Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 277, Issue 15, Pages 12532-12540Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110523200
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Funding
- NIEHS NIH HHS [P30 ES 07784] Funding Source: Medline
- NIGMS NIH HHS [GM 59802] Funding Source: Medline
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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 degreesC, 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).
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