The complete pathway for ERK2-catalyzed reaction. Evidence for an iso random Bi Bi mechanism

In the present study, the enzymatic mechanism of ERK2 is re-examined by a combination of steady-state kinetic studies in the absence and presence of viscosogenic agents. Kinetic studies carried out in various concentrations of sucrose revealed that both k(cat) and k(cat)/K(m) for either ATP or EtsDelta138 were highly sensitive to solvent viscosity, suggesting that the rapid equilibrium assumption is not valid for the phosphorylation of protein substrate by ERK2. Furthermore, the kinetic analysis with the minimal random Bi Bi reaction mechanism is shown to be inconsistent with the principle of the detailed balance. This inconsistent calculation strongly suggests that there is isomerization of the enzyme-substrate ternary complex. The viscosity-dependent steady-state kinetic data are combined to establish a kinetic mechanism for the ERK2-catalyzed reaction that predicts initial reaction velocities under varying concentrations of ATP and substrate. These results complement previous structure-function studies of mitogen-activated protein kinases and provide important insight for mechanistic interpretation of the kinase functions.     

Properties and regulation of a transiently assembled ERK2.Ets-1 signaling complex

ERK2 is a proline-directed protein kinase that displays a high specificity for a single threonine (Thr-38) on the substrate Ets-1, which lies within the consensus sequence 36phi-chi-Thr-Pro39 (where phi is typically a small hydrophobic residue and chi appears to be unrestricted). Thr-38 lies in a long flexible N-terminal tail (residues 1-52), which also contains a second potential phosphorylation site, Ser-26. How Ets-1 binds ERK2 to promote the phosphorylation of Thr-38 while simultaneously discriminating against the phosphorylation of Ser-26 is unclear. To delineate the details of the molecular recognition of Ets-1 by ERK2, the binding of various mutants and truncations of Ets-1 were analyzed by fluorescence anisotropy. The data that were obtained support the notion that the N-terminal tail contains a previously unrecognized docking site that promotes the phosphorylation of Thr-38. This new docking site helps assemble the complex of Ets-1 and ERK2 and makes a similar contribution to the stabilization of the complex as does the pointed domain of Ets-1. The in vitro activation of ERK2 by MKK1 induces a large conformational transition of the activation segment (DFG-APE), but neither induces self-association of ERK2 nor destabilizes the stability of the ERK2.Ets-1 complex. This latter observation suggests that interactions intrinsic to the active site are not important for complex assembly, a notion further supported by the observation that the substitution of a number of different amino acids for Pro-39 does not destabilize the complex. Mutagenesis of ERK2 within loop 13 suggests that Ets-1 binds the substrate-binding groove. These data suggest that ERK2 uses two weak docking interactions to specifically assemble the complex, perhaps in doing so denying Ser-26 access to the active site. Displacement of residues 1-138 of Ets-1 (EtsDelta138) from ERK2 by the peptide N-QKGKPRDLELPLSPSL-C, derived from Elk-1, suggests that Ets-1 engages the D-recruitment site (beta7-beta8 reverse turn and the alphaD-alphaE helix) of ERK2. Displacement of EtsDelta138 from ERK2 by the peptide N-AKLSFQFPS-C derived from Elk-1 shows that EtsDelta138 communicates with the F-recruitment site of ERK2 also.     

Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.     

Steady-state kinetic mechanism of Ras farnesyl:protein transferase.

The steady-state kinetic mechanism of bovine brain farnesyl:protein transferase (FPTase) has been determined using a series of initial velocity studies, including both dead-end substrate and product inhibitor experiments. Reciprocal plots of the initial velocity data intersected on the 1/[s] axis, indicating that a ternary complex forms (sequential mechanism) and suggesting that the binding of one substrate does not affect the binding of the other. The order of substrate addition was probed by determining the patterns of dead-end substrate and product inhibition. Two nonhydrolyzable analogues of farnesyl diphosphate, (alpha-hydroxyfarnesyl)phosphonic acid (1) and [[(farnesylmethyl)hydroxyphosphinyl]methyl]phosphonic acid (2), were both shown to be competitive inhibitors of farnesyl diphosphate and noncompetitive inhibitors of Ras-CVLS. Four nonsubstrate tetrapeptides, CV[D-L]S, CVLS-NH2, N-acetyl-L-penicillamine-VIM, and CIFM, were all shown to be noncompetitive inhibitors of farnesyl diphosphate and competitive inhibitors of Ras-CVLS. These data are consistent with random order of substrate addition. Product inhibition patterns corroborated the results found with the dead-end substrate inhibitors. We conclude that bovine brain FPTase proceeds through a random order sequential mechanism. Determination of steady-state parameters for several physiological Ras-CaaX variants showed that amino acid changes affected the values of KM, but not those of kcat, suggesting that the catalytic efficiencies (kcat/KM) of Ras-CaaX substrates depend largely upon their relative binding affinity for FPTase.     

Expanding the repertoire of an ERK2 recruitment site: cysteine footprinting identifies the D-recruitment site as a mediator of Ets-1 binding

Many substrates of ERK2 contain a D-site, a sequence recognized by ERK2 that is used to promote catalysis. Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing one. This suggests that Ets-1 may contain a novel or cryptic D-site. To investigate this possibility a protein footprinting strategy was developed to elucidate ERK2-ligand interactions. Using this approach, single cysteine reporters were placed in the D-recruitment site (DRS) of ERK2 and the resulting ERK2 proteins subjected to alkylation by iodoacetamide. The ability of residues 1-138 of Ets-1 to protect the cysteines from alkylation was determined. The pattern of protection observed is consistent with Ets-1 occupying a hydrophobic binding site within the DRS of ERK2. Significantly, a peptide derived from the D-site of Elk-1, which is known to bind the DRS, exhibits a similar pattern of cysteine protection. This analysis expands the repertoire of the DRS on ERK2 and suggests that other targeting sequences remain to be identified. Furthermore, cysteine-footprinting is presented as a useful way to interrogate protein-ligand interactions at the resolution of a single amino acid.     

Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.     

Molecular determinants of substrate recognition in hematopoietic protein-tyrosine phosphatase

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183 and Tyr185 in the activation loop. Tyr185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a kcat/Km of 2.6 x 10(6) m(-1) s(-1). In contrast, the kcat/Km values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (approximately 20-fold increase in kcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (approximately 20-fold increase in kcat/Km) by organizing the catalytic residues with respect to Tyr(P)185 for optimal phosphoryl transfer.     

The mechanism of dephosphorylation of extracellular signal-regulated kinase 2 by mitogen-activated protein kinase phosphatase 3.

The mitogen-activated protein (MAP) kinase phosphatase-3 (MKP3) is a dual specificity phosphatase that specifically inactivates one subfamily of MAP kinases, the extracellular signal-regulated kinases (ERKs). Inactivation of MAP kinases occurs by dephosphorylation of Thr(P) and Tyr(P) in the TXY kinase activation motif. To gain insight into the mechanism of ERK2 inactivation by MKP3, we have carried out an analysis of the MKP3-catalyzed dephosphorylation of the phosphorylated ERK2. We find that ERK2/pTpY dephosphorylation by MKP3 involves an ordered, distributive mechanism in which MKP3 binds the bisphosphorylated ERK2/pTpY, dephosphorylates Tyr(P) first, dissociates and releases the monophosphorylated ERK2/pT, which is then subjected to dephosphorylation by a second MKP3, yielding the fully dephosphorylated ERK2. The bisphosphorylated ERK2 is a highly specific substrate for MKP3 with a k(cat)/K(m) of 3.8 x 10(6) m(-1) s(-1), which is more than 6 orders of magnitude higher than that for small molecule aryl phosphates and an ERK2-derived phosphopeptide encompassing the pTEpY motif. This strikingly high substrate specificity displayed by MKP3 may result from a combination of high affinity binding interactions between the N-terminal domain of MKP3 and ERK2 and specific ERK2-induced allosteric activation of the MKP3 C-terminal phosphatase domain.     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.     

Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA

A minimal kinetic mechanism for a trans-acting ribozyme derived from the HDV antigenomic RNA self-cleaving element was established from steady-state, pre-steady-state, single-turnover, and binding kinetics. Rate constants for individual steps, including substrate binding and dissociation, cleavage, and product release and binding, were measured at 37 degrees C at pH 8.0 in 10 mM Mg(2+) using oligonucleotides as either substrates, noncleavable analogues or 3' product mimics. A substrate containing a normal 3',5'-linkage was cleaved with a first-order rate constant (k(2)) of 0.91 min(-)(1). The association rate constant for the substrate to the ribozyme (2.1 x 10(7) M(-)(1) min(-)(1)) was at the lower range of the expected value for RNA duplex formation, and the substrate dissociated with a rate constant (1.4 min(-)(1)) slightly faster than that for cleavage. Thus the binary complex was not at equilibrium with free enzyme and substrate prior to the cleavage step. Following cleavage, product release was kinetically ordered in that the 5' product was released rapidly (>12 min(-)(1)) relative to the 3' product (6.0 x 10(-)(3) min(-)(1)). Rapid 5' product release and lack of a demonstrable binding site for the 5' product could contribute to the difficulty in establishing the ribozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditions as 3' product dissociation was rate-limiting. The equilibrium dissociation constant for the substrate was 24-fold higher than that of the 3' cleavage product. A substrate with a 2',5'-linkage at the cleavage site was cleaved with a rate constant (k(2)) of 1.1 x 10(-)(2) min(-)(1). Thus, whereas cleavage of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2', 5' cleavage followed a Michaelis-Menten mechanism.     

Kinetic studies of fructokinase I of pea seeds

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.     

Interaction of mitogen-activated protein kinases with the kinase interaction motif of the tyrosine phosphatase PTP-SL provides substrate specificity and retains ERK2 in the cytoplasm.

ERK1 and ERK2 associate with the tyrosine phosphatase PTP-SL through a kinase interaction motif (KIM) located in the juxtamembrane region of PTP-SL. A glutathione S-transferase (GST)-PTP-SL fusion protein containing the KIM associated with ERK1 and ERK2 as well as with p38/HOG, but not with the related JNK1 kinase or with protein kinase A or C. Accordingly, ERK2 showed in vitro substrate specificity to phosphorylate GST-PTP-SL in comparison with GST-c-Jun. Furthermore, tyrosine dephosphorylation of ERK2 by the PTP-SLDeltaKIM mutant was impaired. The in vitro association of ERK1/2 with GST-PTP-SL was highly stable; however, low concentrations of nucleotides partially dissociated the ERK1/2.PTP-SL complex. Partial deletions of the KIM abrogated the association of PTP-SL with ERK1/2, indicating that KIM integrity is required for interaction. Amino acid substitution analysis revealed that Arg and Leu residues within the KIM are essential for the interaction and suggested a regulatory role for Ser(231). Finally, coexpression of PTP-SL and ERK2 in COS-7 cells resulted in the retention of ERK2 in the cytoplasm in a KIM-dependent manner. Our results demonstrate that the noncatalytic region of PTP-SL associates with mitogen-activated protein kinases with high affinity and specificity, providing a mechanism for substrate specificity, and suggest a role for PTP-SL in the regulation of mitogen-activated protein kinase translocation to the nucleus upon activation.     

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.