Document Detail


Exploring the role of the active site cysteine in human muscle creatine kinase.
MedLine Citation:
PMID:  16981706     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.
Authors:
Pan-Fen Wang; Allen J Flynn; Mor M Naor; Jan H Jensen; Guanglei Cui; Kenneth M Merz; George L Kenyon; Michael J McLeish
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  45     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2006 Sep 
Date Detail:
Created Date:  2006-09-19     Completed Date:  2006-11-08     Revised Date:  2013-06-07    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  11464-72     Citation Subset:  IM    
Affiliation:
Department of Medicinal Chemistry, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, USA.
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MeSH Terms
Descriptor/Qualifier:
Adenosine Diphosphate / metabolism
Animals
Binding Sites
Computer Simulation
Creatine / metabolism
Creatine Kinase, MM Form / chemistry*,  metabolism*
Cysteine / metabolism*
Humans
Hydrogen-Ion Concentration
Kinetics
Models, Molecular
Mutant Proteins / chemistry,  metabolism
Phosphorylation
Proline / chemistry
Serine / chemistry
Spectrophotometry, Ultraviolet
Structure-Activity Relationship
Torpedo
Grant Support
ID/Acronym/Agency:
GM44974/GM/NIGMS NIH HHS; R01 GM044974-16/GM/NIGMS NIH HHS; R01 GM066859-09/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Mutant Proteins; 147-85-3/Proline; 52-90-4/Cysteine; 56-45-1/Serine; 57-00-1/Creatine; 58-64-0/Adenosine Diphosphate; EC 2.7.3.2/Creatine Kinase, MM Form
Comments/Corrections

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


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