Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

Claudine N. Prowse, Jonathan C. Hagopian, Melanie H. Cobb, Natalie G. Ahn, John Lew

Research output: Contribution to journalArticlepeer-review

53 Scopus citations


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)(η) ± 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 μM-1 s-1, k(cat)/K(m(ATP))(η) = -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 μM- 1 s-1, k(cat)/K(m(MBP))(η) = 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 μM-1 s-1, k(cat)/K(m(ERKtide))(η) = -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)) (≤0.5 μM) is significantly lower than that of the measured K(m(MBP)) (4.2 ± 0.8 μM). Furthermore, MBP binds to the ERK2·ATP complex at least 1500-fold more tightly than does ERKtide (K(d(ERKtide)) ≥ 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide (~600-fold difference) is largely attributable to the slow dissociation rate of MBP (≤ 1.2 s-1) versus that of the synthetic peptide (≥56 s-1), from the ERK2 active site.

Original languageEnglish (US)
Pages (from-to)6258-6266
Number of pages9
Issue number20
StatePublished - May 23 2000

ASJC Scopus subject areas

  • Biochemistry


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