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)) (</=0.5 microM) is significantly lower than that of the measured K(m(MBP)) (4.2 +/- 0.8 microM). Furthermore, MBP binds to the ERK2 x 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 ( approximately 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.
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CN
Graduate Program in Biomedical Sciences, University of California, San Diego, California 92093, USA.
Hagopian
J C
JC
Cobb
M H
MH
Ahn
N G
NG
Lew
J
J
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D002417:Cattle; D006801:Humans; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry*; D004676:Myelin Basic Protein / Q000737:chemistry; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D019151:Peptide Library; D010766:Phosphorylation; D012997:Solvents; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014783:Viscosity
D004676:Myelin Basic Protein; D009842:Oligopeptides; D019151:Peptide Library; D012997:Solvents; D000255:Adenosine Triphosphate; D019950:Mitogen-Activated Protein Kinase 1