The structural, functional, and regulatory properties of the mitogen-activa
ted protein kinases (MAP kinases) have long attracted considerable attentio
n 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. W
e have employed steady-state kinetic and solvent viscosometric techniques t
o characterize the catalytic reaction pathway of the MAP kinase ERK2 with r
espect. to the phosphorylation of a protein substrate, myelin basic protein
(MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effec
t 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)) follo
wed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentra
tion 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 observe
d 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 va
lue for K-d(MBP) (less than or equal to 0.5 mu M) is significantly lower th
an 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 cat
alytic efficiency of MBP in comparison to that of ERKtide (similar to 600-f
old difference) is largely attributable to the slow dissociation rate of MB
P (less than or equal to 1.2 s(-1)) versus that of the synthetic peptide (g
reater than or equal to 56 s(-1)), from the ERK2 active site.