncognate substrates. To clarify the order of substrate binding, the truncated substrate analogue N(2)-(carboxymethyl)-L-arginine was synthesized and demonstrated uncompetitive inhibition versus ATP and competitive patterns versus CEA. These data are consistent with ordered substrate binding, with ATP binding first, an abortive enzyme-DGPC complex, and PP(i) released as the last product. The pH dependence of V and V/K was determined and suggests that residues with a pK of 6.5 and 9.3 must be ionized for optimal activity. These observations were considered in the context of investigations of the homologous primary metabolic enzyme asparagine synthetase B, and a chemical mechanism is proposed that is consistent with the kinetic mechanism.     

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.     

Kinetics and mechanism of angiotensin phosphorylation by the transforming gene product of Rous sarcoma virus

We have studied steady state kinetics of phosphorylation of [Val5]angiotensin II by pp60src, the transforming gene product of Rous sarcoma virus. Results of initial rate studies at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 microM and 0.24 mM, respectively, and Vmax was 1.0 nmol/min/mg. The end product ADP and the ATP analog AMP-PNP were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [delta Phe4]angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations, but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. We also presented evidence that, while pp60src contained essential histidine and/or lysine residues in its active site, the mechanism does not involve a phosphoryl enzyme intermediate.     

Kinetic mechanisms of IkappaB-related kinases (IKK) inducible IKK and TBK-1 differ from IKK-1/IKK-2 heterodimer

Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB 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 IkappaBalpha. 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 IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 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 IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.