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 mu M-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 mu M-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) mu M-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) (less than or equal to 0.5 mu M) is significantly lower than that of the measured K-m(MBP) (4.2 +/- 0.8 mu M). Furthermore, MBP binds to the ERK2.ATP complex at least 1500-fold more tightly than does ERKtide (K-d(ERKtide) greater than or equal to 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide (similar to 600-fold difference) is largely attributable to the slow dissociation rate of MBP (less than or equal to 1.2 s(-1)) versus that of the synthetic peptide (greater than or equal to 56 s(-1)), from the ERK2 active site.