Abstract
No thorough mechanistic study of extracellular signal-regulated protein kinase 2 (ERK2) has appeared in the literature. A recombinant protein termed EtsDelta138, which comprises of residues 1-138 of the transcription factor Ets-1 is an excellent substrate of ERK2 (Waas W. F., and Dalby, K. N. (2001) Protein Exp. Purif. 23, 191-197). The kinetic mechanism of ERK2 was examined, with excess magnesium, by initial velocity measurements, both in the absence and presence of products at 27 degrees C, pH 7.5, and ionic strength 0.1 m (KCl). The velocity data are consistent with a steady-state random-ordered ternary complex mechanism, where both substrates have unhindered access to binding sites on the enzyme. The mechanism and magnitude of product inhibition by monophosphorylated EtsDelta138 is consistent with, but does not prove, the notion that ERK2 forms a discrete interaction with EtsDelta138 in the absence of active site interactions, and that this "docking complex" facilitates intramolecular phosphorylation of the substrate. The approximation of the steady-state data to a rapid equilibrium model strongly suggests that the formation of ERK2.Ets138 complexes are transient in nature with dissociation constants of greater magnitude than the catalytic constant, of k(cat) = 17 s(-1).
Highlights
No thorough mechanistic study of extracellular signal-regulated protein kinase 2 (ERK2) has appeared in the literature
The mechanism and magnitude of product inhibition by monophosphorylated Ets⌬138 is consistent with, but does not prove, the notion that ERK2 forms a discrete interaction with Ets⌬138 in the absence of active site interactions, and that this “docking complex” facilitates intramolecular phosphorylation of the substrate
ERK2 is activated through a protein kinase cascade termed the mitogen-activated protein kinase (MAPK) pathway, usually by Ras, a small guanine nucleotide-binding protein that transduces extracellular signals to the cell nucleus
Summary
Buffers and Reagents—Trizma (Tris) base was purchased from EM Industries (Gibbstown, NJ). Lysis buffer (L1) was 40 mM Tris-HCl, pH 7.0, 0.1% (v/v) -mercaptoethanol, 0.03% (by mass) Brij-30, 1% (by mass) Triton X-100, 0.75 M NaCl, 5 mM imidazole, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM TPCK. The Wash buffer (W1) was 40 mM Tris-HCl, pH 7.5, 0.1% -mercaptoethanol, 0.03% (by mass) Brij-30, 10 mM imidazole, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM TPCK. The elution buffer (E1) was 40 mM Tris-HCl, pH 8.0, 0.1% (v/v) -mercaptoethanol, 0.03% (by mass) Brij 200 mM imidazole. The HPLC buffer (H1) was 20 mM Tris-HCl, pH 8.0, 0.1% (v/v) -mercaptoethanol, 0.02% The activation buffer (P1) was 25 mM HEPES, pH 7.65, 2 mM DTT, 4 mM ATP, 20 mM MgCl2, 0.5 mM EGTA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
