Document Detail


Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface.
MedLine Citation:
PMID:  9070439     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI). The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.
Authors:
F W Herberg; B Zimmermann; M McGlone; S S Taylor
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  6     ISSN:  0961-8368     ISO Abbreviation:  Protein Sci.     Publication Date:  1997 Mar 
Date Detail:
Created Date:  1997-05-27     Completed Date:  1997-05-27     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  569-79     Citation Subset:  IM    
Affiliation:
Abt. für Biochemie Supramolekularer Systeme, Ruhr-Universität Bochum, Germany.
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MeSH Terms
Descriptor/Qualifier:
Catalysis
Cyclic AMP-Dependent Protein Kinases / chemistry,  metabolism*
Enzyme Stability
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Spectrum Analysis
Chemical
Reg. No./Substance:
EC 2.7.11.11/Cyclic AMP-Dependent Protein Kinases
Comments/Corrections

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