Document Detail


Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release.
MedLine Citation:
PMID:  14567689     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.
Authors:
William F Waas; Mark A Rainey; Anna E Szafranska; Kevin N Dalby
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  42     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2003 Oct 
Date Detail:
Created Date:  2003-10-21     Completed Date:  2003-12-02     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  12273-86     Citation Subset:  IM    
Affiliation:
Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Base Sequence
Catalysis
DNA Primers
Dimerization
Kinetics
Mass Spectrometry
Mitogen-Activated Protein Kinase 1 / chemistry,  metabolism*
Molecular Sequence Data
Phosphorylation
Protein Conformation
Grant Support
ID/Acronym/Agency:
ES 07784/ES/NIEHS NIH HHS; GM59802/GM/NIGMS NIH HHS; P30 ES07784/ES/NIEHS NIH HHS; T32 GM08474/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/DNA Primers; EC 2.7.11.24/Mitogen-Activated Protein Kinase 1

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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